WO2010054125A1 - Formulations and uses of 24r, 25-dihydroxyvitamin d3 as an anti-apoptotic - Google Patents

Abstract

The various embodiments of the present disclosure relate generally to formulations and uses of Vitamin D and derivatives thereof for the treatment of osteoarthritis. More particularly, the various embodiments of the present disclosure relate to formulations and uses for 24R, 25-dihydroxyvitamin D₃ as an anti-apoptotic. One aspect of the present invention involves a method of treating osteoarthritis that comprises administering an effective amount of a composition comprising Vitamin D, such as vitamin D₃, or a derivative thereof to a subject for treatment of osteoarthritis.

Description

FORMULATIONS AND USES OF 24R, 25-DIHYDROXYVITAMIN D3 AS AN ANTI-APOPTOTIC

RELATED APPLICATION

This application claims, under 35 U.S. C. § 119(e), the benefit of U.S. Provisional Application Serial No. 61/111,482, filed 05 November 2008, the entire contents and substance of which are hereby incorporated by reference as if fully set forth below.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with U.S. Government support under Grant No. EEC-9731643 awarded by the National Science Foundation. The U.S. Government has certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The various embodiments of the present disclosure relate generally to formulations and uses of Vitamin D and derivatives thereof for the treatment of osteoarthritis. More particularly, the various embodiments of the present disclosure relate to formulations and uses for 24R, 25- dihrdoxyvitamin D₃ as an anti-apoptotic.

BACKGROUND OF THE INVENTION

Arthritis is a prevalent pathology that doctors have diagnosed in over 46 million Americans. The disease causes degradation of the cartilage that protects articulating joint surfaces. Doctors generally characterize osteoarthritis, one form of arthritis, by the symptom of pain in the joints during movement, though it manifests with varying severity for different people. Osteoarthritis tends to progress with age and is becoming more common as the large baby boomer generation ages. Although osteoarthritis is very prevalent, the mechanism of disease is not well understood and no existing treatments address its underlying causes. All current existing clinical treatments simply address the pain and inflammation associated with the degenerating joint. Current treatment regiments for osteoarthritis include some combination of pain relievers (e.g., acetaminophen, non-steroid anti-inflammatory drug (NSAID)), physical therapy, braces, or viscoelastic supplementation The goal of these treatment options is to minimize pain and increase patient mobility for as long as possible so as to delay the necessity of joint replacement surgery. Therefore, while there are various methods to treat pain and inflammation, none of these treatments offer a satisfactory long-term or restorative solution in the absence of surgery.

Accordingly, there is a need for new treatments for osteoarthritis, such as novel pharmaceutical formulations and methods of treatment. It is to the provision of such pharmaceutical formulations and methods of treatment that the various embodiments of the present invention are directed.

BRIEF SUMMARY OF THE INVENTION

The various embodiments of the present disclosure relate generally to formulations and uses of Vitamin D and derivatives thereof for the treatment of osteoarthritis. More particularly, the various embodiments of the present disclosure relate to formulations and uses for 24R, 25- dihrdoxyvitamin D3 as an anti-apoptotic.

An aspect of the present invention comprises a method of treating osteoarthritis, comprising: administering an effective amount of a composition comprising Vitamin D or a derivative thereof to a subject for the treatment of osteoarthritis. The composition comprising Vitamin D or a derivative thereof can comprise one or more of Vitamin D3, Vitamin D₂ or a derivative thereof. In one embodiment, the composition comprising Vitamin D or a derivative thereof comprises 24R, 25 -dihydroxy vitamin D3 or a derivative thereof. The method of treating osteoarthritis can involve an injection of an effective amount of a composition comprising Vitamin D, such as 24R, 25 -dihydroxy vitamin D₃ or a derivative thereof, into a joint. In one embodiment, the composition comprising Vitamin D or a derivative thereof can further comprise a viscosupplement. In another embodiment, the composition comprising Vitamin D or a derivative thereof can further comprise a lysophosphatidic acid.

Another aspect of the present invention comprises a method of inhibiting apoptosis in a cell, comprising: providing to a cell an effective amount of a composition comprising Vitamin D or a derivative thereof; and inhibiting apoptosis of the cell. In an embodiment of the present invention, the cell is a chondrocyte. This method can involve providing a composition comprising one or more of Vitamin D₃, Vitamin D₂, or a derivative thereof, such as 24R, 25- dihydroxyvitamin D₃ or a derivative thereof. This method can also involve the provision of a composition comprising Vitamin D or a derivative thereof and lysophosphatidic acid. Inhibition of apoptosis can be manifested through many cellular pathways and effectors associated with the apoptotic pathway, including but not limited to, reduction in the activity of caspase-3, reduction in the expression of p53, reduction in the activity of a matrix metalloproteinases, and stimulation of extracellular matrix production, among others.

The various embodiments of the present invention comprise a number of compositions and formulations to treat osteoarthritis through reducing the incidence of apoptosis. For example, a composition can comprise 24R, 25-dihydoxyvitamin D₃ and a lubricant. In such an embodiment, a lubricant can comprise one or more of a hyaluronic acid or lubricin. In another example, the composition can comprise 24R, 25-dihydoxyvitamin D₃ and at least one component of synovial fluid. In another example, the composition can comprise synovial fluid and a synthetic 24R, 25-dihydoxyvitamin D₃. Other aspects and features of embodiments of the present invention will become apparent to those of ordinary skill in the art, upon reviewing the following description of specific, exemplary embodiments of the present invention in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF DRAWINGS Figures IA-D illustrate Pi dose-dependently induces 24R,25(OH)₂D₃ sensitivity in

ATDC5 cells.

Figures 2A-D graphically depict 24R,25(OH)₂D₃ dose-dependently responds to Pi pretreatment, and Pi transport is required for Pi-induced 24,25 sensitivity.

Figures 3A-B demonstrate 24R,25(OH)₂D₃ recovers Pi-induced reduction of sulfate incorporation, but neither treatment necessary for collagen type 2 protein expression.

Figure 4 shows ATDC5 mRNA expression for chondrocytic markers during Pi and 24R,25 (OH)₂D₃ treatment.

Figures 5A-F shows time course effects of Pi and 24R,25(OH)₂D₃ on apoptosis and thymidine incorporation. Figures 6A-D demonstrate that resting zone chondrocytes produce LPA and express

LPA receptors.

Figures 7A-F graphically depict LPA increases maturation in resting zone chondrocytes. Figures 8A-C graphically show LPA enhances proliferation.

Figures 9A-F demonstrate that LPA protects cells from phosphate and chelerythrine- induced apoptosis.

Figures 10A-D illustrate that. LPA reduces p53 at the translational level but not at the transcriptional level. Figures 1 IA-C show Bax and Bcl-2 mRNA and protein abundance are regulated by LPA.

Figure 12 is displays a proposed mechanism of LPA signaling in the resting zone.

Figure 13 demonstrates that LPA1/3 signaling is necessary for 24R,25(OH)₂D₃- mediated rescue of Pi-induced apoptosis.

Figure 14 shows 24R,25(OH)₂D₃ reduces caspase-3 activity through PLD, PLC, and calcium signaling.

Figures 15A-B illustrate that Pi modulates ATDC5 responsiveness to 24R,25 (OH)₂D₃.

Figure 16 illustrates mechanisms of 24R,25 (OH)₂D₃ signaling in resting zone growth plate chondrocytes.

DETAILED DESCRIPTION

Throughout this description, various components can be identified as having specific values or parameters, however, these items are provided as exemplary embodiments. Indeed, the exemplary embodiments do not limit the various aspects and concepts of the present invention as many comparable parameters, sizes, ranges, and/or values can be implemented. The terms "first," "second," and the like, "primary," "secondary," and the like, do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. Further, the terms "a," "an," and "the" do not denote a limitation of quantity, but rather denote the presence of "at least one" of the referenced item. The various embodiments of the present invention relate generally to formulations and uses of Vitamin D and derivatives thereof for the treatment of degenerative diseases, such as osteoarthritis, which are characterized at the cellular level by apoptosis.

An aspect of the present invention comprises a method of treating osteoarthritis, comprising: administering an effective amount of a composition comprising Vitamin D or a derivative thereof to a subject for treatment of osteoarthritis. The term "treating" as used herein with regards to osteoarthritis may refer to preventing the condition or disorder, slowing the onset or rate of development of the condition or disorder, reducing the risk of developing the condition or disorder, preventing or delaying the development of at least one symptom associated with the condition or disorder, reducing or ending at least one symptom associated with the condition or disorder, generating a complete or partial regression of the condition or disorder, or some combination thereof. The Vitamin D of the present invention can include, but are not limited to, vitamin D₂ (ergocalciferol) or derivatives thereof, vitamin D₃ (cholecalciferol) or derivatives thereof, and combinations thereof.

Vitamin D₃ has a core structure comprising:

As used herein, a derivative of vitamin D₃ can include any substitution of a hydrogen or a functional group of the core structure with another functional group or chemical moiety. For example, lα, 25 -dihydroxy vitamin D₃ (lα, 25-(OH)₂D₃)

is a derivative of vitamin D₃. Similarly, 24R, 25 -dihydroxy vitamin D₃ (24, 25-(OH)₂D₃) OH

\

is a derivative of vitamin D₃. Another example of a derivative of vitamin D₃ is

lα-(hydroxyl ethyl)-3β,25-dihydroxy vitamin D₃. Yet another example of a derivative of vitamin D₃ is

lβ-(hydroxyl ethyl)-3α,25- dihydroxyvitamin D₃. A derivative of vitamin D₃ can also include any substitution of a backbone carbon atom with another element, functional group, or chemical moiety. For example, another derivative of vitamin D₃ can include

lα-(hydroxymethyl)-3β-hydroxy-20-epi-22-oxa-26,27-dihomovitamin D₃. In another yet example, a derivative of vitamin D3 can include

-(hydroxymethyl)-3α-hydroxy-20-epi-22-oxa-26,27-dihomovitamin D₃. Vitamin D₂ has a core structure comprising

As used herein, a derivative of vitamin D₂ can include any substitution of a hydrogen or a functional group of the core structure with another functional group or chemical moiety. A derivative of vitamin D₂ can also include any substitution of a backbone carbon atom with another element, functional group, or chemical moiety. For example, 24, 25- dihydroxy vitamin D₂ (24, 25-(OH)₂D₂) is a derivative of vitamin D₂.

Embodiments of the methods of treating osteoarthritis can comprise administering a therapeutically effective amount of Vitamin D or a derivative thereof. Administration of a Vitamin D or a derivative thereof may be performed by many known routes of administration, including, but not limited to, topical administration, oral administration, enteral administration, parenteral administration (e.g., epifascial, intraarterial, intracapsular, intracardiac, intracutaneous, intradermal, intramuscular, intraorbital, intraosseous, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, intravesical, parenchymatous, or subcutaneous administration), among others. In an exemplary embodiment of the present invention, a composition comprising Vitamin D or a derivative thereof can be administered by intracapsular injection into a joint.

The phrase "therapeutically effective amount" or "effective amount" as used herein is an amount of a compound that produces a desired therapeutic effect in a subject, such as preventing or treating osteoarthritis or alleviating one or more symptoms associated with osteoarthritis. The precise therapeutically effective amount is an amount of the composition that will yield effective results in terms of efficacy of treatment in a given subject. This amount (i.e., dosage) may vary depending upon a number of factors, including, but not limited to, the characteristics of the composition comprising Vitamin D or a derivative thereof (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease stage, general physical condition, and responsiveness to a given dosage), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. A therapeutically effective dose of a composition comprising Vitamin D or a derivative thereof may be administered daily, weekly, monthly, or over one or more years to treat or prevent osteoarthritis and its related symptoms. An effective dose may comprise from about 0.001 μg to about 1,000 mg/kg subject/day of a composition comprising Vitamin D or a derivative thereof. In another embodiment, an effective dose may comprise from about 0.01 μg to about 100 mg/kg subject/day of a composition comprising Vitamin D or a derivative thereof. In yet another embodiment, an effective dose may comprise from about 0.1 μg to about 10 mg/kg subject/day of a composition comprising Vitamin D or a derivative thereof. The terms "subject," "individual," or "cell" are used interchangeably herein, and refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, primates, humans, cows, dogs, mice, rabbits, pigs, rats, guinea pigs and equine. Tissues, such as cartilage, and cells are also encompassed by this terminology. In an exemplary embodiment of the present invention, a subject comprises a human. In an exemplary embodiment of the present invention, a cell comprises a chondrocyte.

The term "composition" as used herein can refer to one or more compounds or substances, such as a pharmaceutical compound, a therapeutic compound, an active agent, or the like. Thus, the term "composition," "formulation," and "compound" may be used interchangeably throughout this specification. In addition, the term "composition" or "formulation" can refer to more than compounds or substances. A composition or formulation may also comprise one or more pharmaceutical additives including, but not limited to, solubilizers, emulsifiers, lubricants, buffers, preservatives, suspending agents, thickening agents, stabilizers, inert components, and the like.

In an exemplary embodiment of the present invention, a composition comprising Vitamin D or a derivative thereof further comprises a lubricant. More specifically, the lubricant can be a viscosupplement. As used herein, a "viscosupplement" is a substance designed to supplement or substitute for the viscous properties of synovial fluid. In one embodiment, a viscosupplement can comprise one or more components of synovial fluid, which include but are not limited to, hyaluronic acid, lubricin, proteinases, and collagenases, or combinations thereof. In an exemplary embodiment, a viscosupplement can comprise hyaluronic acid (also called hyaluronan or hyaluronate).

In one embodiment of the present invention, a composition can comprise a synovial fluid and 24R, 25-dihydoxyvitamin D₃. In another embodiment, a composition can comprises a synovial fluid and a synthetic 24R, 25-dihydoxyvitamin D₃ In an exemplary embodiment of the present invention, a composition comprising

Vitamin D or a derivative thereof further comprises a lysophosphatidic acid, including but not limited to l-palmitoyl-2-hydroxy-s«-glycero-3-phosphate (16:0 LPA), l-stearoyl-2-hydroxy-sw- glycero-3 -phosphate (18:0 LPA), l-oleoyl-2-hydroxy-s«-glycero-3 -phosphate (18: 1 LPA), or combinations thereof.

In an embodiment of the present invention, methods for treating osteoarthritis can comprise administration of compounds or compositions comprising Vitamin D or an analog thereof and an active agent. As used herein, the term "active agent" can include, without limitation, agents for gene therapy, analgesics, anti-arthritics, anti-asthmatic agents, anticholinergics, anti-convulsants, anti-depressants, anti-diabetic agents, anti-diarrheals, anesthetics, antibiotics, antigens, anti-histamines, anti-infectives, anti-inflammatory agents, antimicrobial agents, anti-migraine preparations, anti-nauseants, anti-neoplasties, anti-parkinsonism drugs, anti-pruritics, anti-psychotics, anti-pyretics, antispasmodics, anorexics, anti-helminthics, antiviral agents, nucleic acids, DNA, RNA, polynucleotides, nucleosides, nucleotides, amino acids, peptides, proteins, carbohydrates, lectins, lipids, fats, fatty acids, viruses, antigens, immunogens, antibodies and fragments thereof, sera, immune stimulants, immune suppressors, sympathomimetics, xanthine derivatives, cardiovascular agents, potassium channel blockers, calcium channel blockers, beta-blockers, alpha-blockers, anti-arrhythmics, anti-hypertensives, diuretics, anti-diuretics, vasodilators comprising general, coronary, peripheral, or cerebral, central nervous system stimulants, vasoconstrictors, gases, growth factors, growth inhibitors, hormones, estradiol, steroids, progesterone and derivatives thereof, testosterone and derivatives thereof, corticosteroids, angiogenic agents, anti-angeogenic agents, hypnotics, immunosuppressives, muscle relaxants, parasympatholytics, psychostimulants, sedatives, tranquilizers, ionized and non-ionized active agents, anti-fungal agents, metals, small molecules, pharmaceuticals, hemotherapeutic agents, wound healing agents, indicators of change in the bio-environment, enzymes, nutrients, vitamins, minerals, coagulation factors, neurochemicals, cellular receptors, radioactive materials, cells, chemical or biological materials or compounds that induce a desired biological or pharmacological effect; and combinations thereof.

Another aspect of the present invention comprises a method of inhibiting apoptosis in a cell, comprising: providing to a cell an effective amount of a composition comprising Vitamin D or a derivative thereof; and inhibiting apoptosis of the cell. As used herein, the terms "inhibiting," "interfering," "preventing," "reducing," "decreasing," or "altering," refer to a difference in degree from a first state, such as an untreated state in a cell, to a second state, such as a treated state in a cell. For example, in the absence of treatment with the methods or compositions of the present invention, an osteoarthritic condition or symptom may occur at first rate or exist at a first state. If a cell is exposed to treatment with the methods or compositions of the present invention, the osteoarthritic condition or symptom occurs at a second rate or exists at a second state, which is altered, lessened, or reduced from the first rate or first state. Thus, the terms "interfering," "preventing," "reducing," "altering," or "inhibiting" may be used interchangeably through this application and may refer to a partial reduction, substantial reduction, near-complete reduction, complete reduction, or an absence of an osteoarthritic condition or symptom, such as apoptosis and the rate thereof.

Accordingly, a method for inhibiting the apoptosis in a cell involves a reduction in the rate or frequency of apoptosis in a cell. This inhibition manifest through many cellular pathways and effectors associated with the apoptotic pathway. For example, the methods and compositions of the present invention can reduce the activity of cellular proteins, such as caspase-3 or a matrix metalloproteinase, or can reduce the expression of a protein, such as p53. As used herein, the phrases "reduce the activity" or "reduce the expression" can refer to both direct and indirect reduction of the activity one or more of the proteins, direct or indirect reduction of the transcription of genes encoding one or more of the proteins, direct or indirect reduction in the translation of mRNAs encoding one or more of the proteins, or direct and indirect reduction in signaling pathways, upstream and/or downstream, of the protein. Furthermore, "reduce the activity" or "reduce the expression" can include partially reduction, substantial reduction, or complete reduction .

Similarly, the methods and compositions of the present invention can stimulate the activity of cellular proteins or cellular processes to inhibit apoptosis, such as stimulation of extracellular matrix production. As used herein, the phrases "stimulate the activity" or "stimulate the expression" can refer to both direct and indirect stimulation of the activity one or more of the proteins, direct or indirect stimulation of the transcription of genes encoding one or more of the proteins, direct or indirect stimulation in the translation of mRNAs encoding one or more of the proteins, or direct and indirect stimulation of signaling pathways, upstream and/or downstream, of the protein.

All patents, patent applications, and references included herein are specifically incorporated by reference in their entireties.

It should be understood, of course, that the foregoing relates only to exemplary embodiments of the present invention and that numerous modifications or alterations may be made therein without departing from the spirit and the scope of the invention as set forth in this disclosure. Therefore, while embodiments of this invention have been described in detail with particular reference to exemplary embodiments, those skilled in the art will understand that variations and modifications can be effected within the scope of the invention as defined in the appended claims. Accordingly, the scope of the various embodiments of the present invention should not be limited to the above discussed embodiments, and should only be defined by the following claims and all equivalents. The present invention is further illustrated by way of the examples contained herein, which are provided for clarity of understanding. The exemplary embodiments should not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention or the scope of the appended claims.

EXAMPLES

EXAMPLE 1: INORGANIC PHOSPHATE MODULATES RESPONSIVENESS TO 24,25(OH)₂D₃ IN CHONDROGENIC ATDC5 CELLS The formation of mammalian long bones occurs through the process of endochondral development, which begins with mesenchymal condensation in the embryo to form cartilaginous limb buds. Primary and secondary centers of ossification develop within the cartilage, ultimately becoming bone. The ends of the bones, the epiphyses, are separated from the metaphyses and diaphysis by a region of cartilage called a growth plate, which is spatially organized into zones defined by the differentiation state of chondrocytes resident in that region of the tissue. Nearest to the epiphysis is the reserve or resting zone. Chondrocytes in this region produce an extracellular matrix enriched in type II collagen and proteoglycan aggregates containing sulfated glycosaminoglycans. In embryonic bone, this region is relatively small as cells are rapidly progressing along the endochondral developmental pathway. In contrast, in post-natal growth plates, the resting zone serves as a chondrocyte reservoir and represents a larger component of the tissue. At the base of the resting zone, chondrocytes appear to align in columns to form the proliferative zone, in which they undergo rapid division, providing the major contribution of the growth plate to longitudinal bone growth. Following proliferation, the cells undergo a prehypertrophic phase, transitioning into hypertrophy, a period in which the cells remodel their extracellular matrix to accommodate their increase in size and to prepare the matrix for calcification. During this phase, many of the hypertrophic chondrocytes also undergo apoptosis, which causes the growth plate to retain a consistent length despite continued growth of the bone. This process depends upon coordinated mineralization of the matrix. In conditions like vitamin D and phosphate deficient rickets, where the growth plate fails to become calcified, the hypertrophic zone continues to increase in length.

Previous work examining mouse and rat growth plates has shown that two metabolites of vitamin D, 24,25 -dihydroxy vitamin D3 [24,25(OH)₂D₃] and 1,25-dihydroxy vitamin D3 [1,25(OH)₂D₃], each play a role in regulating the process of endochondral development. Chondrocytes from the resting zone exhibit specific sensitivity to 24,25(OH)₂D₃, whereas cells in the growth zone no longer exhibit the same responses to 24,25(OH)₂D₃ but have acquired specific sensitivity to 1,25(OH)₂D₃. 24R,25(OH)₂D₃ stimulates extracellular matrix production by resting zone cells, increasing production of sulfated glycosaminoglycans. In addition, it causes resting zone chondrocytes to produce extracellular matrix vesicles containing neutral metalloproteinases and reduces total matrix vesicle metalloproteinase activity in vitro and in vivo. In contrast, lα,25(OH)₂D3 inhibits DNA synthesis in prehypertrophic and hypertrophic chondrocytes and reduces synthesis of sulfated proteoglycans, while increasing production of alkaline phosphatase-enriched matrix vesicles that contain increased metalloproteinase activity. Moreover, lα,25(OH)₂D₃ acts directly on matrix vesicles produced by these cells, activating resident phospholipases, causing loss of membrane integrity and release of matrix processing enzymes. These observations suggest that 24R,25(OH)₂D₃ enhances matrix production and maintenance of resting zone cartilage, whereas 1(1,25(OH)₂Ds modulates the rate and extent of matrix degradation during chondrocyte hypertrophy. Interestingly, lα,25 (OH)₂D₃ induces production of 24R,25(OH)₂D₃ by growth zone chondrocytes, suggesting cross-talk among cells at different maturation states in endochondral development.

Inorganic phosphate (Pi) has also been implicated in the differentiation of the growth plate by acting as a signal affecting the differentiation of mineralization-competent cells. The extracellular concentration of Pi is relatively high in the extracellular matrix produced by hypertrophic chondrocytes, in part due to the increased activity of matrix vesicle alkaline phosphatase. Studies examining the effects of exogenous Pi on chondrocyte phenotype in post- fetal growth plates show that Pi can induce apoptosis. This suggests a feed-back loop in which lα,25(OH)₂D₃ activates matrix vesicle alkaline phosphatase, releasing Pi into the matrix and Pi then acts back on the chondrocytes to induce apoptosis. It is less clear how Pi might interact with 24R,25 (OH)₂D₃. To address this question, we took advantage of the embryonic ATDC5 cell model. This prechondrocyte cell line offers a useful culture system for studying the progression of endochondral development. When confluent cultures of ATDC5 cells are grown in high insulin media, they form cartilage nodules that exhibit the differentiation sequence typical of long bone growth plates. lα,25(OH)₂D₃ has been shown to inhibit proliferation and differentiation of ATDC5 cells, but it is not known if these cells are regulated by 24,25(OH)₂D₃. Interestingly, Pi has been shown to be a regulator of chondrogenic differentiation and apoptosis in these cells, including upregulation of collagen type X, a marker of maturation in the hypertrophic zone of the growth plate. Pi was also shown to regulate expression of matrix GIa protein (MGP) via ERK1/2 in both ATDC5 cells and primary growth plate organ cultures [24]. MGP is an inhibitor of matrix calcification, suggesting that Pi may induce production of factors that retard endochondral ossification like 24R,25(OH)₂D₃, as well as production of factors that stimulate chondrocyte maturation and apoptosis. The purpose of the present study was to determine if Pi treatment causes ATDC5 cells to become responsive to lα,25(OH)₂D₃ or 24R,25(OH)₂D₃ and if so, what are the consequences to endochondral maturation of the cells. The physiological importance of Pi is supported by the observation that active ion transport through the membrane is required. METHODS AND MATERIALS

Cell Culture. ATDC5 cells were cultured in a maintenance medium consisting of a 1 : 1 ratio of DMEM/F12 media (Cellgro, Manassas, VA) with 5% fetal bovine serum (FBS) (Hyclone, Logan, UT), 10 μg/ml human transferrin (Sigma Chemical Company, St. Louis, MO), 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA), and 3 x 10^~8 M sodium selenite (Sigma). After reaching confluence cells were cultured with differentiation media, which is identical to maintenance media with the addition of 10 μg/ml bovine insulin (Sigma) and 50 μg/ml ascorbic acid (Sigma). At 10 days post-confluence, cells were cultured for 24 hours in differentiation media supplemented with Pi (0 to 20 mM beyond media basal level ) and 10% FBS (10% FBS was used to ensure sufficient serum proteins such as fetuin that help regulate pathologic precipitation of calcium phosphate crystals). To make Pi-supplemented media, a more concentrated volume of Pi was dissolved in warm DMEM/F12 (37°C) using molar ratios of 4 moles of dibasic sodium phosphate Na₂HPO₃ (Sigma) to one mole of monobasic sodium phosphate NaH₂PO₃(Sigma). When the phosphate salts were completely dissolved, the pH was adjusted to 7.4, and the solution was filter sterilized. An appropriate aliquot from the concentrated Pi solution was added to the media preparation to result in the desired final concentration. Control cultures were also treated on day 10 with differentiation media with 10% FBS. Some experiments also included concurrent treatment with the Pi transporter inhibitor phosphonoformic acid (PFA) (sodium phosphonoformate tribasic hexahydrate) (Sigma) to test the effect of phosphate transport inhibition. Cells were returned to differentiation media with 5% FBS on day 11 for treatment with 24R,25(OH)₂D₃ or 1,25(OH)₂D₃ or ethanol vehicle (Sigma).

Cell Number. Effects of Pi and 24R,25 (OH)₂D₃ on proliferation were determined by measuring cell number at harvest and also as a function of DNA synthesis (described below). To measure cell number, ATDC5 cells were treated with Pi for 24 hours followed by treatment with 24R,25(OH)₂D₃ for 24 hours. At harvest, cells were washed twice with DMEM and trypsinized (Invitrogen) for 10 minutes. Cells were resuspended in saline, and counted on a Beckman Coulter Zl particle counter. Alkaline Phosphatase Activity. Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, alkaline]-specific activity was used as an indication of chondrocyte differentiation. Harvested cells were suspended in 0.05% Triton-X. After 3 freeze-thaw cycles to lyse the cells, alkaline phosphatase activity in the cell lysates was determined and normalized to protein content using the Macro BCA Protein Assay Kit (Pierce, Rockford, IL). r³⁵S]-Sulfate Incorporation. To determine the role of inorganic phosphate in mediating the effects of 24R,25(OH)₂D₃ on extra-cellular matrix production, proteoglycan synthesis was assessed by measuring [³⁵S]-sulfate incorporation as described previously [31, 32]. Four hours prior to harvest, [³⁵S]-sulfate (Perkin Elmer) was added to the cultures. Cell layers were collected and dialyzed to remove any unbound [³⁵S]-sulfate. Radiolabeled incorporated into the cell layer was expressed as disintegration per minute normalized to protein levels for each sample.

Histology. To verify that the ATDC5 cells produced a cartilage extracellular matrix, cultures were examined for collagen II protein by immunohistochemistry. Cells were seeded into four well chamber slides. Once the cells reached confluence they were cultured with differentiation media and were treated with 20 mM Pi on Day 10 and 10^"7 M 24,25(OH)₂D₃ on Day 11, respectively, or with the appropriate vehicle. The cells were fixed for 30 min in 4% formalin in PBS, after which the cells were rinsed three times in PBS and stored in 75% absolute ethanol. At time of staining, the fixed cultures were etched with 0.25% pepsin to expose the antigen, followed by PBS washes. Cell monolayers were blocked with 2% horse serum in PBS. Cells were then treated with the mouse anti-collagen II antibody (Hybridoma Bank, University of Iowa). After more PBS washes a biotinylated horse anti-mouse IgG antibody was applied. An alkaline phosphatase ABC kit was used to visualize the biotin. Samples were counterstained with haematoxylin, and visualized with light microscopy. RNA Extraction and RT-PCR. Cellular RNA was harvested using the Trizol® reagent kit (Invitrogen). RNA samples were converted to cDNA using the Omniscript RT kit (Qiagen, Valencia, CA) and then PCR was performed using HotStar Taq Master Mix Kit (Qiagen). PCR product was visualized using gel electrophoresis in 5% TBE Ready Gels (Biorad, Hercules, CA) and visualized on a Versadoc Model 1000 (Biorad). To better visualize qualitative differences between groups, densities of the visualized bands were measured using Quantity One 4.4.1 Software (Biorad). The gene specific primers (MWG Biotech, Huntsville, AL) used to amplify mRNA were as follows: aggrecan - 5'ATC ACA GCC ACC ACT TCC 3' (sense) and 5' CTC CAC TCA CAG ATG TTA TAC C 3' (anti-sense), collagen type 1 - 5' GGC TCC TGC TCC TCT TAG 3' (sense) and 5' TCT TCT GAG TTT GGT GAT ACG 3' (anti-sense), collagen type II - 5' GCG GTC CTA ACG GTG TCA G 3' (sense) and 5' ACC AGC CTT CTC GTC ATA CC 3' (anti-sense), collagen type X - 5' GCA CCT ACT GCT GGG TAA GC 3' (sense) and 5' GCC AGG TCT CAA TGG TCC TA 3' (anti-sense), cartilage oligomeric matrix protein (COMP) - 5' CCA CTG ATG ATG ACT ATG C 3' (sense) and 5' GAT GTA GCC AAC TTG AGG 3' (anti-sense), SOX9 - 5' GAA CGA GAG CGA GAA GAG ACC 3' (sense) and 5' GGC GGA CCC TGA GAT TGC 3' (anti-sense), and glyceraldehyde phosphate dehydrogenase (GAPDH) - 5' TTC AAC GGC ACA GTC AAG G 3' (sense) and 5' TCT CGC TCC TGG AAG ATG G 3' (anti-sense). The negative control was RNA from mouse liver tissue and the positive control was RNA from mouse cartilage (Zyagen, San Diego, CA).

DNA Synthesis. The effects of inorganic phosphate and 24R,25 (OH)₂D₃ on DNA synthesis were determined by measuring [³H]-thymidine incorporation into trichloroacetic acid (TCA, Sigma) insoluble cell precipitates as previously described. ATDC5 cells were treated for twenty-four hours with Pi followed by treatment with 24R,25(OH)₂D₃. Two hours prior to harvest, [³H]-thymidine (Perkin Elmer, Waltham, MA) was added. Radioactivity in TCA- precipitable material was measured by liquid scintillation spectroscopy.

Assays for Apoptosis. DNA Fragmentation: Cells were pre-labeled with [ H]- thymidine (Perkin Elmer) for 4 hours and then treated with Pi for 24 hours followed by 24R,25 (OH)₂D₃ for 24 hours, or with Pi followed by vehicle. Cell monolayers were washed with DMEM three times to remove any residual unincorporated [³H]-thymidine and cells were lysed with TE buffer (1OmM Tris-HCl, ImM EDTA, 0.2% Triton X-IOO) for 30 minutes. Cell lysates were centrifuged at 13,00Og for 15 minutes to separate intact DNA from fragmented DNA. The amount of incorporated [³H] -thymidine was determined in each fraction to establish the total amount of [³H]-DNA. Caspase-3 Activity: Caspase-3 activity was assessed using the colorimetric CaspACE™

Assay System (Promega, Madison, WI). Cells were harvested 24h post treatment with 200μl cell lysis buffer followed by two 10 second periods of sonication. After harvest, 2μl of the caspase-3 selective substrate DEVD-pNA were added to each well containing lOOμl of cell lysate and incubated at 37⁰C for 4h. DEVD-pNA cleavage into the colorimetric product pNA was measured at 405nm. Caspase-3 activity was normalized to protein content as determined by the Pierce Macro BCA Protein Assay Kit.

Statistical Analysis. Data are presented as means + standard error of the mean (SEM) for six independent cultures for each variable. The results for individual experiments are shown. To ensure validity of the results, all quantitative experiments were repeated at least two or more times. Data were analyzed with ANOVA followed by Bonferroni's modification of Student's T-test. Differences in means were considered to be statistically significant if the P value was less than 0.05. RESULTS

Pi treatment alone did not affect cell number except at the highest concentration (20 mM) tested (Figure IA). 24R,25(OH)₂D₃ caused a small but significant decrease in the control cultures and further decreased the effects of 2OmM Pi. The expanded dose response (Figure IB) confirmed that the effects of Pi on response to 24R,25(OH)₂D₃. Pi reduced ATDC5 cell number at 2OmM. Effects of 24R,25(OFf)₂D₃ depended on Pi concentration and were dose- dependent from 19.5 to 20.25 mM with the greatest effect at 2OmM.

Pi had a biphasic effect on alkaline phosphatase activity in the ATDC5 cell lysates, with an increase over control levels at 2OmM Pi (Figure 1C). Effects of lα,25(OH)₂D₃ and 24R,25(OH)₂D₃ on alkaline phosphatase were also sensitive to Pi pretreatment. lα,25(OH)₂D₃ and 24R,25(OH)₂D₃ reduced enzyme activity in control cultures. At 5 mM Pi, only 10^"8M lα,25(OH)₂D₃ reduced enzyme activity over that seen in Pi treated cells. At 15 mM Pi, both 24R,25(OH)₂D₃ and lα,25(OH)₂D₃ stimulated activity but at 2OmM Pi, 24R,25(OH)₂D₃ caused a 30% increase in alkaline phosphatase. The stimulatory effect of 2OmM Pi on 24R,25(OH)₂D₃-dependent alkaline phosphatase activity was confirmed in the expanded dose- response study (Figure ID).

The response of ATDC5 cells to 24R,25(OH)₂D₃ was dose-dependent following pretreatment with 20 mM Pi. The reduction in cell number was greatest at 10^"7M (Figure 2A) and the stimulatory effect of alkaline phosphatase was greatest at 10^"7 M 24R,25(OH)₂D₃ (Figure 2B). The effects of 20 mM Pi were specific based on their inhibition by phosphonoformic acid (PFA), which is a specific competitive inhibitor of the type III sodium dependent phosphate transporter Glvr-1. PFA caused a dose-dependent decrease in the Pi- induced reduction in cell number (Figure 2C) and a dose-dependent decrease in Pi-activated alkaline phosphatase (Figure 2D).

Treatment with 2OmM Pi on Day 10 reduced [³⁵S]-sulfate incorporation at the end of Day 11, but this was restored to control levels when Pi was followed by treatment with 10^"7 M 24R,25(OH)₂D₃ (Figure 3A). ATDC5 cells produced an extracellular matrix containing type II collagen, regardless of the treatment regimen. Cell layers stained positively with anti-type II collagen antibody whether they were untreated or treated with Pi followed by 24R,25(OH)₂D₃ (Figure 3B). Semiquantitative analysis by RT-PCR showed that mRNA expression of chondrogenic markers was affected (Figure 4). Pi treatment reduced collagen II mRNA, but dramatically increased collagen X mRNA. During direct exposure to Pi there was an increase in aggrecan and decrease in COMP mRNAs. By itself, 24R,25(OH)₂D₃ had minimal effect on any markers, but the steroid rescued collagen II mRNA and enhanced collagen X mRNA after pretreatment with Pi.

Pi caused an increase in ATDC5 apoptosis. Pi increased DNA fragmentation (Figure 5A), increased caspase-3 activity (Figure 5B), and reduced DNA synthesis (Figure 5C) by the end of Pi treatment. The stimulatory effect of Pi on apoptosis was reversed by subsequent treatment with 24R,25 (OH)₂D₃. 24R,25(OH)₂D₃ blocked DNA fragmentation in Pi-treated cells (Figure 5D), decreased caspase-3 activity (Figure 5E), and increased DNA synthesis (Figure 5F).

DISCUSSION

The results presented here demonstrate that exogenous Pi is a potent inducer of endochondral development, not only for hypertrophic cells as has been reported previously, but also for prechondrocytes. In response to relatively high levels of Pi, ATDC5 cells exhibited increased levels of mRNA for type II collagen and aggregan. These cells also exhibited markers of endochondral development, including reduced expression of the early differentiation marker Sox 9, reduced cell numbers and increased alkaline phosphatase specific activity as well as elevated expression of the later-stage marker of hypertrophic chondrocytes, collagen type X. Others have reported a dose dependent increase in collagen X in ATDC5 cells treated with Pi in the range of 3-3Oum, supporting our finding. Moreover, our results confirm that the effects of Pi on endochondral development were specific and were dependent on active transport of the ion because treatment of the cells with the phosphate transporter inhibitor phosphonoformic acid blocked the Pi-induced responses.

Interestingly, the Pi-induced chondrocytes were sensitive to both lα,25(OH)₂D₃ and 24R,25(OH)₂D₃ with respect to reduced cell number and increased alkaline phosphatase at Pi concentrations below 20 mM, but in cultures treated with 20 mM Pi, there was a very specific enhancement of response to the 24R,25(OH)₂D₃ metabolite of vitamin D3. This was unanticipated since studies using rat and mouse costochondral growth plate chondrocytes have shown that resting zone cells are the primary target for 24R,25(OH)₂D₃, whereas prehypertrophic and hypertrophic chondrocytes are primary targets for lα,25 (OH)₂D₃. Moreover, 20 mM Pi induced sensitivity of the ATDC5 cells to 10^"7 M 24R,25(OH)₂D₃, which is the concentration at which costochondral resting zone cells exhibit maximal responses to the seco-steroid and similar to the level of endogenous 24R,25(OH)₂D₃ produced by these cells when stimulated in culture.

These observations suggest that 24R,25(OH)₂D₃ may serve to protect the early endochondral chondrocytes from premature terminal differentiation due to high levels of exogenous Pi. Our results support this hypothesis. 24R,25(OH)₂D₃ blocked the inhibitory effect of Pi on [³⁵S]-sulfate incorporation. Moreover, it blocked the stimulatory effects of Pi on apoptosis, based on two different indicators of cell death. 24R,25(OH)₂D₃ increased DNA synthesis, reduced DNA fragmentation, and reduced caspase-3 activity in Pi-treated ATDC5 cells.

The fact that the effects of Pi treatment on sensitivity to 24R,25(OH)₂D₃ were so narrowly focused in terms of dose may indicate that one or more critical conditions must be met with precision to invoke the need for response to the steroid during endochondral ossification in the embryo. The spatial and temporal sequence of events in embryonic bone formation differs from post-fetal bone growth. Thus, Pi and 24R,25(OH)₂D₃ may act together to reduce proliferation and begin the process of hypertrophy, but as alkaline phosphatase increases generating higher levels of exogenous Pi, 24R,25(OH)₂D₃ acts as a brake on the apoptotic process induced by the active uptake of Pi.

In summary, our study demonstrates the value of the ATDC5 prechondrocyte model for studying factors that modulate endochondral ossification, as noted by others [19, 24, 36]. Our results confirm the importance of exogenous Pi in regulating the differentiation and maturation of chondrocytes in endochondral development. Most importantly, they show that Pi treatment induces sensitivity to vitamin D metabolites 24R,25(OH)₂D₃ and lα,25(OH)₂D₃ in a dose- dependent manner and at the higher concentrations of Pi, the cells become specifically responsive to 24R,25(OH)₂D₃. This metabolite acts with Pi to reduce cell number and increase endochondral differentiation, but at the same time it blocks the activation of apoptosis, suggesting a role for modulating the rate of terminal differentiation in embryonic bone formation.

EXAMPLE 2: LYSOPHOSPHATIDIC ACID SIGNALING PROMOTES

PROLIFERATION, DIFFERENTIATION, AND CELL SURVIVAL IN RAT GROWTH PLATE CHONDROCYTES Lysophosphatidic acid (LPA 18: 1; 1 -oleoyl-2-hydroxy-sn-glycero-3 -phosphate) is a bioactive lysophospholipid that consists of a single fatty acid chain and is produced by activated platelets and cancer cell types. LPA is derived from a number precursor lipids including phosphatidic acid (PA) which is generated by the metabolism of phosphatidylcholine (PC) by phospholipase D (PLD). LPA exerts its effects on cells by activating the cell surface G-protein coupled receptors (GPCRs) LPA1/Edg2, LPA2/Edg4, LPA3/Edg7, LPA4/GPR23, and LPA5/GPR92. These receptors collectively stimulate the G_αi, G_αq, G_αs, and G_αi2/i3 signaling pathways. In addition to G-protein coupled receptors, LPA has been shown to activate the nuclear fatty acid receptor peroxisome proliferator-activated receptor gamma (PPAR-γ). LPA signaling has been implicated in a wide array of cellular processes including wound healing and smooth muscle contraction as well as cell proliferation, survival, and migration. These latter functions support a role for LPA signaling in cancer progression, where LPA has been shown to promote tumorigenesis by enhancing adhesion, migration, and invasion.

LPA appears to be involved in regulation of bone and cartilage. LPA has been shown to regulate osteoblasts and chondrocytes are also sensitive to the lipid mediator. These studies suggest that LPA may also be involved in endochondral ossification, a process involving the formation of bone upon a cartilage template and the mechanism by which long bones in children and adolescents lengthen. This template is the result of growth, maturation, and calcification of growth plate cartilage, which is regulated in part by the vitamin D metabolites 1,25-dihydroxy vitamin D3 [lα,25(OH)₂D₃] and 24,25-dihydroxy vitamin D3 [24R,25(OH)₂D₃]. LPA acts synergistically with lα,25 (OH)₂D₃ to promote osteoblast differentiation, providing evidence of a relationship between this metabolite and LPA signaling, but it is not known if there is a relationship between LPA and 24R,25(OH)₂D₃.

The resting zone of the growth plate provides a reservoir of chondrocytes that will eventually undergo terminal differentiation, hypertrophy, and apoptosis as the growth plate matures. The cells in the resting zone are surrounded by a proteoglycan-rich extracellular matrix and apoptosis is a relatively infrequent event. These cells respond in particular to the 24R,25(OH)₂D₃, resulting in increased cell maturation, matrix synthesis, and cell survival. 24R,25 (OH)₂D₃ acts on resting zone chondrocytes via a PLD-dependent mechanism and many 24R,25(OH)₂D₃-mediated effects in resting zone chondrocytes have been shown to be dependent upon PLD activation.

These observations implicate LPA as a second messenger during the promotion of cell maturation and survival in chondrocytes by 24R,25 (OH)₂D₃. However, the downstream targets by which LPA exerts its effect on growth plate chondrocytes are unknown. One possibility is that LPA acts by modulating the abundance of the tumor-suppressor p53. LPA has been shown to promote the degradation of p53 in several cancer cell types, resulting in increased cell survival. Reduction of p53 protein abundance is necessary in osteoblast maturation suggesting that LPA-mediated decreases in p53 may be important in the maintenance of cartilage tissue as well.

The purpose of this study was to investigate the role of LPA signaling in the maintenance of the growth plate resting zone. Specifically, we assessed how LPA regulates proliferation, maturation, and apoptotic cell death in growth plate chondrocytes using resting zone cells isolated from adult rat costochondral growth plate cartilage as our model system. We found that LPA enhances two markers of chondrocyte maturation: alkaline phosphatase enzymatic activity and [³⁵S]-sulfate incorporation. In addition, LPA was found to be a potent stimulator of proliferation. Lastly, LPA protects resting zone chondrocytes from apoptotic cell death by decreasing the abundance of the tumor suppressor p53 to alter p53 target gene expression and protein abundance. Collectively, these data suggest that LPA signaling promotes cellular proliferation, maturation and survival in resting zone chondrocytes demonstrating a novel physiological function of LPA signaling and providing evidence that LPA produced by the cells in response to 24R,25 (OH)₂D₃ stimulation may act to mediate its effects on resting zone chondrocytes. MATERIALS AND METHODS

Reagents. 18: 1 LPA (l-oleoyl-2-hydroxy-s«-glycero-3 -phosphate), OMPT ((2S)-I- oleoyl-2-O-methyl-glycero-3-phosphothionate), and VPC32183(S) ((S)-phosphoric acid mono- {2-octadec-9-enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl]-propyl} ester) were purchased from Avanti Polar Lipids (Alabaster, AL). All lipid species were reconstituted in 1% charcoal- stripped bovine serum albumin (BSA) prior to treatment of cells. Unless otherwise stated, all other reagents were acquired from VWR International (West Chester, PA).

Cell Culture. The culture system used in this study has been previously described in detail. Chondrocytes were obtained from the resting zone (reserve zone) of costochondral cartilage from 125-g male Sprague-Dawley rats and cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1% antibiotics, and 50 μg/ml ascorbic acid (GIBCO-BRL, Gaithersburg, MD). Primary cells were cultured until fourth passage prior to experimental analysis. LPA Isoforms. The abundance of LPA isoforms in lysates and media collected from the resting zone chondrocyte cultures was determined by liquid chromatography electrospray ionization tandem mass spectrometry (LC ESI MS/MS). Resting zone chondrocytes were cultured in T75 flasks and grown until confluence. Cells were then treated for 30 minutes with starvation media (1% FBS) containing 1% bovine serum albumin (BSA) and 24R,25(OH)₂D₃ (10^~9,10^~8, or 10^"7M) (BioMol, Plymouth Meeting, PA) or vehicle alone. After the treatment period, ImI of conditioned media was collected and cell monolayers were harvested using 0.05M sodium hydroxide (NaOH). Media and lysate samples were spiked with 1 pmol of 17:0 LPA (internal standard) prior to lipid extraction via the Bligh-Dyer method [38]. LC ESI MS/MS analysis was conducted at the Georgia Institute of Technology Bioanalytical Mass Spectrometry Facility using a Shimadzu HPLC pump and a Q-TRAP 4000 (Applied Biosystems, Foster City, CA). Reverse phase chromatography was performed using a Supelco Cl 8 column with a flow rate of 500μl/min and an injection volume of lOμl. The isocratic buffers were 75:25 methanokwater (v/v) and methanol each with 5mM TEAA. Eluted samples were then analyzed on the QTRAP 4000 and LPA isoforms were compared to LPA standards supplied by Avanti Polar Lipids. Peak areas of LPA isoforms were normalized to 17:0 peaks to account for differences in lipid extraction efficiency.

LPA Receptor, Bax, Bcl-2, and p53 Expression. mRNA was harvested from resting zone chondrocytes using Trizol (Invitrogen, Carlsburg, CA) and reverse transcriptase polymerase chain reaction (RT-PCR) was used to identify the presence of the LPA receptors LPA 1-5 and PPAR-γ. The following sequence specific primers were used: LPAl sense: 5'- GGTTCTCTACGCTCACATC-3', LPAl antisense 5 '-GCAGTAGCAAGACCAATCC-S ', LPA2 sense: 5 '-CACCACCTCACAGCCATCC-S ', LPA 2 antisense: 5'- AGACATCCACAGCACTCAGC-3', LPA3 sense: 5 '-CTACAACAGGAGCAACAC-S ', LPA3 antisense: 5'-CCAGCAGGTAGTAGAAGG-S ', LP A4 sense: 5'- ACAACTTTAACCGCCACTGG-3', LP A4 antisense: 5'-ATTCCTCCTGGTC CTGATGG-3', LPA5 sense: 5'-ACCTTGGTGTTCCCTATAATGC-S ', LPA5 antisense: 5'- AGCCAGAGCGTTGAGAGG-3', PPAR-γ sense: 5 '-CCGAAGAACCATCCGATTGAAG-S ', and PPAR-γ antisense: 5'-CTCCGCCAACAGCTTCTCC-S ' . In order to determine the effect of LPA on p53, Bax, and Bcl-2 mRNA expression, cells were treated with 0, 0.01, 0.1, and lμM LPA for 6 h prior to harvesting the mRNA with Trizol. The following primers were used to amplify p53, Bax, and Bcl-2: p53 sense: 5 '-CCGTCCCAGAAGGTTGCC-S ', p53 antisense: 5'-CGC TGC TCC GAA GGT GAT-3', Bax sense: 5 '-TTTGTTACAGGGTTTCATCC-S ', Bax antisense: 5 '-CCAGTTCATCTCCAATTCG-S ', Bcl-2 sense: 5'- CTCGTGGCTGTCTCTGAAG-3', Bcl-2 antisense: 5'-TCTGCTGACCTCACTTGTG-S ' . Glyceraldehyde-3 -phosphate dehydrogenase (GADPH) was amplified as a control in each experiment: GAPDH sense: 5'-ATGCAGGGATGATGTTC-S', GAPDH antisense: 5'- TGCACCA CCAACTGCTTAG-3'.

Chondrocyte Maturation Assays. Confluent cultures were treated with LPA (0.0 InM to lμM) for the times indicated below. To determine if the LPA 1/3 receptor was involved, cultures were also treated with LPA in the presence or absence of the LPA 1/3 -selective agonist (2S)-l-oleoyl-2-O-methyl-glycero-3-phosphothionate (OMPT) (Avanti Lipids, Alabaster, AL) (0. InM to lμM) or the LPA 1/3 -selective antagonist (S)-phosphoric acid mono-(2-octadec-9- enoylamino-3-[4-(pyridine-2-ylmethoxy)-phenyl] -propyl) ester (VPC32183(S)) (Avanti Lipids) (0.01 μM to lμM). Chondrocyte maturation was assessed by examining alkaline phosphatase specific activity and [³⁵S]-sulfate incorporation.

To determine that 24R,25 (OH)₂D₃ elicited its effects via an LPA-dependent mechanism, confluent cultures of resting zone cells were cultured for 24 hours in the presence and absence of the LPA1/3 selective antagonist VPC32183(S). Alkaline phosphatase specific activity was measured as described below.

Alkaline Phosphatase Specific Activity. Initial experiments determined the optimal time course by treating confluent cultures with lμM LPA for 3, 6, 12, 18, and 24 hours. Subsequent experiments were performed after treating the cells for 24 hours. Following treatment, cell monolayers were lysed using 0.1% Triton X followed by sonication of each sample for 30 seconds. Alkaline phosphate activity was measured in cell layer lysates as a function of release ofpαrα-nitrophenol from pαrα-nitrophenylphosphate at pH 10.2. Activity was normalized to the protein concentration of the lysates, determined using the macro-BCA assay (Macro BCA, Pierce Chemical Co., Rockford, IL).

[³⁵S1-Sulfate Incorporation. Mature chondrocytes produce a proteoglycan-rich extracellular matrix that is characterized by sulfated glycosaminoglycans. To assess the effects of LPA on chondrocyte maturation, confluent cells were labeled with [³⁵S]-sulfate 3 hours prior to harvest. At harvest, the conditioned media were removed, the cell layers (cells and matrix) were collected, and the amount of [³⁵S]-sulfate incorporated was determined as a function of protein in the cell layer.

DNA Synthesis. To determine if LPA regulated chondrocyte proliferation, DNA synthesis was assessed by measuring the incorporation of radio-labeled thymidine. Cells were grown to subconfluence and treated with DMEM containing 1% FBS for 48 hours to induce quiescence. Cells were then treated with LPA (O.lnM to lμM) in the presence or absence of OMPT (O. lnM to lμM) or VPC32183(S) (0.0 lμM to lμM) for 24 hours. Prior to harvest, cell were labeled for 3 hours with [³H]-thymidine. The monolayers were washed three times with phosphate buffer solution (PBS) to remove unincorporated [ H]. Cells were then fixed with cold 5% trichloroacetic acid followed by lysis with 1% sodium dodecyl sulfate. The amount of [³H] activity was determined in each sample to determine the total amount of incorporated radio-labeled thymidine.

Apoptosis Assays. The role of LPA in chondrocyte survival was assessed by examining its ability to reduce apoptosis induced by two apoptogens, inorganic phosphate and chelerythrine. Apoptotic cell death was determined by measuring by caspase-3 activity, TUNEL staining, and DNA fragmentation. Confluent cultures of resting zone chondrocytes were treated with either 10^"5M chelerythrine or 7.5mM monobasic sodium phosphate to induce apoptosis. LPA (0.01 μM, 0.1 μM, or 1 μM), VPC32183(S) (0.01 μM, 0.1 μM, or 1 μM), or the vehicle was added to the cultures.

Caspase-3 Activity: Caspase-3 activity was determined using the Colorimetric CaspACE™ Assay System from Promega (Madison, WI). Cells were harvested 24h post treatment with 200μl cell lysis buffer followed by two 10 s periods of sonication. After harvest, 2μl of the caspase-3 selective substrate DEVD-pNA were added to each well containing lOOμl of cell lysate and incubated at 37⁰C for 4h. DEVD-pNA cleavage into the colorimetric product pNA was measured at 405nm. Caspase-3 activity was normalized to protein content as determined by the Pierce Macro BCA Protein Assay Kit.

DNA Fragmentation: Cells were labeled with [³H] -thymidine for 4 h prior to treatment. At the end of the treatment period, cell monolayers were washed with DMEM three times to remove unincorporated [³H] and cells were lysed with TE buffer (1OmM Tris-HCl, ImM EDTA, 0.2% Triton X-100) for 30 minutes. Cell lysates were centrifuged at 13,000g for 15 minutes to separate intact DNA from fragmented DNA. The amount of incorporated [³H] -thymidine was determined in each fraction to establish the total amount of fragmented DNA.

TUNEL Staining: DNA nicking was measured using the In Situ Cell Death Detection Kit (Roche Applied Science, Indianapolis, IN). After treatment, cells were fixed using 4% paraformaldehyde in PBS for lhour. To detect nicks, cells were incubated with horse radish peroxidase-conjugated dUTP for lhour at 37 ⁰C. Nicks were visualized with DAB substrate (3,3-diaminobenzidinetetrahydrochloride) also purchased from Roche. Regulation of p53. Confluent cultures in T75 flasks were treated for 6 h with 0, 0.01, 0.1, and lμM LPA. After treatment, the cell monolayer was washed twice with PBS and harvested with RIPA buffer. mRNA for p53 was determined as described above. The abundance of p53 protein in both the whole cell lysate and nuclear and cytoplasmic fractions was determined by ELISA (p53 pan ELISA, Roche) and normalized to total cellular or total fraction protein as determined by Pierce Macro BCA Protein Assay Kit. Nuclear and cytoplasmic fractions were isolated by centrifuging whole cell lysates for 10 minutes at 13,000 rpm. The resulting supernatants (cytoplasmic fraction) were collected and the pellets (nuclear fraction) were resuspended in 500μL RIPA buffer. To assess changes in p53-mediated transcription, luciferase reporter gene assays were conducted as previously described [44]. Cells were transfected with two plasmids: one containing p53 -controlled firefly luciferase (pp53_TA-Luc, Clonetech, Mountain View, CA) and the other carrying constitutively expressed Renilla luciferase (pLR-TK, Promega, Madison, WI). 24 h after transfection, cells were treated with 0, 0.01, 0.1, and lμM LPA for 16 h and luciferase activity was measured using the Dual Luciferase Reporter Assay kit (Promega, Madison, WI).

Abundance of Bax, Bcl-2, and p21 Protein. Western blots were performed to determine the effect of LPA on the protein abundance of Bax, Bcl-2, and p21. Cell culture lysates were prepared from confluent resting zone cells and were resolved on 10% SDS-polyacrylamide gels. Blots of the gels were probed with monoclonal antibodies against Bax (Δ 21, Santa Cruz Biotechnology, Inc.), Bcl-2 (DC 21, Santa Cruz Biotechnology, Inc.), p21 (BD Pharmingen, San Jose, CA), or GAPDH (MAB374 Chemicon, Billerica, MA). Immunoreactive bands were detected using 1 :5,000 dilutions of horseradish peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA), and visualized using enhanced chemiluminescence (Super-Signal WestPico Chemiluminescent Substrate (Pierce Biotechnology, Rockford, IL). mRNA for Bax and Bcl-2 were performed as described above.

Statistical Analysis. Each experiment had six independent cultures per variable to ensure sufficient power to detect statistically significant differences. All experiments were conducted multiple times to validate the observations, but data from a single representative experiment are shown in the figures and are expressed as means + SEM. Statistical analysis was conducted using ANOVA analysis followed by Student's T-test with a Bonferroni modification. Differences in means were considered to be statistically significant if the p value was less than 0.05. RESULTS

Resting Zone Chondrocytes Produce LPA and Express LPA Receptors. LC ESI

MS/MS showed that three LPA isoforms, 16:0 LPA, 18: 1 LPA, and 18:0 LPA, were present in both the cell monolayer (Fig. IA) and conditioned media (Fig. IB), indicating the existence of both intracellular and extracellular LPA in cultures of RC cells. 24R,25(OH)₂D₃ increased the abundance of 16:0 and 18: 1 LPA in the media, but not in the cell monolayer. The effect of

24R,25 (OH)₂D₃ on both LPA forms was dose-dependent and was greatest in cultures treated with 10^"8 M. RT-PCR using sequence-specific primers detected the presence of the five cell surface G-protein coupled receptors, LPA 1-5, and the nuclear receptor PPAR-gamma (Fig. 1C). Distinct bands for LPAl, LP A3, and LP A4 were observed. Bands for LP A2 and LPA5 were present but to a lesser extent.

24R,25(OH)₂D3 exerted its effects on chondrocyte maturation via an LPA1/3 dependent mechanism. The LPA 1/3 -selective antagonist VPC32183(S) attenuated 24R,25(OH)2D3- mediated increases in alkaline phosphatase activity (Fig. ID). Exogenous LPA Enhances Chondrocyte Maturation. Initial time course experiments showed that LPA increased alkaline phosphatase specific activity at 24 hours, but no LPA effect was observed prior to this time point (data not shown). For this reason, all future maturation experiments were conducted following a 24 hour exposure to the lipid mediator. Both LPA and the LPA 1/3 -selective agonist OMPT increased alkaline phosphatase activity in the resting zone chondrocytes in a dose dependent manner at concentrations ranging from 0.0 lμM to lμM (Fig. 2A and 2C). The same concentrations of LPA and OMPT also increased [³⁵S]-sulfate incorporation (Fig. 2B and 2D). Furthermore, VPC32183(S) attenuated LPA-mediated increases in both alkaline phosphatase activity and [³⁵S]-sulfate incorporation in a dose dependent manner (Fig 2E and 2F), indicating that the effects of LPA stimulation are dependent upon activation of LPAl and/or LP A3.

LPA Increases DNA Synthesis in a Dose-Dependent Manner. Treatment of pre- confluent cells culture with lμM LPA or OMPT enhanced DNA synthesis 100% over control cultures (Fig. 3A and 3B). Inhibition of LPAl and LPA3 with VPC321283(S) inhibited LPA- mediated increases in proliferation (Fig. 3C). These data demonstrate LPA promotes proliferation in resting zone chondrocytes through activation of LPAl and/or LP A3.

LPA Reduces the Stimulatory Effects of Phosphate and Chelerythrine Apoptosis. Both phosphate and chelerythrine increased DNA fragmentation relative to the control in a dose- dependent manner (Fig. 4A and 4B). LPA doses ranging from 0.0 lμM to lμM completely and partially rescued phosphate and chelerythrine-induced DNA fragmentation, respectively. Similarly, LPA reduced DNA nicking induced by both apoptogens, evidenced by reduced TUNEL staining (Fig 4C). Inorganic phosphate and chelerythrine also increased caspase-3 activity and LPA reduced this marker of apoptosis as well (Fig. 4D, 4E). The rescue of Pi- induced caspase-3 activity by LPA was attenuated by VPC32183 (S) (4F).

LPA Promotes Cell Survival via p53 Signaling. Control cultures of resting zone chondrocytes expressed p53 mRNA (Fig. 5A) and protein (Fig. 5B). Treatment with LPA had no effect on p53 mRNA at 6 hours, but there was a decrease in p53 protein at this time point. Nuclear p53 protein was decreased by LPA, whereas, cytoplasmic p53 did not change in response to the treatment (Fig 5C). Both p53-mediated transcription (Fig. 5D) and the abundance of the p53-target gene p21 (data not shown) were decreased by LPA. Moreover, LPA decreased both the mRNA expression (Fig. 6A) and protein abundance (Fig. 6B) of Bax. Conversely, both Bcl-2 mRNA and protein abundance were increased by LPA (Fig. 6A, 6B). DISCUSSION Our results indicate that LPA is an autocrine regulator in the growth plate resting zone.

Resting zone chondrocytes contain intracellular LPA and secrete extracellular LPA. The LPA isoforms identified, 16:0, 18: 1, and 18:0, are the most biologically relevant of the LPA isoforms. In addition, LPA receptors are present in RC cells, demonstrating that they have the potential to respond to this phospholipid metabolite. Moreover, 24R,25(OH)₂D3 increased the extracellular abundance of LPA 16:0 and LPA 18: 1 and the LPA 1/3 -selective antagonist VPC32183(S) attenuated 24R,25(OH)₂D3-mediated maturation, suggesting that LPA may act as downstream mediators of vitamin D metabolite effects on resting zone cells.

This study demonstrates that LPA signaling plays a role in the maintenance of the resting zone cartilage by promoting the survival of the chondrocyte pool. At least two mechanisms are involved in the inhibition of apoptosis. LPA acts via reduced p53 and its downstream mediator p21, reduced Bax and increased Bcl-2. This is particularly important because resting zone cells serve as the pool for the growth zone and premature cell death in the resting zone could result in premature closure of the growth plate and limb shortening. In addition, LPA stimulated DNA synthesis, suggesting that LPA is involved in maintaining the pool of resting zone chondrocytes via proliferation. LPA has been shown to act as a mitogen in other systems, as well as in primary rat articular chondrocytes. However, it decreased proliferation of T/C-28a2 cells, a human articular chondrocyte-like cell line, raising the possibility that its effects are cell specific. In addition to its stimulatory effects on DNA synthesis, LPA increased [³⁵S]-sulfate incorporation, suggesting an increase in the synthesis of a sulfated proteoglycan extracellular matrix around the newly generated chondrocytes. Alkaline phosphatase specific activity also increased, supporting the hypothesis that the chondrocytes were producing a mature matrix containing alkaline phosphatase-rich extracellular matrix vesicles.

Our results strongly support a physiological role for LPA in promoting chondrocyte survival in the resting zone. We demonstrated by four different methods (DNA fragmentation, TUNEL staining, caspase-3 activity, and Bcl-2/Bax ratio) that LPA signaling reduced the induction of apoptosis by two agents shown previously to stimulate the apoptotic pathway in resting zone chondrocytes in vitro. Moreover, the ability of LPA to rescue the apoptotic effect of Pi was attenuated by the LPA1/3 receptor antagonist, indicating that LPA1/3 signaling was responsible. The timing of programmed cell death is crucial in the maintenance of the growth plate. Inhibition of apoptotic signaling in hypertrophic chondrocytes prevents their terminal differentiation, resulting in lengthening of the growth plate as is typically seen in the vitamin D- deficient rickets. Phosphate plays an important role in this process, but it isn't known if the phosphate content is also a regulator of apoptosis in the resting zone.

Cell proliferation is associated with an increase in protein kinase C (PKC) in many cell types, and LPA reduced the effects of PKC inhibition by chelerythrine. Previously we have showed that 24R,25(OH)₂D3 stimulates PKC and cell proliferation in resting zone chondrocytes via a PLD-dependent mechanism. Our results suggest that LPA may mediate this response.

The LPA-induced decrease in the cellular abundance of the tumor suppressor p53 may be involved as well. The reduction of p53 correlates enhanced cell survival, indicating that the inhibition of p53 is the mechanism of LPA-mediated protection against cell death. The inhibition of p53 has also been implicated in the maturation of osteoblasts, suggesting that LPA-mediated decreases in p53 may be significant in chondrocyte maturation in addition to enhancing survival in these cells. LPA altered p53-mediated transcription and the expression of the p53-target genes p21, Bax and Bcl-2 at the transcriptional and translational level. The change in the cellular Bax to Bcl-2 ratio would result in the inhibition of the release of cytochrome c from the mitochondria, halting the initiation of the apoptotic proteolytic caspase cascade. This is supported by our finding that LPA inhibits chelerythrine and phosphate- induced caspase-3 activity via an LPA 1/3 mediated mechanism. Collectively, our results define a pathway for LPA-mediated enhancement of cell survival and chondrocyte maturation by which LPA decreases the abundance of p53 to alter p53-target gene expression resulting in the inhibition of caspase activity.

In summary, LPA was found to be a stimulator of resting zone chondrocyte proliferation and maturation and an inhibitor of chondrocyte apoptosis. This confirms a physiological role for LPA as a regulator of growth plate cartilage, and suggests that LPA produced via 24R,25(OH)₂D3-stimulated PLD activity may mediate the actions of the secosteroid in growth plate resting zone chondrocytes. Additionally, this establishes LPA as a potential therapeutic regulatory agent in controlling the processes of endochondral bone formation during long bone growth and development and during fracture repair.

EXAMPLE 3: 24R,25-DIHYDROXYVITAMIN D3 [24R,25(OH)₂D₃] CONTROLS GROWTH PLATE DEVELOPMENT BY INHIBITING APOPTOSIS IN THE RESERVE ZONE AND STIMULATING RESPONSE TO lα,25(OH)2D3 IN HYPERTROPHIC CELLS INTRODUCTION Chondrocytes isolated from the resting zone of rat growth plates respond preferentially to the vitamin D metabolite 24R,25-dihydroxyvitamin D3 [24,25(OH)₂Ds], with increased alkaline phosphatase activity and [³⁵S]-incorporation, decreased DNA synthesis, and increased neutral matrix metalloproteinase (MMP) activity. Similar observations have been made in avian growth plate chondrocytes. 24,25(OH)₂D3-mediated effects are maintained in mice lacking functional nuclear vitamin D receptors (n VDRd) and are not inhibited by Ab99, a blocking antibody targeted against the 1,25-dihydroxyvitamin D3 [1,25(OH)₂Ds] membrane receptor protein disulfide isomerase family A, member 3 (PDIA3). Additionally, the effects of 24,25 (OH)₂D3 are rapid, inducing protein kinase C (PKC) activation in as little as 9 minutes, with peak activity at 90 minutes. These observations indicate that 24R,25(OH)₂D₃ acts through a membrane-associated receptor (mVDR_24;25) that is distinct from the lα,25(OH)₂D₃- responsivie PDIA3 and functional nVDRs, but do not rule out the possibility that 24R,25 (OH)₂D₃ is acting via a VDR variant.

Rapid Actions of 24.25(OH)₉D₃ in the Resting Zone. The actions of 24,25(OH)₂D₃ in the resting zone chondrocytes are mediated through rapid activation of PKC. 24,25(OH)₂D₃ increases the abundance of diacylglycerol (DAG), an activator of many PKC isoforms. PKC activation by 24,25(OH)₂D₃ is maintained in the presence of chemical inhibitors targeted against either phosphatidylcholine (PC)- or phosphatidylinositol (PΙ)-specific phospholipase C (PLC), indicating that the source of diacylglycerol (DAG) is not due to the action of this enzyme. Inhibition of tyrosine kinase signaling also does not attenuate rapid actions of 24,25(OH)₂D₃, eliminating tyrosine kinases as a source of PKC activation. Instead, activation of PKC by 24,25(OH)₂D₃ is dependent upon DAG derived from phosphatide acid (PA) generated the actions of by phospholipase D (PLD), specifically PLD2. A second phospholipid-dependent mechanism also contributes to the rapid actions of

24,25(OH)₂D₃. Inhibition of phospholipase A2 (PLA₂) enhances PKC activation by 24,25(OH)₂D₃, whereas PLA2 activating protein (PLAA) decreases this. This is in contrast to 1,25(OH)₂D₃, which elicits its rapid effects via PLA₂-activation. Inhibition of PLA₂ by 24,25(OH)₂D₃ results in a rapid decrease in arachidonic acid (AA) abundance and cyclooxygenase-1 (COX-I) activity. Following the initial decrease in PLA2, 24R,25(OH)₂D₃ upregulates arachidonic acid turnover, altering fluidity of the plasma membrane, and increasing the production of prostaglandins El and E2 (PGEl, PGE2) to induce protein kinase A (PKA) activity. Inhibition of PKA mitigates 24,25(OH)₂D₃-induced rapid signaling and chondrocyte maturation, demonstrating the importance of this signaling pathway. Together, activated PKC and PKA promote MEK and ERK1/2 signaling in response 24,25(OH)₂D₃ to induce changes in gene transcription to promote chondrocyte maturation.

The Role of Lysophosphatidic Acid Signaling. Recently our group has focused the role of lysophospholipids in 24R,25(OH)₂D₃-mediated effects in the growth plate. As previously mentioned, 24R,25(OH)₂D₃-induced PLD activity results in the production of DAG, which stimulates PKC. Another consequence of PLD activation is the production of lysophosphatidic acid (LPA), a bioactive lysophospholipid that has recently been implicated in the regulation of bone and cartilage. These findings implicated LPA as a second messenger in 24,25(OH)₂D₃- directed signaling. We found that 24,25(OH)₂D₃ increased the abundance of extracellular LPA and LPA receptor 1 (LPAl) mRNA. Additionally, inhibition of LPAl and LP A3 attenuated 24,25(OH)₂D₃-induced chondrocyte maturation and cell survival. Resting zone chondrocytes responded to LPA with increased DNA synthesis, alkaline phosphatase activity, and [³⁵S]- incorporation. Furthermore, LPA protected chondrocytes against inorganic phosphate (Pi)- induced apoptosis by activating the phosphoinositol 3 -kinase (PI₃K) and murine double minute 2 (mdm2) signaling, resulting in the degradation of p53 and a decrease in p53 -mediated transcription. Interestingly, this is the same mechanism by which LPA enhances cell survival in cancer cells. We have also observed that Gβγ-mediated PLC activation also contributes to the inhibition of Pi-induced apoptosis by 24,25(OH)₂D₃ (Hurst-Kennedy, Boyan, et al, unpublished data). The stimulation of the pro-survival actions of LPA by 24,25(OEThDs establishes an anti- apoptotic function for the metabolite.

24,25 (OH)?D₃_and Phosphate-Induced Apoptosis. Pi induces apoptosis in terminally differentiated chondrocytes, allowing for the invasion of blood vessels and the deposition of new bone. Recently, we have observed that resting zone chondrocytes also undergo apoptosis in response to Pi as evidenced by an increase in DNA fragmentation and caspase-3 activity in response to Pi in male and female resting zone chondrocyte cultures. Normally the Pi content of the resting zone cartilage matrix is comparatively low whereas in the hypertrophic cell zone, marked increases in l,25(OH)₂D₃-dependent alkaline phosphatase result in high Pi content. 24,25(OH)₂Ds causes a small increase in alkaline phosphatase, which may cause an increase in local Pi. Perhaps more importantly, 24,25(OH)₂D₃ stimulates resting zone cells to produce 1, 25(OH)₂D₃ by increasing expression of 1 -hydroxylase. This suggests that Pi-induced apoptosis is dependent on the chondrocyte microenvironment rather than differentiation state.

We have also observed Pi-induced apoptosis in ATDC5 cells, a mouse chondrogenic cell line that exhibits a resting zone chondrocyte-like phenotype as evidenced by expression of collagens II and X, Sox9, and cartilage oligomeric matrix protein (COMP) [2, 29]. ATDC5 cells respond to 24,25(OH)₂D₃ with increased alkaline phosphatase activity and decreased cell number. Treatment with Pi increases DNA fragmentation and caspase-3 activity, both of which are mitigated by 24,25(OH)₂D₃. Furthermore, 24,25(OH)₂D₃ attenuates Pi-induced decreases in DNA synthesis and [³⁵S]-incorporation. Taken together, these results support the hypothesis that 24R,25(OH)₂D₃ enhances cell survivability in the presence of Pi. Additionally, these data suggest the existence of an inhibitory feedback loop in the resting zone between Pi and 24,25(OH)₂D₃.

MATERIALS AND METHODS Cell Culture. Chondrocytes were obtained from the resting zone (reserve zone) of costochondral cartilage from 125-g male Sprague-Dawley rats and cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 1% penicillin-streptomycin (Invitrogcn. Carlsbad, CA), and 50 μg/ml ascorbic acid (GIBCO-BRL, Gaithersburg, MD). Experiments were conducted using fourth passage cells. ATDC5 cells were cultured in a maintenance medium consisting of a 1: 1 ratio υf

DMEM/F12 media (Cellgro, Manassas, VA) with 5% FBS, 10 rag/ml human transferrin (Sigma Chemical Company, St. Louis, MO), 1% antibiotics, and 3xlO^~sM sodium sclenite (Sigma). After reaching confluence cells were cultured with differentiation media, which is identical to maintenance media with the addition of 10 mg/ml bovine insulin CSigma) and 50 μg/ml ascorbic acid (Sigma). At 10 days post-confluence, cells were cultured for 24 h in differentiation media supplemented with Pi (0-20 mM beyond media basal level) and 10% FBS (10% FBS was used to ensure sufficient serum proteins such as fetuin that help regulate pathologic precipitation of calcium phosphate crystals).

Caspase-3 Activity. The role of 24R,25(OH)₂D₃ in resting zone chondrocyte survival was assessed by examining its ability to reduce caspase-3 activity induced by inorganic phosphate (Pi). Confluent cultures of resting zone chondrocytes were treated for 24h with 7.5mM monobasic sodium phosphate to induce apoptosis. At the same time, cells were treated with 24R,25(OH)₂D₃ (10^"7M). VPC32183(S) (10^"8-10^"6M, Avanti Polar Lipids, Alabaster, AL) was used to inhibit LPA1/3 signaling; U73122 (lOμM, Sigma, St. Louis, MO) were used to inhibit PC-PLC. Thapsigargin (3μM, Sigma, St. Louis, MO) was used to block release of calcium from the endoplasmic reticulum, while wortmannin (lOμM, Calbiochem, Gibbstown, NJ) was used to inhibit PLD and PI₃K signaling. Caspase-3 activity was determined using the colorimetric CaspACE™ Assay System from Promega (Madison, WI). Cells were harvested 24h post treatment with 200μl cell lysis buffer followed by two 10 s periods of sonication. After harvest, 2μl of the caspase-3 selective substrate DEVD-pNA were added to each well containing lOOμl of cell lysate and incubated at 37⁰C for 4h. DEVD-pNA cleavage into the colorimetric product pNA was measured at 405nm. Caspase-3 activity was normalized to protein content as determined by the Pierce Macro BCA Protein Assay Kit.

Chondrocyte Maturation. Alkaline phosphatase [orthophosphoric monoester phosphohydro- lase, alkaline] -specific activity was used as an indication of chondrocyte differentiation. Confluent cultures of ATDC5 cells were treated with vehicle alone (control) or treated with 24R,25(OH)₂Di (10^"7M) and/or 2OmM Pi. Harvested cells were suspended in 0.05% Triton-X followed by three freeze-thaw cycles to lyse the cells. Alkaline phosphate activity was measured in cell layer lysates as a function of release of pαrα-nitrophenol from pαrα-nitrophenylphosphate at pH 10.2. Activity was normalized to the protein concentration of the lysates, determined using the macro-BCA assay (Macro BCA, Pierce Chemical Co., Rockford, IL). DNA Fragmentation. Regulation of Pi-induccd apoptosis by 24R, 25 (OFTs₂D₃ in ATDC5 cells was assessed by examining DNA fragmentation. Confluent cultures of ATDC5 cells w^rcre treated with vehicle alone (control), 24R,25(OH)_?Di ( 10^"7M), Pi (2OmM), or a combination of the aforementioned. Cells were labeled with [³H] -thymidine for 4 h prior to treatment. At the end of the treatment period, cell monolayers were washed with DMEM three times to remove unincorporated [³H] and cells were lysed with TE buffer (1OmM Tris-HCl, ImM EDTA, 0.2% Triton X-100) for 30 minutes. Cell lysates were centrifuged at 13,000g for 15 minutes to separate intact DNA from fragmented DNA. The amount of incorporated [³H] -thymidine was determined in each fraction to establish the total amount of fragmented DNA.

Statistical Analysis. Each experiment had six independent cultures per variable to ensure sufficient power to detect statistically significant differences. All experiments were conducted multiple times to validate the observations, but data from a single representative experiment are shown in the figures and are expressed as means + SEM. Statistical analysis was conducted using ANOVA analysis followed by Student's T-test with a Bonferroni modification. Differences in means were considered to be statistically significant if the p value was less than 0.05. RESULTS

24R.25(OH)₂D₃ Inhibits Apoptosis in the Resting Zone Through LPA. PLC. PLD. and Calcium Signaling. Pi treatment increased caspase-3 activity in the male rat resting zone chondrocytes relative to untreated control (Fig. 1). The addition of

24R,25(OH)2D3,25(OH)₂D₃ reduced caspase-3 activity to basal level. The LPA1/3 receptor antagonist VPC32183(S) inhibited 24R,25(OH)₂D₃-mediated rescue of Pi-induced apoptosis in a dose dependent manner. The PC-PLC inhibitor U73122, the intracellular calcium inhibitor thapsigargin, and the PLD/PI₃K inhibitor wortmannin also inhibited the reduction of caspase-3 activity by 24R,25(OH)₂D₃ (Fig. 2).

Pi Modulates ATDC5 Responsiveness to 24R.25 (OH)₂D₃. 24R,25(OH)₂D₃ did not induce an increase in alkaline phosphatase activity in ATDC5 cells that were not pre-treated with Pi (Fig. 3A). However, ATDC5 cells that were pre-treated with Pi did exhibit an increase in alkaline phosphatase activity in response to 24R,25 (OH)₂D₃. Pi treatment increased DNA fragmentation relative to untreated control (Fig. 3B). 24R25(OH)₂D₃ attenuated the increase in DNA fragmentation levels caused by Pi. SUMMARY

In summary, 24R,25(OH)₂D₃ regulates less mature growth plate chondrocytes through rapid activation of mVDR_24;25. This results in PLA2 and PLD-mediated phospholipid metabolism and activation of PKC to induce chondrocyte maturation. Our findings demonstrate that 24R,25(OH)₂D₃ also protects chondrocytes from apoptosis induced by Pi in their microenvironment. Collectively, this suggests that 24R,25(OH)₂D₃ stabilizes chondrocytes in the resting zone by inhibiting degradation characteristic of apoptotic hypertrophic chondrocytes. This implies that 24R,25 (OH)₂D₃ modulates growth plate development by controlling the rate and extent of chondrocyte transition from resting zone to growth zone phenotype.

Claims

CLAIMSWhat is claimed is:

1. A method of treating osteoarthritis, comprising: administering a therapeutically effective amount of a composition comprising Vitamin D or a derivative thereof to a subject for treatment of osteoarthritis.

2. The method of treating osteoarthritis of Claim 1, wherein the composition comprising Vitamin D or a derivative thereof comprises one or more of Vitamin D3, Vitamin D₂ or a derivative thereof.

3. The method of treating osteoarthritis of Claim 1, wherein the composition comprising Vitamin D or a derivative thereof comprises

or a derivative thereof.

4. The method of treating osteoarthritis of Claim 3, wherein the composition comprising Vitamin D or a derivative thereof comprises

, OH

OH

or a derivative thereof.

5. The method of treating osteoarthritis of Claim 1, wherein the administering a therapeutically effective amount of a composition comprises injecting a therapeutically effective amount of a composition into a joint.

6. The method of treating osteoarthritis of Claim 5, wherein the composition comprising Vitamin D or a derivative thereof comprises

OH

HO' or a derivative thereof.

7. The method of treating osteoarthritis of Claim 6, wherein the composition comprising Vitamin D or a derivative thereof further comprises a viscosupplement.

8. The method of treating osteoarthritis of Claim 1, wherein the composition comprising Vitamin D or a derivative thereof further comprises a lysophosphatidic acid.

9. A method of inhibiting apoptosis in a cell, comprising: providing to a cell a therapeutically effective amount of a composition comprising Vitamin D or a derivative thereof; and inhibiting apoptosis of the cell.

10. The method of inhibiting apoptosis in a cell of Claim 9, wherein the cell is a chondrocyte.

11. The method of inhibiting apoptosis in a cell of Claim 9, wherein the composition comprising Vitamin D or a derivative thereof comprises one or more of Vitamin D₃, Vitamin D₂, or a derivative thereof.

12. The method of inhibiting apoptosis in a cell of Claim 9, wherein the composition comprising Vitamin D or a derivative thereof comprises

OT a derivative thereof.

13. The method of inhibiting apoptosis in a cell of Claim 12, wherein the composition comprising Vitamin D or a derivative thereof comprises

, OH

,-*

OH

.U \ H

HO' "-' or a derivative thereof.

14. The method of inhibiting apoptosis in a cell of Claim 9, wherein the composition comprising Vitamin D or a derivative thereof further comprises a lysophosphatidic acid.

15. The method of inhibiting apoptosis in a cell of Claim 9, wherein inhibiting apoptosis of the cell comprises reducing the activity of caspase-3.

16. The method of inhibiting apoptosis in a cell of Claim 9, wherein inhibiting apoptosis of the cell comprises reducing the expression of p53.

17. The method of inhibiting apoptosis in a cell of Claim 9, wherein inhibiting apoptosis of the cell comprises reducing the activity of a matrix metalloproteinases.

18. The method of inhibiting apoptosis in a cell of Claim 9, wherein inhibiting apoptosis of the cell comprises stimulating extracellular matrix production.

19. A composition comprising 24R, 25-dihydoxyvitamin D₃ and a lubricant.

20. The composition of Claim 19, wherein the lubricant comprises at least one component of synovial fluid.

21. The composition of Claim 20, wherein the 24R, 25-dihydoxyvitamin D₃ is a synthetic 24R, 25-dihydoxyvitamin D₃.

22. The composition of Claim 19, wherein the lubricant comprises one or more of a hyaluronic acid or lubricin.

23. A composition comprising 24R, 25-dihydoxyvitamin D₃ and a lysophosphatidic acid.

24. The composition of Claim 23, wherein the lysophosphatidic acid comprises 1-palmitoyl- 2-hydroxy-s«-glycero-3-phosphate.

25. The composition of Claim 23, wherein the lysophosphatidic acid comprises l-oleoyl-2- hydroxy-SM-glycero-3 -phosphate