WO1995014104A1 - Procede d'identification d'une substance apte a induire la formation osseuse - Google Patents

Procede d'identification d'une substance apte a induire la formation osseuse Download PDF

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Publication number
WO1995014104A1
WO1995014104A1 PCT/US1994/013215 US9413215W WO9514104A1 WO 1995014104 A1 WO1995014104 A1 WO 1995014104A1 US 9413215 W US9413215 W US 9413215W WO 9514104 A1 WO9514104 A1 WO 9514104A1
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cells
protein
substance
kidney
mrna
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PCT/US1994/013215
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English (en)
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H. William Harris
Ana Paredes
Anabel Piqueras
Michael Somers
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Children's Medical Center Corporation
Creative Biomolecules, Inc.
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Priority to AU11801/95A priority Critical patent/AU1180195A/en
Publication of WO1995014104A1 publication Critical patent/WO1995014104A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • Osteogenic protein-1 is a member of the transforming growth factor (TGF) - ⁇ family which induces de novo bone formation by stimulating collagen synthesis and proliferation and differentiation of osteoblasts.
  • TGF transforming growth factor
  • the OP-1 protein was initially isolated in demineralized bone matrix.
  • the murine OP-1 gene encoding the homolog of human OP-1 (Ozkaynak, E., et a_l. EMBO J.
  • OP-1 in the kidney suggests its potential role as a link between this organ and cellular elements in the skeleton, and may explain an earlier observation (Huggins, Arch. Surer. , 22:377-408 (1931)) regarding the capability of the urinary tract epithelium to induce new bone formation.
  • the present invention relates to Applicants' characterization of mRNA expression and protein synthesis of osteogenic protein-1 (OP-1) in kidney sections. Additionally, by screening a variety of cultured renal epithelial cell lines, Applicants have found a cell line with markedly high baseline OP-1 mRNA expression. As a result of Applicants' work, presented herein, it has been determined, for the first time, that OP-1 mRNA transcripts and proteins are present in the renal medulla and that inner medullary collecting duct (IMCD) cells synthesize abundant OP-1 protein.
  • IMCD inner medullary collecting duct
  • identification of a substance capable of inducing new bone formation is determined by evaluating the effect the substance has on inducing OP-1 mRNA expression in mammalian cells .
  • Mammalian cells are grown in culture and the substance to be tested (i.e., the test substance) is introduced into the cell culture medium, or into the cell.
  • the test substance is introduced in an amount which would produce a detectable increase in the expression of OP-1 mRNA if the substance possesses OP-1 mRNA inducing properties.
  • the cells are maintained in culture under conditions which support cell growth and RNA transcription, for a period of time sufficient for RNA transcription to occur.
  • the cellular RNA is then isolated, and the amount of OP-1 mRNA expressed by the cultured cells is determined.
  • the amount of OP-1 mRNA expressed by the cultured cells in the presence of test substance is compared to the amount of OP-1 mRNA expressed by cultured cells under similar conditions, but without the presence of the test substance, to determine the effectiveness of the test substance to induce OP-1 mRNA.
  • identification of a substance capable of inducing new bone formation is determined by evaluating the effect the substance has on inducing the synthesis OP-1 protein in mammalian cells.
  • Mammalian cells are grown in culture and the test substance is introduced into the cell culture medium, or into the cell.
  • the test substance is introduced in an amount which would produce a significant increase in the synthesis of OP-1 protein if the substance possesses OP-1 protein inducing properties.
  • the cells are maintained in culture under conditions which support cell growth and protein synthesis and the amount of OP-1 protein synthesized by the cultured cells is determined.
  • the amount of OP-1 protein synthesized by the cultured cells in the presence of test substance is compared to the amount of OP-1 protein synthesized by cultured cells under similar conditions, but without the presence of the test substance, to determine the effectiveness of the test substance to induce OP-1 protein synthesis.
  • the present invention provides a method of therapeutically treating a patient with bone disease, such as osteoporosis, to induce bone growth (bone morphogenesis) .
  • This method comprises administering to the patient an effective amount of a substance capable of inducing bone growth by stimulating OP-1 mRNA expression, or OP-1 protein synthesis.
  • the present invention provides a method of therapeutically treating a patient with a bone disease, characterized by overproduction of bone (i.e., osteopetrosis) to suppress bone formation.
  • This method comprises administering to the patient an effective amount of a substance capable of suppressing bone growth by suppressing OP-1 mRNA expression, or OP-1 protein synthesis.
  • the present invention provides a rapid and sensitive method for screening candidate substances (test substances) to determine their effect on OP-1 mRNA expression, or OP-1 protein synthesis and secretion, and thus, determine the effect of the substance on bone formation. It also provides a model system to study the interaction of OP-1 protein with other known regulators of bone homeostasis, such as vitamin D, calcitonin, parathyroid hormone or growth factors such as TGF- ⁇ , and other substances like mineralocorticoids, and the response of OP-1 production to changes in pH, calcium, phosphorous concentration. Finally, the present invention provides a therapeutic treatment for patients suffering from bone disease.
  • Figure IA and IB depicts the murine OP-1 cDNA sequence (SEQ ID NO: 1) and encoded amino acid sequence (SEQ ID NO: 2) .
  • Figure 2 is a photograph of a Northern blot showing the results of Poly A * RNA from kidney (cortex, medulla and papilla) , lung, liver, brain, ovary and small intestine probed with OP-1 cDNA.
  • Figure 4 is a photograph of an immunoblot showing the results of Western blot analysis showing that 12G3 recognizes OP-1 protein.
  • the arrows denote the positions of molecular weight standards expressed as molecular mass X 10 3 .
  • Figure 5 is a photograph of an immunoblot showing that mouse monoclonal antibody 12G3 identifies multiple OP-1 protein bands in homogenates from rat kidney and cultured mouse inner medullary collecting duct (IMCD) cells.
  • the small arrowheads point to the top (T) and bottom (B) of the gel.
  • Figure 6A-6D is a series of micrographs showing that mouse monoclonal 12G3 localizes OP-1 staining to cells in kidney glomeruli, tubules and vessels in rat kidney.
  • Figure 7A-H is a series of micrographs showing localization of OP-1 expression in mouse kidney by in si tu hybridization.
  • Figures 8A-B are photographs of a Northern blot showing the results of northern analysis of MDCK and IMCD cells probed with OP-1 cDNA.
  • Applicants have determined, by expression studies in dissected kidney sections and immunohistochemistry analysis, that the renal medulla contains high levels of the OP-1 protein. Specifically, Applicants have demonstrated that OP-1 mRNA is highly expressed in inner medullary collecting duct cells. Based on Applicants results, a rapid and sensitive method for screening candidate substances to determine their effect on OP-1 mRNA expression, or OP-1 protein synthesis, and thus, determining the effect of the substance on bone formation in now provided.
  • RNA extracted from kidney sections were analyzed by Northern blot analysis using a 32 P-labeled OP-1 cDNA probe (SEQ ID NO: 1) .
  • the probe shown in Figure 1, b.p. 370-1050, is a 680 b.p. fragment that covers two thirds of the pro-domain and one third of the mature murine OP-1 cDNA, immediately upstream to the highly conserved 7-cysteine domain present in all members of the TGF-jS superfamily (Ozkaynak, E., et. al. , Biochem. Biophvs. Res.
  • the relative mRNA expression for the 2.4 and 4.0 kb transcripts was respectively higher in the renal medulla (77.6 ⁇ 11.2% and 65.0 ⁇ 8.3% ) followed by cortex (72.7 ⁇ 12.2% and 41.6 ⁇ 7.8% ) and papilla (62.7 ⁇ 8.3% and 53.9 ⁇ 11.9%) .
  • Brain showed the lowest signal (9.0 ⁇ 3.3 and 8.3 ⁇ 3.0) whereas ovary (18.8 ⁇ 5.2 and 15.1 ⁇ 3.1) elicited the highest extrarenal expression.
  • Results for liver (15.2 ⁇ 5.2 and 8.2 + 2.0) , intestine (12.4 ⁇ 3.7 and 8.1 + 2.3) and lung (10.8 + 3.0 and 8.2 ⁇ 2.0) were more variable.
  • the OP-1 cDNA predicts a primary translation polypeptide of 49 kD that includes a signal secretory sequence (29 A.A or -3.5 kD) , a pro domain (-270 A.A. or 31 kD) containing two polybasic proteolytic processing sites and a TGF-/3 domain (138 A.A. or 16 kD) .
  • OP-1 is a glycoprotein (Sampath, T.K., et . al . . , J. Biol . Chem. , 265:13198-13205 (1990) ; Sampath, T.K., et al. , J. Biol. Chem. , 267:20352-20362 (1992)) possessing 4 distinct potential glycosylation sites that would increase the apparent molecular mass of any of these OP-1 polypeptides.
  • a monoclonal antibody, antibody 12G3, was produced as described in detail in Example 2.
  • 0.5 ugs of spent media derived from CHO cells expressing recombinant human OP-1 was probed. (Sampath, T.K., et al., J. Biol. Chem. , 267:20352-20362 (1992) ; Jones, W.K., et . al . . , Bone and Mineral Research, S348.
  • monoclonal 12G3 recognized 3 bands of 65 kD, 44 kD and broad band of 27-19 kD. As reported previously (Jones, W.K., et . al. , Bone and Mineral Research, S348. (1993)) . These bands correspond to the glycosylated primary translation OP-1 polypeptide (65 kD) , pro-OP-1 (44 kD) and mature OP-1 (27-19 kD) proteins. These data demonstrate that 12G3 recognizes an epitope present on both primary and mature OP-1 proteins.
  • the overall pattern of OP-1 bands was similar in homogenates of renal cortex and medulla (lanes 2 and 3, of Figure 5) .
  • the 53 kD was less intense in the renal papilla (lane 4, of Figure 5) as compared to renal cortical and medullary homogenates.
  • Several of the bands identified in kidney homogenates by 12G3 possess molecular masses identical to that described for OP-1 species harvested from OP-1 producing CHO cells shown in Figure 4.
  • the major OP-1 bands of 45 kD, 19 kD and 17 kD are identical to those displayed in Figure 3 and likely correspond to pro-OP-1 protein as well as the 2 mature glycosylated OP-1 products.
  • Previous data (Sampath, T.K., et . al..
  • kidney derived cell line to further study OP-1 expression
  • confluent cells from 3 types of cultured epithelial cells were screened by northern and immunoblot analyses using the cDNA and 12G3 antibody OP-1 probes described above.
  • Opossum kidney (OK) cells, canine (MDCK cells and an SV-40 transformed mouse IMCD cell line each form polarized epithelia. Both OP and MDCK cells have been utilized extensively in studies of epithelial cell polarity and transport.
  • the IMCD cell line is derived from the cells of the IMCD and it retains many characteristics of this nephron segment including increased transepithelial resistance in response to dexamethasone, inhibition of sodium flux by amiloride and atrial natriuretic peptide and the ability to adapt to hypertonicity (Rauchman, M.I., Am. J. Physiol . , 265-271 (1993) ) .
  • both MDCK and IMCD cell poly A + RNA contained both 4.0 and 2.4 kb OP-1 transcripts as shown in Figure 8.
  • OP-1 expression by MDCK cells confirms previous reports using a canine OP-1 cDNA probe (Ishibashi, K. , et, al . , Biochem. and Biophysical Research Comm. , 193:235-239 (1993)) .
  • OP-1 expression by IMCD cells was consistently larger when compared to MDCK cells (Left Panel) even under conditions of reduced stringency to account for species differences.
  • Immunoblot analysis using 12G3 also detected the presence of 4 OP-1 species in IMCD cell homogenates (lane 5, Figure 5) that were identical to the pattern exhibited by rat kidney homogenates (lanes 1-4, Figure 5) .
  • the major IMCD OP-1 bands included bands of 36 kD, 19 kD, 17 kD and 15 kD.
  • IMCD cells did display 12G3 reactive bands of 53 kD and 45 kD, both were less intense as compared to cortical (lane 2) and medullary (lane 3) homogenates.
  • the abundance of OP-1 protein species in IMCD cells is reflected in the fact that lane 5 contains approximately one half the total protein content of lanes 1-4 of Figure 5.
  • tubular epithelial cells in the renal medulla are an important source of the OP-1 protein.
  • Applicants have determined that renal epithelial cells, in particular, tubular epithelial cells in renal medulla express high levels of OP-1 mRNA and OP-1 protein.
  • a method is now available to identify substances capable of inducing bone formation by determining the effect a substance has on OP-1 mRNA expression, or OP-1 protein synthesis.
  • effect on OP-1 mRNA expression means effect on the transcription of genetic information from DNA to mRNA.
  • effect on OP-1 protein synthesis means the effect on the translation of mRNA to protein molecules, as well as effects on the biological activity of the protein molecule, or secretion of protein molecules from the cell.
  • OP-1 protein is a potent bone morphogen member of the transforming growth factor beta (TGF- ⁇ ) superfamily and induces de novo bone formation by stimulating collagen synthesis and the proliferation and differentiation of osteoblasts.
  • TGF- ⁇ transforming growth factor beta
  • downregulation of OP-1 mRNA expression, or a decrease in OP-1 protein synthesis provides a means to suppress new bone formation.
  • substances that affect OP-1 mRNA expression, or OP-1 protein synthesis also affect bone formation.
  • the regulation of expression of OP-1 mRNA can be accomplished by any method that regulates the transcription of OP-1 DNA to mRNA.
  • Upregulation of the expression of OP-1 mRNA can be accomplished, for example, by a transcriptional activator factor, such as an ancillary protein necessary to initiate the transcriptional process. Upregulation can also be accomplished, for example, by removal of a transcriptional factor which prevents initiation of the transcriptional process.
  • the upregulation of OP-1 mRNA expression can be accomplished by introducing into the cell an antisense oligonucleotide which binds to a gene encoding a regulatory protein, that, when present, binds to OP-1 DNA and prevents initiation of transcription of OP-1 DNA to OP-1 mRNA.
  • Removal of this regulatory protein thus upregulates the expression of OP-1 mRNA expression.
  • removal of the regulatory protein can be accomplished by introducing into the cell and an antibody, or antibodies, that bind to the regulatory protein and, thus, block, or neutralize, its activity. Removal of this regulatory protein initiates transcription of OP-1 DNA to mRNA, and, thus, induces new bone formation.
  • OP-1 mRNA can also be downregulated by any method that which regulates the transcription of OP-1 DNA to mRNA. Downregulation of mRNA expression can be accomplished, for example, by sense, or antisense, DNA complementary to a portion of OP-1 DNA, which hybridizes with all, or a portion, of the OP-1 gene, thus, preventing transcription.
  • the regulation of synthesis of OP-1 protein can be accomplished by any method that regulates protein synthesis, or regulates the biological activity of the protein, or the secretion of protein from a cell.
  • the synthesis of OP-1 protein can be regulated by substances that affect the translation of mRNA to protein, such as the binding of an antisense oligonucleotide to OP- 1 mRNA, thus preventing translation.
  • the biological activity of OP-1 protein can be altered by a substance that interferes with the maturation (e.g., biological activity) of the protein, such as cleavage of the pro region, or glycosylation of the protein.
  • regulation can occur by substances that stimulate, or inhibit secretion of OP-1 protein from the cell.
  • the method for the identification of substances capable of inducing new bone formation is performed as follows.
  • Renal medullary cells are grown under standard laboratory conditions at pH 7.4, at 37°C in 5% C0 2 , in Dulbecco's Modified Eagle's Medium (DMEM) as described in Example 1. Renal medullary cells can be obtained from a variety of sources. These include, for example, dissecting mammalian kidneys and establishing primary renal medullary cell cultures, or using a cell line such as the Madin-Darby canine kidney (MDCK) cell line. Applicants have demonstrated that inner medullary collecting duct (IMDC) cells exhibit significantly higher expression levels of OP-1 mRNA and OP-1 protein than other kidney cells.
  • IMDC inner medullary collecting duct
  • IMDC cells suitable for use in the present invention, is the mIMCD-3 cell line derived from a transgenic SV-40 mouse as described in Gullans, S.R., et al. , Am. J. Phvsiol . , 265 (1993) , the teachings of which are incorporated herein by reference.
  • other culture media can be used to practice this assay. Selection of suitable culture medium will depend on the ability of the medium to support growth of renal cells and to provide an environment which permits the test substance to detectably affect OP-1 mRNA expression or OP-1 protein synthesis, and thus, induce bone formation.
  • Test substances can be oligonucleotides, peptides, proteins, small molecules or organic molecules, including molecules that alter the pH, or ionic strength, of the cellular environment.
  • a test substance can be a peptide hormone (e.g., parathyroid hormone, anti-diuretic hormone, or atrial naturetic peptide) or a steroid hormone such as vitamin D, or one of its active metabolites.
  • Mineralocorticoids can also be tested in IMCD cells.
  • Test substances can also be antisense oligonucleotides or antibodies. Accordingly, a test substance can be any substance reasonably believed to affect bone formation. However, the candidate substance must be sufficiently soluble in an aqueous medium to allow a sufficient amount to dissolve so that the effect of the substance on OP-1 mRNA expression, or OP-1 protein synthesis, can be determined.
  • test substance is then introduced into the culture medium, in sufficient amount and in a form that allows the test substance to affect the expression of OP-1 mRNA or synthesis of OP-1 protein.
  • a sufficient amount of the test substance (e.g., an effective amount) is determined for each individual substance to be tested.
  • 10 ⁇ 7 M atrial natriuretic peptide (ANP) inhibits luminal-to-basal transport of Na in IMCD cells. (Gullans, S.R., Am. J. Phvsiol . , 265 (1993) .
  • 10 "7 M ANP would be a reasonable amount of ANP to be tested in the method of the present invention.
  • This substance would also be tested in a greater or lesser amount than 10 "7 M to determine the optimum dose of ANP effective to induce bone formation.
  • the test substance must be in a form so as to allow the substance to affect the OP-1 mRNA expression or OP-1 protein synthesis.
  • the test substance is sufficiently soluble to allow an effective amount of substance to dissolve in the aqueous culture medium and subsequently to enter into the IMCD cell.
  • the test substance can be introduced into the culture medium, or the cell via other routes, such as encapsulation by a liposome, or by other standard methods used to introduce substances into culture media and cultured cells.
  • test culture is incubated under conditions (e.g., time, temperature, C0 2 content) suitable for growth of the cells. Incubation conditions and duration will depend on the nature of the individual substance being tested.
  • a control culture is run in tandem with the test culture, under similar conditions, but without the presence of the test substance.
  • total RNA is isolated from the cultured cells and prepared for Northern blot analysis as described in Example 1. After fractionation of mRNA on a 1% agarose formaldehyde gel using standard techniques, the mRNA is transferred to a membrane suitable for Northern blot analysis and hybridized with a 32 P-labeled OP-1 cDNA probe under conditions of high stringency.
  • the probe can be all or a portion of the murine OP-1 cDNA sequences described in Ozkaynak, E., et al. , Biochem. Biophys . Res. Commun. , 30:116-123 (1991) .
  • the OP-1 cDNA probe can be all or a portion of the human OP-1 cDNA sequence (Ozkaynak, E., et al. , EMBO J. , 9:2085- 2093 (1990) , deposited in the EMBL data library under accession number X51801) .
  • Other OP-1 oligonucleotide probes can also be used in the Northern blot analysis.
  • a labeled OP-1 RNA oligonucleotide probe can be used.
  • the probes can be of a length different from the cDNA probe used in Example 1. However, the probe must be of a length sufficient to specifically hybridize with OP-1 mRNA under conditions of high stringency.
  • the probes can also vary in nucleotide sequence from the probe used in Example 1. However, the probe sequence must contain sufficient number of similar nucleotides to permit specific hybridization of the probe to OP-1 mRNA under conditions of high stringency.
  • the oligonucleotide probes used in this embodiment of the present invention can be labeled with radioactive molecules, such as 32 P. mRNA on the membrane which hybridized with the 32 P-labeled OP-1 oligonucleotide probe is then visualized by autoradiography, and quantified, for example, by laser scanning densitometry as described in Example 1. Alternatively, the probes can be labeled with non-radioactive molecules, such as peroxidase, biotin or digoxigenin. mRNA hybridized with non-radioactive OP-1 oligonucleotide probes is then visualized by color development by enzyme assay, binding to labeled strep- avidin, or chemiluminescence.
  • radioactive molecules such as 32 P.
  • the amount of OP-1 mRNA expressed in the test culture is compared to the amount of mRNA expressed in the control culture (i.e., cells cultured without the presence of the test culture) . If the amount of OP-1 mRNA expressed in the test culture is higher than the amount of OP-1 mRNA expressed in the control culture, the presence of the test substance induced OP-1 mRNA expression, and thus, is capable of inducing bone formation. If the amount of OP-1 mRNA expressed in the test culture is less than the amount of mRNA expressed in the control culture, the presence of the test substance suppressed OP-1 mRNA expression, and thus, is capable of suppressing bone formation.
  • the synthesis of OP-1 protein is determined.
  • Cultured cells can be prepared, denatured by SDS and heat treatment, and subjected to SDS polyacrylamide (PAGE) electrophoresis as described in Example 2. After separation on the basis of size, the separated proteins are transferred to a membrane suitable for Western blot analysis, and incubated with antibody specific for OP-1 protein (e.g., an OP-1 specific monoclonal, polyclonal antibody, or an antibody mix) .
  • antibody specific for OP-1 protein e.g., an OP-1 specific monoclonal, polyclonal antibody, or an antibody mix
  • OP-1 protein secreted into the culture medium can be measured by standard laboratory techniques (e.g., ELISA or RIA) , also using antibodies specific for OP-1.
  • the amount of OP-1 protein synthesized in the test culture i.e., cells cultured in the presence of test substance
  • the control culture i.e., cells cultured without the presence of the test culture
  • the amount of OP-1 protein synthesized in the test culture is higher than the amount of OP-1 protein synthesized in the control culture, the presence of the test substance induced OP-l protein synthesis, and thus, is capable of inducing bone formation.
  • the amount of OP-1 protein synthesized in the test- culture is less than the amount of OP-1 protein synthesized in the control culture, the presence of the test substance suppressed OP-1 protein synthesis, and thus, is capable of suppressing bone formation.
  • the biological activity of the OP-1 protein synthesized, or secreted can be measured using, for example, the in vivo bone matrix implant bioassay described in Rueger, D.C., et. al. , J. Biol . Chem. , 265:13198-13205 (1990) , the teachings of which are incorporated herein by reference.
  • This assay measures the bone-inducing activity of OP-1 protein on bone matrix implanted in rats.
  • a test substance affects the biological activity of the OP-1 protein (e.g., by affecting maturation of the protein, or glycosylation of the protein) the increase, or decrease in biological activity can be determined.
  • Peptide hormones classically involved in bone metabolism could modulate OP-1 expression and production by the kidney via activation of the protein kinase A pathway, to stimulate the level of OP-1 mRNA expression.
  • IMCD cells expressed OP-1 at such high levels it is reasonable to believe that renal OP-1 production may be an integral part of a mechanism distinct from parathyroid hormone (PTH) and vitamin D by which the kidney influences skeletal maturation and growth.
  • PTH parathyroid hormone
  • the mIMCD-3 cell line, together with the assay described herein, are particularly useful as a model system to study the pathway of bone morphogenesis regulated by OP-1.
  • the IMCD cell line was developed from the terminal inner medullary collecting duct of a large T antigen SV40 mouse and it retains many characteristics of this nephron segment, including increased transepithelial resistance in response to dexamethasone, inhibition of sodium flux by amiloride and atrial natriuretic peptide, and the ability to adapt to hypertonicity. Receptors to calcitriol or PTH have not been found in inner medulla. (Karashima, H., _ al. , Kidney Int . , 29:98 (1986) ; Alexander, E. and Schwartz, J. , American J. of Kid.
  • OP-1 protein may serve as a link between ion sensing cells in the inner medulla and cellular elements of the skeleton.
  • Substances identified in the method of the present invention can be used to therapeutically treat a patient with bone disease, such as osteoporosis, to induce bone growth (bone morphogenesis) .
  • This method comprises administering to the patient an effective amount of a substance capable of inducing bone growth by inducing OP-1 mRNA expression, or OP-1 protein synthesis.
  • Administration of such bone-inducing substances can be by medically accepted techniques, including intravenous, subcutaneous, or oral administration.
  • Appropriate amounts, or effective dosages will, of course, vary from individual to individual and by type of disease and/or severity of the disease. Appropriate dosages can be calculated by those skilled in the art taking such factors into account.
  • the present invention provides a method of therapeutically treating a patient with bone disease, such as osteopetrosis, to suppress an increase in bone formation.
  • This method comprises administering to the patient an effective amount of a substance capable of suppressing bone growth by suppressing OP-1 mRNA expression, or OP-1 protein synthesis.
  • ovary tissue had the highest OP-1 mRNA expression.
  • a possible role of the OP-1 synthesized by the ovary in the maintenance of bone homeostasis is also reasonable, especially in the relationship of age related OP-1 mRNA expression and OP-1 protein synthesis (e.g., post- menopausal osteoporosis) .
  • the method of the present invention can also be used to identify substances capable of inducing bone formation by their effect on OP-1 mRNA expression, or OP-1 protein synthesis in ovary cells.
  • IMCD cells of the kidney may make a unique form of OP-1 that does not cause calcification immediately but would have to be processed to become active in calcification processes.
  • IMCD OP-1 may have other functions unique to the kidney itself. Since IMCD cells secrete OP-1, these cells can be used in the described methods to further elucidate these functions. Unique properties of IMCD-derived OP-1 would have considerable value. For example, because these cells are epithelial cells, it is reasonable to predict that OP-
  • OP-1 is only secreted on one side of the layer of cells, either the apical side (urine facing) or basolateral side (blood facing) of epithelial plasma membranes.
  • apical side urine facing
  • basolateral side blood facing
  • OP-1 could reflect one form of OP-1 going to one surface and the targeting of another form to the opposite surface.
  • OP-1 secreted by renal epithelial cells is also present in the urine.
  • Assay systems using IMCD cell supernatants can be designed to measure the urine and hence body stores, of OP-1 using OP-1 specific monoclonal antibodies such as 12G3. If renal IMCD cells make a specialized form of OP-1 this form could be distinguished from OP-1 made by other regions of the kidney as well as that filtered by glomeruli from the serum. Such an assay could distinguish between the various forms of OP-1. This information would be of value, for example, to distinguish which patients might benefit from treatment with OP-1 and/or stimulation of synthesis of OP-1 exogenous as opposed to patients requiring other therapy.
  • OP-1 may also function as a "repair" molecule in mammalian kidney tissue. As discussed above, is has been demonstrated that OP-1 mRNA and protein expression is
  • kidney endothelial cells In addition, studies of unrelated proteins have provided examples of proteins synthesized by the kidney that have both autocrine (acting at a distance) and paracrine (acting locally) effects.
  • Several proteins derived from the kidney have been demonstrated to possess effects on other organs as well as serving to modulate cellular growth within the kidney. Renin/angiotensin proteins alter systemic blood pressure and acts as a factor for kidney growth (Ichikawa, I. and Harris, R.C., Kidney International, 40:583-596 (1991)) .
  • Endothelin produced by kidney endothelial cells and exerts both local and systemic vasoconstrictive effects and effects proliferation of glomerular endothelial cells (Kon, V. and K.
  • EGF Epidermal growth factor
  • a model assay system is now available to identify substances capable of inducing new bone formation by the evaluation of the effect of test substances on the expression of OP-1 mRNA expression and OP-1 protein synthesis in inner medullary collecting duct cells.
  • Significant levels of OP-1 mRNA transcripts and proteins are present in the renal medulla. Therefore, renal OP-1 production in the inner medulla may act as an alternative pathway to the classic PTH and vitamin D mechanisms by which kidney modulates bone homeostasis.
  • OP-1 may also act in the repair of normal and abnormal kidney tissue.
  • Animals Young adult female Sprague-Dawley rats approximately 300 gms (2-4 months old) were purchased from Charles River Laboratories (Wilmington, MA) . After euthanasia under general anesthesia, organs were flushed with ice-cold saline via intracardiac puncture. Renal cortex, medulla and papilla were immediately dissected. Equal amounts of lung, liver, brain, small intestine and ovary were also extracted.
  • opossum kidney (OK) cells a gift from Dr. M.H. Montrose, Johns Hopkins University, MD
  • Madin Darby Conni Kidney (MDCK) cells obtained from American Type Culture Collection (Rockville, MD)
  • IMCD inner medullary collecting duct
  • the cells were grown to confluency on tissue culture dishes (Nunclon Delta; Nunc, Inc; Naperville, IL) at pH 7.4 in incubators at 37°C with a 5% C0 2 95% air atmosphere.
  • OK and IMCD cells were fed on alternate days in Dulbecco's Modified Eagle's Medium (DMEM) -F12 (GIBCO, Gaithersburg, MD) supplemented with 29 mM bicarbonate, 2mM glutamate, 100 IU/cc penicillin, 100 pg/cc of streptomycin and 10% (OK cells) or 5% (IMCD cells) fetal calf serum.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MDCK cells were fed on alternate days with a standard MEM culture medium (GIBCO, Gaithersburg, MD) supplemented with Earles Salt, 2 mM glutamate, 10% fetal calf serum, 100 IU/cc penicillin and 100 pg/cc of streptomycin.
  • GEBCO Gaithersburg, MD
  • Probes A 680 bp fragment (SEQ ID NO: 1) containing two thirds of the pro domain and one third of the mature murine OP-1 cDNA (nucleotides 370-1050 immediately upstream to the highly conserved 7-cysteine domain present in all members of the TGF-/3 superfamily) was used as a probe for Northern analysis. (Ozkaynak, E., et al. , Biochem. Biophvs. Res. Commun., 30:116-123 (1991)) . USB Sequenase Random Prime DNA labeling Kit (United States Biochemical, Cleveland, OH) and c_ 32 P dCTP (New England Nuclear, Boston, MA) were used for standard labeling reactions. A 40 bp oligonucleotide of mouse ⁇ - act in (Oncogene Science, Uniondale, NY) was used as control gene.
  • RNA Preparation Total RNA was isolated from animal tissues (100 mg/assay) and cultured cells (90-100% confluency, 100 x 20 mm plate/assay) using the guanidine isothyocyanate/phenol-chloroform method (Chomczynski, P. and Sacchi, N. , Anal . Bioche . , 162:156-159 (1987)) . Poly A + was selected by oligo dT cellulose using the mRNA MicroFastTrack Kit from Invitrogen, San Diego, CA.
  • membranes were prehybridized with 50% deionized Formamide (Sigma) , 10% Dextran Sulfate, 1% sodium dodecyl sulfate (SDS) , 1 M NaCI and 100 pg/ml of denatured sonicated salmon sperm DNA (Sigma) for 2 hours at 42°C in a hybridization oven. Using the same solution, the membranes were then hybridized for 20 hours at 42 ⁇ c with 1 x 10 6 cpm/ml of the 32 P-labeled OP-1 cDNA probe.
  • Membranes were washed for 30 minutes with 0.lx SSC-O.lx SDS at 65°C and then subjected to autoradiography at -80oc for 24 hours using Kodak XAR-5 film and an intensifying screen (Lightning Plus; Eastman Kodak, Rochester, NY) . To control for mRNA content and quality, the blots were stripped and subsequently hybridized with mouse /--actin. The intensity of the signal was quantified by laser scanning densitometer (model SL-504-XL; Biomed Instruments,
  • the ratio OP-1 mouse -actin signal was calculated for each tissue and the highest value within an animal was assignated as 100%. The percentages of mRNA expression for the rest of the tissues were figured out by comparison of their ratios to the highest one.
  • the selection of the IgG j monoclonal anti-OP-1 antibody 12G3 was accomplished by injecting recombinant human OP-01 and fusing the spleen cells with nurine myeloma cells, followed by screening using Western blots of oxidized and reduced OP-1 (Vukicevic, S., et, al. , Biochem. Biophys. Res. Commun. 198, 693-700 (1994))
  • the injected recombinant human OP-1 used in the generation of 12G3 was composed of amino acids 293-431 where amino terminal methionine of pre-pro OP-1 is defined as residue #1 (Ozkaynak, E., et al. , EMBO J. 9, 2085-2093 (1993) ) .
  • Immunocytochemistry Following an overnight incubation in 3% normal goat serum at 4° C, rat kidney sections were incubated in primary monoclonal antibody 12G3, that was diluted in PBS, 1% fetal calf serum and 5 0.1% sodium azide for 60 minutes at room temperature. Subsequently sections were exposed to a peroxidase- conjugated goat anti-mouse immunoglobulin (Jackson Immunoresearch, Westgrove, PA) in the same diluent, at a dilution of 1:40 for an additional 60 minutes. The slides 0 received sequential washes of 5 minutes duration in PBS then PBS containing 0.2% gelatin after each incubation.
  • the specimens were exposed to 5 minute intervals of 0.1 M sodium acetate buffer (pH 5.2) and 0.25 mg/ml 3-amino 9-ethylcarbazole 5 (AEC) (Sigma Chem. Co., St. Louis, MO) in 2% N-N dimethylformamide (Sigma Chem. Co., St. Louis, MO) followed by 0.1 M sodium acetate buffer (pH 5.2) with 0.03% hydrogen peroxide. The specimens were finally washed in tap water for 5 minutes, counterstained with 0 hematoxylin, and mounted in glycerol gelatin (Sigma Chem. Co. , St. Louis, MO) .
  • AEC 3-amino 9-ethylcarbazole 5
  • a 3 P-labelled antisense RNA probe was prepared by linearizing Bluescript (Stratagene, La Jolla, CA) containing the 680 bp OP-1 cDNA with Hind III followed by transcription with T7 polymerase (Promega Inc., Madison, WI) in the presence of [ 32 P] CTP (800
  • RNA probe As a control, 32 P-labelled sense RNA probe was prepared by linearizing the same vector with BAM HI followed by transcription with T3 polymerase under identical conditions. Paraffin sections of adult C57BL/6 mouse kidney were subjected to in si tu hybridization as described in Arceci, R.J., et al., Proc. Natl. Acad. Sci. USA, 86:8818-8822 (1989) using [ 3 P] CTP-labeled sense (Control) or antisense (Experimental) RNA probes transcribed form Bluescript-OP-1 vector as described above. After autoradiography, the slides were stained with hematoxylin/eosin prior to photographic analysis.
  • Lys lie Pro Glu Gly Glu Arg Val Thr Ala Ala Glu Phe Arg lie Tyr 165 170 175
  • Lys Asp Tyr lie Arg Glu Arg Phe Asp Asn Glu Thr Phe Gin lie Thr 180 185 190

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Abstract

Procédé rapide et sensible de sélection de substances candidates pour déterminer leur effet sur l'expression de l'ARNm de la protéine ostéogène-1 (OP-1), ou sur la syntèse de la protéine OP-1, et permettant ainsi de déterminer l'effet desdites substances sur la formation osseuse. L'invention se rappporte également à des procédés de traitement de sujets souffrant d'affections osseuses.
PCT/US1994/013215 1993-11-16 1994-11-16 Procede d'identification d'une substance apte a induire la formation osseuse WO1995014104A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
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WO1995033831A1 (fr) * 1994-06-07 1995-12-14 Creative Biomolecules, Inc. Procedes et compositions de modulation de l'expression de proteines morphogenetiques
WO1997005285A2 (fr) * 1995-07-26 1997-02-13 Creative Biomolecules, Inc. Procedes et compositions d'identification d'analogues de morphogenes
WO1997005241A2 (fr) * 1995-07-26 1997-02-13 Creative Biomolecules, Inc. Procedes et compositions d'identification d'analogues de morphogenes
WO1998054572A1 (fr) * 1997-05-30 1998-12-03 Creative Biomolecules, Inc. Procedes pour evaluer la morphogenese des tissus et leur activite
US6071695A (en) * 1992-02-21 2000-06-06 Creative Biomolecules, Inc. Methods and products for identification of modulators of osteogenic protein-1 gene expression
US6090544A (en) * 1995-07-26 2000-07-18 Creative Biomolecules, Inc. Methods and compositions for identifying morphogen analogs
US6103491A (en) * 1995-07-26 2000-08-15 Creative Biomolecules, Inc. Methods and compositions for identifying morphogen analogs
US7147839B2 (en) 1998-05-29 2006-12-12 Curis, Inc. Methods for evaluating tissue morphogenesis and activity
US7306903B1 (en) 1995-07-26 2007-12-11 Curis, Inc. Methods and compositions for identifying morphogen analogs

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US4857456A (en) * 1985-04-30 1989-08-15 The Regents Of The University Of California Assay of Bone morphogenetic protein (BMP) and anti-BMP antibody for the diagnosis of bone disorders
WO1993005172A1 (fr) * 1991-08-30 1993-03-18 Creative Biomolecules, Inc. Procede de triage de proteines morphogeniques
WO1994003600A1 (fr) * 1992-07-31 1994-02-17 Creative Biomolecules, Inc. Complexe soluble de proteines morphogeniques et sa composition

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US4857456A (en) * 1985-04-30 1989-08-15 The Regents Of The University Of California Assay of Bone morphogenetic protein (BMP) and anti-BMP antibody for the diagnosis of bone disorders
WO1993005172A1 (fr) * 1991-08-30 1993-03-18 Creative Biomolecules, Inc. Procede de triage de proteines morphogeniques
WO1994003600A1 (fr) * 1992-07-31 1994-02-17 Creative Biomolecules, Inc. Complexe soluble de proteines morphogeniques et sa composition

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Title
T.K. SAMPATH ET AL.: "Recombinant human osteogenic protein (hOP-1) induces new bone formation in vivo with a specific activity comparable with natural bovine osteogenic protein and stimulates osteoblast proliferation and differentiation in vitro", J. BIOL. CHEM., vol. 267, no. 28, 5 October 1992 (1992-10-05), AM. SOC. MOL. BIOL., INC.,BALTIMORE, US;, pages 20352 - 20362 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6071695A (en) * 1992-02-21 2000-06-06 Creative Biomolecules, Inc. Methods and products for identification of modulators of osteogenic protein-1 gene expression
WO1995033831A1 (fr) * 1994-06-07 1995-12-14 Creative Biomolecules, Inc. Procedes et compositions de modulation de l'expression de proteines morphogenetiques
WO1997005241A3 (fr) * 1995-07-26 1997-05-22 Creative Biomolecules Inc Procedes et compositions d'identification d'analogues de morphogenes
US7306903B1 (en) 1995-07-26 2007-12-11 Curis, Inc. Methods and compositions for identifying morphogen analogs
WO1997005241A2 (fr) * 1995-07-26 1997-02-13 Creative Biomolecules, Inc. Procedes et compositions d'identification d'analogues de morphogenes
US5834188A (en) * 1995-07-26 1998-11-10 Creative Biomolecule, Inc. Methods and compositions for identifying morphogen analogs
WO1997005285A3 (fr) * 1995-07-26 1997-03-06 Creative Biomolecules Inc Procedes et compositions d'identification d'analogues de morphogenes
US5932716A (en) * 1995-07-26 1999-08-03 Creative Biomolecules, Inc. Morphogen-responsive regulatory elements
WO1997005285A2 (fr) * 1995-07-26 1997-02-13 Creative Biomolecules, Inc. Procedes et compositions d'identification d'analogues de morphogenes
US6090544A (en) * 1995-07-26 2000-07-18 Creative Biomolecules, Inc. Methods and compositions for identifying morphogen analogs
US6103491A (en) * 1995-07-26 2000-08-15 Creative Biomolecules, Inc. Methods and compositions for identifying morphogen analogs
US6110460A (en) * 1995-07-26 2000-08-29 Creative Biomolecules, Inc. Methods of using morphogen analogs
WO1998054572A1 (fr) * 1997-05-30 1998-12-03 Creative Biomolecules, Inc. Procedes pour evaluer la morphogenese des tissus et leur activite
EP1988395A1 (fr) * 1997-05-30 2008-11-05 Curis, Inc. Procédés d'évaluation de la morphogénèse de tissus et activité
EP2309261A1 (fr) * 1997-05-30 2011-04-13 Stryker Corporation Procédés d'évaluation de la morphogénèse de tissus et activité
US7147839B2 (en) 1998-05-29 2006-12-12 Curis, Inc. Methods for evaluating tissue morphogenesis and activity

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