KR100734429B1 - Signals and molecular species involved in senescence - Google Patents

Abstract

The present invention provides a method for detecting senescent cells, comprising the steps of: (a) measuring a relative change in a young cell of a signal or molecular species involved in signal transduction; And (b) treating the senescent cells with an effective amount of an inhibitor of adenylyl cyclase or an inhibitor of protein kinase A, and (c) a composition.

Description

SIGNALS AND MOLECULAR SPECIES INVOLVED IN SENESCENCE             

The present invention relates to a signal or molecular species involved in aging, and more particularly to a signal or molecular species involved in aging of cells and their use.

Cell aging plays an important role in complex biological processes, including development, maturation and tumor formation, and many attempts have been made to understand the basic characteristics of cell aging (Peacocke and Campisi, 1991; Smith and Pereira-Smith, 1996 ). One characteristic of cell aging is hypoesponsiveness to growth factors and mitogen.

The reduced reactivity of senescent cells to growth factors is explained, at least in part, by quantitative and qualitative changes associated with aging of growth factor receptors. A series of cultures in vitro has been reported to reduce the number of high- and low-affinity epidermal growth factor (EGF) receptors in human omental microvascular endothelial cells (Matsuda et al., 1992). The reduced reactivity of certain chondrocytes derived from animals to EGF appears to be due to a decrease in the number of EGF receptors (Ribault et al., 1998). Aging-related decreases in platelet-derived growth factor (PDGF) binding sites or PDGF receptors have also been reported to occur in several cellular systems, including human smooth muscle cells (Mori et al., 1993; Aoyagi et al., 1995). .

In contrast, some aging cells in culture have a functioning growth factor receptor system and have a normal number of receptors with normal binding affinity for growth factors (Paulsson et al ., 1986; Gerhard et al ., 1991; Hensler and Pereira-Smith, 1995; Park et al ., 2000). In such cases, post-receptor signaling pathways in cellular senescence may account for the reduced reactivity to growth factors. Upregulation of carveolin and / or downregulation of ampiphycin may explain the responsiveness of aged fibroblasts to EGF stimulation (Park et al ., 2000, 2001). In addition, defects in the calcium-phospholipid-dependent protein kinase C (PKC) pathway have been suggested to be involved in the mitosis of senescent cells (Pascale et al ., 1998; Venable and Obeid, 1999). Since phosphatidylcholine-specific phospholipase D (PLD) is activated by PKC, and then phosphoric acid acts, phosphohydrolase forms a delayed, more sustained diacylglycerol, which leads to the sustained activation of PKC. It may be the cause, and the activity of the PLD may be changed during the aging process. Indeed, Venable et al . (1994) reported a defect in the serum-stimulated PLD / PKC pathway upon aging of cells.

In contrast to previous reports showing a decrease in agonist-stimulated signal transduction, some reports show an increase in signal transduction. Bradykinin increases IP ₃ formation in fibroblasts from normal aged donors and Alzheimer's disease donors (Huang et al ., 1991) and PLD activity in human aged fibroblasts rather than unaged fibroblasts Increase (Meacci et al ., 1995). After exposure to beta-amyloid, a major component of senile plaques in Alzheimer's disease, human lymphocytes showed an elevated mitogen-induced Ca ²⁺ response (Eckert et al ., 1994). This result suggests that aging is agonist-specific in signal transduction.

PDGF delivers mitogen signals through plasma membrane-binding receptors with protein tyrosine kinase activity, and lysophosphatidic acid (LPA) is extracellular messenger via guanine nucleotide binding protein (G-protein). act as a messenger). Since both PDGF and LPA induce the same signal transduction phenomena, including intracellular Ca ²⁺ migration, actin polymerization, and the production of phosphatidic acid in human diploid fibroblasts, the inventors of the present invention have two other major mitogens. The reactivity of aged or nearly aged cells with agonists PDGF and LPA was compared.

Lysophosphatidic acid (LPA), on the other hand, is a lipid mediator with various biological activities such as cell shape change, chemotaxis, proliferation and differentiation (Moolenaar et al., 1997; An et al., 1998c). LPA is produced by phospholipase cleavage of membrane phospholipids in stimulated cells, especially activated platelets (Gaits et al., 1997). Intracellular biochemical signal transduction phenomena that mediate the effects of LPA include increased cytoplasmic calcium concentration, stimulation of phospholipase, phosphatidylinositol 3-kinase, Ras-Raf-MAP kinase cascade and adenylyl cyclase (AC) (Moolenaar et al., 1997). Recently, it has been identified that the cell surface G-protein-binding receptors of LPA are a family of endothelial cell differentiation genes (EDGs) (LPA receptors reviewed in Contos et al., 2000; Fukushima et al., 2001). The major members of the EDG family that interact with LPA are known EDG-2 (Hecht et al., 1996), EDG-4 (An et al., 1998a) and EDG-7 (Bandoh et al., 1999). LPA is also a low affinity agonist of EDG-1 (Lee et al., 1998).

Specific responses of G-protein-binding receptors to ligands will be possible when suitable G-proteins are bound to the receptors (Figler et al., 1996; Moolenaar, 1997). EDG-2 binds to Gi, which is native to pertussis toxin, EDG-4 binds to both Gi and Gq (An et al., 1998), and EDG7 binds to pertussis toxin-insensitive G-protein (s). It is believed to bind to Gq (Bandoh et al., 1999; Im et al., 2000). Gq protein can mediate inositol 1,4,5-triphosphate (IP ₃ ) production and subsequent Ca ⁺⁺ migration, while Gi can mediate the inhibition of AC. This complex linkage of LPA-signal systems with various factors suggests the possibility of age-dependent functional degeneration.

Cell cAMP can be synthesized by activated AC and hydrolyzed by cyclic nucleotide phosphodiesterase (PDE). Increasing cAMP content activates cAMP-dependent protein kinase (PKA), which phosphorylates cellular proteins and regulates gene expression by activation of cAMP response element binding protein (CREB), resulting in cell proliferation, differentiation and metabolism And a number of cellular responses, including neuronal reactions (Taussig and Gilman, 1995).

It has been reported previously that PGE1-induced cAMP accumulation and subsequent phosphorylation of CREB by protein kinase A is markedly reduced in senescent cells (Chin et al., 1996). However, cAMP signal transduction induced by porsporin or interferon-γ-inducible protein-10 (IP-10) can be maintained relatively in aged human diploid fibroblasts Hs68 (Shiraha et al., 2000 ). Increased cAMP in serum-treated late-passage-human embryonic lung fibroblasts (Polgar et al., 1978) and isoproterenol treated aging IMR-90 lung lung fibroblasts (Ethier et al., 1992) Irritation was observed. In VSMC cultured from aged rats, the response was substantially increased compared to VSMC cultured from younger rats, and cAMP response to isoproterenol was decreased in fibroblasts cultured from aged rats (Chin and Hoffman, 1991). ). These results suggest that the effect of aging on cAMP signal transduction is agonist specific and cell specific.

Patent applications relating to nucleic acids and proteins involved in the aging process are disclosed in WO 99/52929 and WO 01/23615.

As described above, various theories have been proposed, it is required to clarify the cell aging phenomena, and specific biomarkers for identifying aging cells and biomolecules that can regulate cell aging are required.

In particular, reversing aging and restoring normal physiological function are expected to be important for the treatment of certain diseases associated with aging, such as Werner Syndrome and Hutchinson-Gilford Syndrome.

Throughout this specification, numerous patent documents and articles are referenced and their citations are indicated in parentheses. The disclosures of cited patent documents and articles are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the contents of the present invention are more clearly explained.

In this context, the present inventors have made intensive research efforts to identify signals and subsidiary species involved in aging, and have discovered novel signals and molecular species useful for detecting aging. Surprisingly, some molecular species have been found to be useful in regulating aging of cells.

Accordingly, it is an object of the present invention to provide a method for detecting aged cells.

Another object of the present invention to provide a composition for regulating cell aging.

It is another object of the present invention to provide a method for regulating cellular senescence in a patient in need of cellular senescence control.

Another object of the present invention is to provide a method for identifying a substance affecting aging of a cell.

Other objects and advantages of the present invention will become apparent from the following detailed description taken in conjunction with the appended claims and drawings.

The present invention relates to novel signals and molecular species involved in aging of cells. The signal and molecular species may indicate whether or not the cells aging in a reliable range.

I. Detection of Aging Cells and Methods of Identification of Substances Affecting Cell Aging

According to one aspect of the invention, the invention comprises measuring a relative change in a young cell of a signal or molecular species involved in signal transduction, wherein the change in the signal or molecular species is (a) a Ca ²⁺ variation reduction in oscillation; (b) reduced expression of F-actin; (c) reduced activity of phospholipase C; (d) decreased activity of phospholipase D; (e) decreased expression or phosphorylation of platelet-derived growth factor receptors; (f) reduced phosphorylation of phospholipase C-γ1; (g) decreased expression of phospholipase D1; (h) decreased expression of EDG-2; (i) decreased expression of EDG-7; (j) decreased expression of Gi1; (k) decreased expression of Gi2; (l) decreased expression of Gi3; (m) increased adenylyl cyclase activity or expression; (n) decreased activity and expression of phosphodiesterases; (o) increased activity of protein kinase C; (p) increased activity and expression of protein kinase A; (q) increased phosphorylation of CREB; And (r) provides a method for detecting senescent cells, characterized in that at least one selected from the group consisting of increased cAMP content.

According to another aspect of the invention, the invention provides a method of identifying a substance affecting aging of a cell comprising the steps of: (a) culturing the cell in the presence of the substance to be tested; And (b) measuring the relative changes to the young cells of the signal or molecular species involved in signal transduction. In step (b), the change in the signal or molecular species is reduced by (a) Ca ²⁺ variation; (b) reduced expression of F-actin; (c) reduced activity of phospholipase C; (d) decreased activity of phospholipase D; (e) decreased expression or phosphorylation of platelet-derived growth factor receptors; (f) reduced phosphorylation of phospholipase C-γ1; (g) decreased expression of phospholipase D1; (h) decreased expression of EDG-2; (i) decreased expression of EDG-7; (j) decreased expression of Gi1; (k) decreased expression of Gi2; (l) decreased expression of Gi3; (m) increased adenylyl cyclase activity or expression; (n) decreased activity and expression of phosphodiesterases; (o) increased activity of protein kinase C; (p) increased activity and expression of protein kinase A; (q) increased phosphorylation of CREB; And (r) increasing the cAMP content.

The methods of the present invention employ signals or molecular species (eg, ions and proteins) that are involved in signal transduction. In the present invention, the level of signal or molecular species measured for an unknown cell sample is compared with that of young cells. Decreased or increased levels of signals or molecular species on young cells allow detection of senescent cells and the identification of substances affecting cellular senescence.

As used herein, the term "senescene" has the same meaning as "aging". As used herein, the term near senescent means that the state of the cell is substantially the same as the state of aging. For example, nearly aged cells show arrest of cell growth and changes in aging-like morphology. In the context of cells, the term "presenescent" means young cells. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art. For example, terms used herein can be found in Benjamin Lewin, Genes VII (Oxford University Press (2000); and Kendrew et al., The Encyclopedia of Molecular Biology (Blackwell Science Ltd. (1994)).

According to a preferred embodiment of the invention, the signals or molecular species of (a)-(g) are involved in signal transduction caused by platelet-induced growth factors (hereinafter referred to herein as PDGF). In a more preferred embodiment, the signals and molecular species of (h)-(r) are involved in signal transduction caused by lysophosphatidic acid (hereinafter referred to herein as LPA).

According to a preferred embodiment of the present invention, cells suitable for the present invention are derived from mammalian cells such as human cells. More preferably, the cells used in the present invention are fibroblasts.

In order to detect senescent cells, when measuring the activity or expression of adenylyl cyclase (adenyyl cyclase) Adenyl containing adenylyl cyclase II, adenylyl cyclase IV and adenylyl cyclase VI It is desirable to target specific isoforms of nilyl cyclase. In addition, in order to detect aged cells, when measuring the expression of phosphodiesterases, it is preferable to target phosphodiesterase 4B. In order to detect aged cells, when measuring the expression of protein kinase A, it is preferable to target the protein kinase subunits Cα, RIα or RIβ subunits.

In a specific example of the method of the invention, the measuring step is carried out by western blotting method. General methods of western blotting are disclosed in Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 108-121, CRC Press. Antibodies used for western blotting can be obtained by methods known to those skilled in the art (Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988)). The antibody is a polyclonal or monoclonal antibody. Monoclonal antibodies can be prepared via known methods, such as those disclosed in US Pat. No. 4,196,265. Antibodies can be labeled with radioactive, fluorescent, biological or enzymatic tags or labels for more convenient detection.

Aging can be easily detected by comparing western blotting bands derived from senescent cells to those derived from young cells. Moreover, the method of the present invention can be carried out quantitatively. For example, bands resulting from western blotting can be converted into quantitative data using a densitometer.

In a specific example of the method of the invention, the measuring step, in particular the measuring of the expression level of a particular protein, can be carried out by northern blotting or reverse transcription-polymerase chain reaction (RT-PCR), preferably RT-PCR to be. General procedures of the northern blotting method are disclosed in Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC Press. In addition, general procedures for RT-PCR are disclosed in J. Sambrook, et al., Molecular Cloning, 2: 8.86-8.95, CSHL Press (2001).

II. Aging control method and composition of cells

According to another aspect of the present invention, the present invention provides a method for aging a cell comprising treating an senescent cell with an effective amount of an inhibitor of adenylyl cyclase, an inhibitor of protein kinase A, an inhibitor of protein kinase C or an activator of a Gi protein. Provide an adjustment method.

In addition, the present invention requires controlling the senescence of cells comprising administering to a patient an effective amount of an inhibitor of adenylyl cyclase, an inhibitor of protein kinase A, an inhibitor of protein kinase C or an activator of Gi protein. It provides a method for regulating patient's cell aging.

These proteins are involved in Gi-mediated signal transduction in vivo .

The inventors of the present invention have found that cell aging can be regulated by modulating the activity and expression of proteins involved in Gi-mediated signal transduction.

The method of the present invention can be applied to any senescent cell, but more important cells for the purpose of treatment include: (a) cells with replicative capacity in the central nervous system: Alzheimer's disease, Parkinson's disease, Huntington's disease and Astrosite, endothelial cells and fibroblasts that play an important role in aging-related diseases such as stroke; (b) Cells with restored cloning capacity in the integument: skin atropies, elastolysis and skin wrinkles, sebaceous gland hyperplasia, senile sunspots, hair whitening and hair loss, chronic skin ulcers and aging- Fibroblasts, sebaceous gland cells, melanocytes, keratinocytes, Langerhans cells, and hair follicle cells, which play an important role in aging-related diseases of the envelope, such as attenuation of related wound healing ability; (c) cells with limited replication capacity in articular cartilage: for example, chondrocytes with important dynamics in degenerative joint disease, and lacunal and synovial fibroblasts; (d) cells with limited replication capacity in bone : Osteoblasts, myeloid stromal fibroblasts and bone precursor cells, for example, which play an important role in osteoporosis; (e) Cells with limited replication capacity in the immune system: for example, B and T lymphocytes, monosites, neutrophils, eosinophils, basophilic leukocytes, NK cells and their respective precursors, which play an important role in aging-related immune system weakness cell; (f) Cells with limited replication capacity in the vascular system: epidermal cells, smooth muscle cells and adventitial fibroblasts that play an important role in the aging-related diseases of the vascular system, such as atherosclerosis, calcification, thrombus and aneurysms, and (g) Cells with limited replication capacity in the eye: for example, pigmented epithelial and vascular endothelial cells that play a role in macular degeneration.

According to another aspect of the invention, the invention provides a composition for regulating cellular senescence of senescent cells comprising an effective amount of an inhibitor of adenylyl cyclase, an inhibitor of protein kinase A, an inhibitor of protein kinase C or an activator of Gi protein. to provide.

Based on the novel findings described above, the inventors of the present invention have studied targets that are effective in regulating cellular senescence. As a result, it was found that adenylyl cyclase, protein kinase A, protein kinase C and Gi proteins are suitable targets for the regulation of cell aging. As mentioned above, the targets exhibited increased or decreased expression and / or activity in young cells, ie, senescent cells.

In the present invention, suitable inhibitors of adenylyl cyclase are not particularly limited and include inhibitors known in the art. For example, the inhibitor may be selected from 2 ', 5'-dideoxyadenosine, cis-N- (2-phenylcyclopentyl) azacyclotridec-1-en-2-amine ( cis-N- (2-phenylcyclopentyl) azacyclotridec-1-en-2-amine), and 9- (tetrahydro-2'-furyl) adenine (9- (tetrahydro-2'-furyl) adenine). The inhibitor is commercially available from Calbiochem. An effective amount of the adenylyl cyclase inhibitor necessary for regulating aging of the cells is 1 to 500 μM.

Suitable inhibitors of protein kinase A in the present invention are adenosine 3 ', 5'-cyclic phosphorothiolate, 8-bromo-adenosine 3', 5'-cyclic monophosphoro Thiolate (8-bromo-adenosine 3 ', 5'-cyclic monophosphorothioate), 4-cyano-3-methylisoquinoline, 1- (5-isoquinolinesulfonyl) -2 Methylpiperazine (1- (5-isoquinolinesulfonyl) -2-methylpiperazine), N- [2- (methylamino) ethyl] -5-isoquirrolinesulfonamide (N- (2-aminoethyl) -5-isoquinolinesulfonamide) , Isoquinolinesulfonamide, N- (2-aminoethyl) -5-isoquinolinesulfonamide, N- [2-((p-bromosinnami) amino) ethyl] -5-isoquinolinesulfonamide (N- [2-((p-bromocinnamy) amino) ethyl] -5-isoquinolinesulfonamide) and (5-isoquinolinesulfonyl) piperazine ((5-isoquinolinesul fonyl) piperazine) (available from Calbiochem) Including but not limited to.

Inhibitors of protein kinase C in the present invention are 2- [1- (3-dimethylaminopropyl) -1H-indol-3-yl] -3- (1H-indol-3-yl) -maleimide (2- [1 -(3-dimethylaminopropyl) -1H-indol-3-yl] -3- (1H-indol-3-yl) -maleimide), 2- [1- [2- (1-methylpyrrolidino) ethyl]- 1H-indol-3-yl] -3- (1H-indol-3-yl) maleimide (2- [1- [2- (1-methylpyrrolidino) ethyl] -1H-indol-3-yl] -3- (1H-indol-3-yl) maleimide), 2- [1- (3-aminopropyl) -1H-indol-3-yl] -3- (1H-indol-3-yl) maleimide) (2- [1- (3-aminopropyl) -1H-indol-3-yl] -3- (1H-indol-3-yl) maleimide), 2,3-bis (1H-indol-3-yl) maleimide (2 , 3-bis (1H-indol-3-yl) maleimide) and 2,3-bis (1H-indol-3-yl) -N-methylmaleimide 2,3-bis (1H-indol-3-yl) -N-methylmaleimide (commercially available from Calbiochem), including but not limited to. An effective amount of protein kinase C inhibitor for aging regulation is 1 to 500 μM.

Activators of Gi protein in the present invention include N ₆ - cyclopentyl adenosine (N ₆ -cyclopentyladenosine), 5- chloro -N ₆ - adenosine _{(5-chloro-N 6 -adenosine} ), 2- [p- (2- carboxyethyl Phenethylamino] -5'-N-ethylcarboxamidoadenosine (2- [p- (2-carboxyethyl) phenethylamino] -5'-N-ethylcarboxamidoadenosine), oxymetazoline, prazosin , 2- [2- [4- (2-methoxyphenyl) -1-piperazinyl] ethyl] -4,4-dimethyl- (2H, 4H) -isoquinoline-1,3-dione (2- [ 2- [4- (2-methoxyphenyl) -1-piperazinyl] ethyl] -4,4-dimethyl- (2H, 4H) -isoquinoline-1,3-dione), cannibinol, MGSA (melanoma growth Stimulatory activity; sigma), 3-aminopropylphosphinic acid, galanin, quisqualate, sumatriptan, melatonin, (5,7,8) -(-)-N-methyl- [7- (1-pyrrolidinyl) -1-oxaspiro (4,5) dec-8-yl] benzeneacetamide ((5,7,8)-(- ) -N-methyl- [7- (1-pyrrolidinyl) -1-oxaspiro (4,5) de c-8-yl] benzeneacetamide) and pertussis toxin. An effective amount of protein kinase A inhibitor for aging regulation is 1 to 500 μM.

According to a preferred embodiment of the invention, cells suitable for the invention are derived from mammalian cells such as human cells. More preferably, the cells in the present invention are fibroblasts.

1 shows the differential change in growth factor-stimulated calcium release in aged fibroblasts.

Presenescent (passage 7: A and C) and aged (passage 27: B and D) fibroblasts were grown on coverslips, serum depleted, serum-free 4 μM fluoro- before aging Incubate for 40 min in 3-AM medium. For real-time measurement of signal transduction molecules, the stained cells were passed through a micro-spray system with 50 ng / ml of platelet-derived growth factors (A and B) or 1 μg / ml of lysophosphadiic acid (C and D). Treated. Changes in intracellular Ca ²⁺ at the single cell level were analyzed by laser scanning confocal microscopy and the results are expressed in relative fluorescence intensity (RF1).

2 shows the differential changes in growth factor-induced actin polymerization in aging fibroblasts. Pre-aging fibroblasts (passage 10: Pa 10) and aging fibroblasts (passage 27: Pa 27) were grown on coverslips and serum depleted, followed by excipient (C), 50 ng / ml platelet- Treatment with derived growth factor (PDGF) or 1 μg / ml lysophosphatidic acid (LPA). Cells were fixed, permeabilized, stained with NBD-palasidine and photographed.

3 shows a decrease in growth factor-induced inositol phosphate production in nearly aged fibroblasts. Overnight labeled with 0.5 μCi / ml myo- [ ³ H] inositol without serum feeding subconfluent pre-aging cells (passage 6: Pa 6) and nearly aged (passage 23: Pa 23) cells Then, excipients (C), 50 ng / ml platelet-derived growth factor (PDGF) or 1 μg / ml lysophosphatidic acid (LPA) were stimulated for 20 minutes in the presence of 20 mM LiCl. Accumulated total [ ³ H] inositol phosphate was measured as described in Materials and Methods. Data is presented as the level of inositol phosphate (IPs) divided by the sum of inositol (Ins) and inositol phosphate (IPs), the data being the average of the experiments performed in three replicates.

4 shows a reduction of growth factor-induced phosphatidylbutanol formation in nearly aged fibroblasts. Semi-dense pre-aging cells (passage 8: Pa 8) and nearly aged (passage 24: Pa 24) cells were overnight labeled with [ ³ H] mylistic acid without serum supply. Cells were then stimulated for 20 minutes using excipient (C), platelet-derived growth factor (PDGF) or lysophosphatidic acid (LPA) in the presence of 0.5% 1-butanol. [ ³ H] phosphatidylbutanol (PtdBut) accumulation was measured as described in Materials and Methods. Representative experimental results of the two repetitions.

5 shows the reduction of growth factor-induced thymidine incorporation in nearly aged fibroblasts. Semi-dense pre-aging fibroblasts (passage 8: Pa 8) and nearly aged (passage 24: Pa 24) fibroblasts were depleted of serum for 2 days and platelet-derived growth factor (PDGF) of 10-200 ng / ml ) Or 1-70 μg / ml lysophosphatidic acid (LPA) was treated for 16 hours. The amount of [ ³ H] thymidine injected into DNA for 4 hours was measured. Data is the average of three counts in cpm. A is a representative experimental result of three experiments. The fold increase for untreated controls is calculated and shown in B.

6 shows a decrease in protein tyrosine phosphorylation stimulated by platelet-derived growth factors in nearly aged fibroblasts. Semi-dense pre-aging cells (passage 6: Pa 6), medium cells (passage 18, Pa 18) and almost aged (passage 25: Pa 25) cells were not serum fed for 2 days, then Cells were treated with excipient (C) or 50 ng / ml platelet-derived growth factor (PG). After growth factor treatment cells were lysed for 5 minutes and tyrosine phosphorylated proteins were visualized by SDS-PAGE and Western blot analysis using polyclonal anti-phosphotyrosine antibodies. Two major tyrosine-phosphorylated bands showing platelet-derived growth factor receptor (PDGFR) and phospholipase C-γ1 (PLC-γ1) could be observed.

7 shows changes in the levels of phospholipase C-γ1 and platelet-derived growth factor receptors during the aging process. Pre-aging cells (passage 6, 8 or 10: Pa 6, Pa 8, Pa 10), intermediate cells (passage 18, Pa 18) and nearly aged cells (passage 25 or 27: Pa 25 or Pa 27) Lysis in lysis buffer containing 1% Igepal CA630 nonionic solvent (detergent). Equal amounts of protein in total cell lysate were analyzed by 8% SDS-PAGE and Western blot and were polyclonal anti-phospholipase C-γ1 (PLC-γ1: A) or anti-platelet-derived growth factor receptor A / B antibody (PDGFR: B) was used. Main platelet-derived growth factor receptor type B in fibroblasts was visualized using the ECL detection system.

8 shows changes in levels of protein kinase C and phospholipase D1 protein during the aging process. Pre-aging cells (passage 6 or 8: Pa 6, Pa 8), intermediate cells (passage 18, Pa 18), and nearly aged cells (passage 25 or 27: Pa 25 or Pa 27) were 1% Igepal CA630 Lysis was performed in lysis buffer containing a detergent. To determine the level of protein kinase C, the same amount of protein in total cell lysate was analyzed by 10% SDS-PAGE and Western blot and polyclonal anti-PKCs antibodies (PKCs: A) were used. Phospholipase D1 was immunoprecipitated in cell lysates using anti-phospholipase D1 antibody (0.5 mg protein / ml) and analyzed by 8% SDS-PAGE and Western blot using anti-phospholipase D1 antibody It was. Phospholipase D1 protein bands and unknown nonspecific bands (p15) were visualized using the ECL detection system (PLD1: B).

9 shows LPA-induced changes in cellular cAMP in pre-aging fibroblasts and nearly aged fibroblasts. Semi-dense pre-aging fibroblasts (passage 8: Pa 8) and nearly aged fibroblasts (passage 24: Pa 24) were depleted serum for 2 days and treated with LPA: A, 30 min 1-70 μg / Ml LPA; B, indicated time 30 μg / ml LPA. The level of cAMP in the acid extract was measured by cAMP binding assay. Data is the average of three replicates.

10 shows LPA-dependent PKA activity and CREB phosphorylation in pre-aging fibroblasts and nearly aged fibroblasts. Semi-dense pre-aging fibroblasts (passage 8: Pa 8) and nearly aged fibroblasts (passage 24: Pa 24) were depleted serum for 2 days and treated with 30 μg / ml LPA for the indicated time. PKA activity (A) in cell lysates was measured using kemptide and [γ- ³² P] ATP as substrate. Radioactivity injected into the camptide was measured by beta-counter and the activity of the enzyme was calculated as pmol / min / mg protein. Data is the average of 3 replicates. Levels of phospho-CREB (B) were determined by Western blot analysis using anti-phospho-CREB antibodies.

Figure 11 shows LPA-dependent adenylyl cyclase and phosphodiesterase activity in pre-aging fibroblasts and nearly aged fibroblasts. Semi-dense pre-aging fibroblasts (passage 8: Pa 8) and nearly aged fibroblasts (passage 24: Pa 24) were depleted serum for 2 days and treated with 30 μg / ml LPA for the indicated time. The activity of adenylyl cyclase (A) and the phosphodiesterase (B) were measured as described in Materials and Methods on fresh membranes and whole cell lysates. Enzyme activity was calculated as pmol / μg / min. Data is the average of 3 replicates.

12 shows differential changes in mRNA levels of LPA receptors during aging. Total RNA was extracted from pre-aging cells (Pa 7) and nearly aged cells (Pa 27) using the acid guanidinium thiocyanate phenol-chloroform extraction method and mRNA levels were quasi-as described in Materials and Methods. Comparison was made by quantitative RT-PCR. RNA mixtures (R1-R4) containing 2 μg total RNA in a 12 μl volume were transcribed and amplified by PCR. RT-PCR products of LPA receptors (EDG-1, 2, 4, 7) were isolated on 1.2% agarose gels, photographed (A) and analyzed by densitometry (B). Three replicates were performed and similar results were obtained. Representative experimental results are disclosed.

Figure 13 shows the differential changes in mRNA levels of G protein during the aging process. Total RNA was extracted from pre-aging cells (Pa 7) and nearly aged cells (Pa 27). RT-PCR products of Gq and Gi (Gi1, Gi2, Gi3) were separated on 1.2% agarose gels, photographed (A) and analyzed by densitometry (B). Three replicates were performed and similar results were obtained. Representative experimental results are disclosed.

FIG. 14 shows expression of AC, PDE and PKA heterologous mRNAs in pre- and near aging human diploid fibroblasts. Total RNA was extracted from pre-aging cells (Pa 9, Y) and nearly aged cells (Pa 28, O) using the acid guanidinium thiocyanate phenol-chloroform extraction method, and mRNA levels were determined in materials and methods. Comparison was made by sub-quantitative RT-PCR as described. RNA mixtures (R1-R4) containing 2 μg total RNA in a 12 μl volume were transcribed and amplified by PCR. The RT-PCR product of the AC isoform was separated on a 1.2% agarose gel, photographed (A) and analyzed by densitometer (B). Three replicates were performed and similar results were obtained. Representative experimental results are disclosed.

15 shows cAMP content change by LPA in the presence of PKC inhibitor. Semi-dense nearly-aged fibroblasts (passage 26, Pa 26) were depleted of serum for 2 days and 30 μg / ml LPA was applied for 10 μM PKC inhibitor bisindolylmaleiamide (bIM) for the indicated time. Treatment was in the absence or presence. The level of cAMP in the acid extract was measured by cAMP binding method. Average data for three replicates were disclosed.

FIG. 16 shows PKC activation by LPA in pre- and near aging human diploid fibroblasts. Semi-dense aging (passage 10, Pa 10) and nearly aged (passage 29, Pa 29) fibroblasts were depleted of serum for 2 days and treated with 30 μg / ml LPA for the indicated time. Aggregation and migration of PKC proteins into the plasma membrane showed PKC activation. PKC-α was detected using polyclonal anti-PKC-α antibody and FITC-conjugated secondary antibody under confocal microscopy system.

FIG. 17 shows ERK activation by LPA in pre- and near aging human diploid fibroblasts. Serum depleted (passage 10, Pa 10) and nearly aged (passage 29, Pa 29) fibroblasts were treated with 30 μg / ml LPA for the indicated time. To confirm ERK activation, phospho-ERK levels in the cytoplasmic fraction of each cell were measured by Western blot using anti-phospho-ERK antibodies.

18 shows changes in LPA-induced thymidine infusion and cell proliferation in nearly aged fibroblasts versus pre-aging fibroblasts. Semi-dense aging (passage 8, Pa 8) and nearly aged (passage 24, Pa 24) fibroblasts were depleted of serum for 2 days and treated with LPA (1-70 μg / ml) for 16 hours. [ ³ H] thymidine injected into DNA for 4 hours was measured as described. Data is the average of three counts in cpm units of an experiment run independently. Representative experimental results of three experiments are shown in A. FIG. The converted fold increase for the untreated control was calculated and shown in B.

19 shows ERK activation of LPA-induced cell proliferation in pre- and near aging human diploid fibroblasts. Optical density at 570 nm is shown (A). The actual viable cell number is shown in (B).

20 shows the results of viable cell counts dependent on adenylyl cyclase inhibitor (ACI) and / or LPA in senescent cells. O and Y refer to aged cells and young cells, respectively.

21 shows the results of FACS analysis. The analysis evaluated the effect of LPA on cell cycle progression in aged cells in association with the effect of cAMP control. Con represents a control and ACI represents an adenylyl cyclase inhibitor.

The following examples are intended to illustrate the invention in more detail, and should not be construed as limiting the scope of the invention described in the claims.

Test substance and test method

Experimental substance

Dulbeccos modified Eagles medium (DMEM), platelet-derived growth factor (PDGF) lysophosphatidic acid (LPA), dibutyric cAMP (dbcAMP) and IGEPAL CA630 were purchased from Sigma; Fetal serum, antibiotics including penicillin and streptomycin and the SuperScript II reverse transcriptase kit were purchased from Gibco / BRL Life Technologies, Inc .; Polyclonal anti-phospholipase D1 antibody (Box45-ESTP4 for immunoprecipitation, NC-STP4 for Western blot analysis) was donated by Dr. Paek Gill of Pohang University of Science and Technology; Polyclonal anti-platelet-derived growth factor (PDGF) type A / B receptors and protein kinase C-α antibodies were purchased from Upstate Biotechnology; Polyclonal anti-Gi and Gq antibodies were purchased from Santa Cruz Biotechnology; PCR reagents containing Taq polymerase were purchased from Takara. ECL detection kits, cAMP assay kits, [γ- ³² P] ATP and [ ³ H] myritic acid were purchased from Amersham Pharmacia Biotech. [ ³ H] thymidine is purchased from NEN; Phosphatidylbutanol was purchased from Avanti; Kemptide is purchased from UBI; Immobilon PVDF was purchased from Millipore; NBD-palasidine [N- (7-Nitrobenz-2-oxa-1,3-diazol-4-yl) phallacidin] was purchased from Mocula Probe (Eugene, OR, USA).

Cell culture

Foreskin fibroblasts were isolated according to Voice and Implications (1983). The isolated cells were cultured in DMEM containing 10% fetal bovine serum and antibiotics. Primary culture included smaller and more tightly packed cells during the initial stages of culture. Generally, cells of passage 10 or less were considered presenescent cells. As the cells were passaged, they grew in size and slowed in passage 10-20. Cells over passage 27 completely stopped growing and began to exhibit characteristic phenomena such as previously reported replicative senescence (Yeo et al ., 2000a and b). To determine if the cells are aging, we measure the beta-galactosidase activity or a membrane permeable substrate of the enzyme, which produces a blue insoluble product inside the cell. Stained with gal. The number of cells stained with X-gal increased with age and most cells of passage 27-28 were stained. In addition, cultures from passages 23-28 stopped cell growth and showed aging-like morphological changes, but 70-80% of cells stained with X-gal. This phenomenon is referred to as near-senescent. Although nearly aged cultures did not become completely uniform, we compared the changes in these cultures with cells of passages 6-10. To identify growth factor-stimulated signaling phenomena, cells were cultured in a culture medium at 60-70% semi-dense for 1-2 days, and then in serum free medium (SFM) for 2 days. The cultures were then quiescent to serum-depleted state. The stabilized cells were treated with various agonists as mentioned in the brief description of the figures. To label cells, radiochemicals were treated during incubation with SFM.

Cellular free (Ca) ²⁺ ] measurement of i

[Ca ²⁺ ] i was measured using a laser scanning confocal microscope as described in Jun et al . (2000). Stabilized fibroblasts cultured on glass cover slips in 6-well plates were treated with 4 μM of fluo-3-acetoxymethyl ester (fluoro-3-AM) in medium without serum and without phenol red. Stain for 40 minutes and wash three times with serum-free medium. Stained cells on each cover slip were mounted in a perfusion chamber (MPS-1000, Seoul Engineering, Korea), and placed on a confocal laser scanning microscope (Carl Zeiss LSM 410) followed by a 488 nm excitation argon laser and 515 Scanning was performed every 5 seconds with a nm long pass emission filter. Growth factors were added to the cells using an automated pump system (self-rigidized). All scanned images are “Time Series Program” for measuring simultaneous relative fluorescence intensities mounted on a Carl Zeiss LSM 410 confocal system to analyze [Ca ²⁺ ] i changes at the single cell level. It was analyzed using.

Confocal Microscopy Measurement of Actin Polymerization

F-actin was stained as described in Shin et al . (1999). Stabilized cells cultured on glass cover slips in a multi-well culture plate were treated with agonists for 30 minutes, and the cells were fixed for 30 minutes in ice with 3.7% paraformaldehyde dissolved in PBS. Then permeate on ice with 0.2% Triton X-100 dissolved in PBS for 15 minutes, and the cells were stained for 30 minutes at room temperature with 0.165 M NBD-palasidine dissolved in PBS. The stained cells were washed three times with PBS for 15 minutes and then the cover slip was mounted on slide glass using Gelvatol prepared by mixing 100 ml of 23% polyvinyl alcohol dissolved in PBS with 50 ml glycerol. I was. The cells were then observed under confocal fluorescence microscopy.

Measurement of phosphatidylbutanol formation as a label of phospholipase D activity

Stabilized cells were labeled overnight with [ ³ H] myrilic acid (1 μCi / ml) and then washed with PBS. The formation of phosphatidylbutanol was measured as described in Yeo et al . (1994). The cells were pre-reacted for 15 minutes with 0.5% (v / v) 1-butanol and then treated with agonists for 20 minutes in the presence of 0.5% 1-butanol. Total lipids were extracted and separated by thin layer chromatography. A band of phosphatidylbutanol was separated from the plate and its radioactivity counted in a liquid scintillation cocktail.

Western blot analysis

Expression levels of signaling proteins were analyzed by Western blot analysis as described in Yeo et al ., 2000a. 150 mM NaCl, 2 mM EDTA, 1 mM EGTA, 1 mM Na ₃ VO ₄ , 10 mM NaF, 1 mM DTT, 1 mM PMSF, 25 μg / ml Lupeptin, 25 containing 50 mM Tris-HCl, pH 7.5 Total cell lysates were prepared using lysis buffer containing μg / ml aprotinin, 5 mM benzamidine, and 1% nonidet P-40. Protein concentration of the cell lysate was measured according to the manufacturer's policy using the BCA protein quantification kit. Cell lysates containing the same amount of protein were separated via SDS-polyacrylamide gel electrophoresis and transferred to an immobilon PVDF membrane. The blots were blocked using a blocking solution containing 5% skim milk powder and 0.1% Tween 20 and then treated with antibodies in the blocking solution overnight. The blot was washed and then reacted with horseradish peroxidase-conjugated anti-rabbit IgG (1: 5000) and the immune precipitate formed was visualized according to the manufacturer's policy using an enhanced chemiluminescence (ECL) system. To confirm the level of phospholipase D 1/2, immunoprecipitated proteins obtained using 10 μl of anti-phospholipase D 1 anti-serum (Box45, ESTP4) were subjected to polyclonal anti-phospholipase. Analyze by Western blot for D 1 antibody (diluted 1: 2 with NC STP4 anti-serum).

Determination of Total Inositol Phosphate

Stylized depleted 0.1% bovine serum albumin, and in the absence of inositol DMEM containing antibiotics, 0.5 μCi / ㎖ Maio-aging before and little aging cell serum for 2 days by using a ^[3 H] inositol was labeled overnight . The cells were then washed three times with medium containing 0.1% bovine serum albumin in serum free medium. The cells were preincubated for 1 hour at 37 ° C. in the medium and allowed to stand for 10 minutes with the addition of 20 mM LiCl before reacting with 50 ng / ml PDGF or 1 μg / ml LPA. After 20 minutes, the cells were washed twice with ice-cold PBS and lysed with 25 mM formic acid. Total inositol phosphate was measured according to Yeo et al ., 1994.

Measurement of Cyclic AMP (cAMP) Accumulation

Cells cultured in 12 well-plates were serum-depleted by incubating for 2 days with SFM. The cells were equilibrated in fresh SFM medium for 1 hour and then treated with LPA as described in the detailed description of the figures. The reaction was stopped by removing the medium and adding 2.5 M perchloric acid. Acidic extracts were stored at -20 ° C until use. By literature as competitive binding (Jang and Juhnn (2001)) [3 H] cAMP to a cAMP binding protein on the cAMP- dependent protein kinase according to the RIα, such as the extract of the acid after neutralization with a sheet of 4.2 M KOH cAMP The content was measured. After extraction, cells were lysed with 0.1 N NaOH and analyzed for protein content using Bradford protein assay reagent. cAMP content was normalized to the amount of acid-insoluble protein.

Determination of Adenylyl Cyclase Activity

Cells seeded in 10-cm culture plates were serum-depleted and treated with LPA as described in the detailed description of the drawings. Cells were harvested and tissue milled in ice-cooled tissue grinding buffer containing 0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 2 mM EGTA and protease inhibitor. The tissue mill was centrifuged at 20,000 × g for 20 minutes at 4 ° C. The pellet was washed twice with this buffer, then suspended in 10 mM Tris-HCl, pH 7.4 and 10 mM MgCl ₂ and subjected to adenylyl cyclase assay according to Jan et al. (Jang et al. 2001). Membrane protein (15 μg / ml) was added to 40 mM Tris, pH 7.4, 0.2 mM EGTA, 100 mM NaCl, 10 mM MgCl ₂ , 0.5 mM ATP, 5 μg / ml phosphocreatin, 5 IU / ml creatine phosphokinase, The reaction mixture (100 μl) containing 10 μM GTP and 0.5 mM IBMX was reacted at 30 ° C. for 10 minutes. The reaction was stopped by the addition of 2.5 M perchloric acid. The amount of cAMP produced was measured by cAMP binding assay.

Determination of Phosphodiesterase Activity

Phosphodiesterase (PDE) analysis was performed according to Jan and Jun (Jang and Juhnn, 2001). 15 μg of cytosolic protein was reacted at 37 ° C. for 15 minutes in 200 μl of reaction mixture containing 50 mM Tris-HCl, pH 7.4, 3 mM MgCl ₂ , 60 pmol cAMP, 1 mM 5′-AMP. The reaction was then stopped by the addition of 2.0 M perchloric acid and the amount of cAMP remaining was measured by cAMP binding assay.

Measurement of Protein Kinase A Activity

LPA-treated cells were harvested and then tissue milled in an ice-cooled tissue grinding buffer containing 0.25 M sucrose, 10 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 2 mM EGTA and protease inhibitor. The tissue mill was centrifuged at 20,000 × g at 4 ° C. for 20 minutes. Protein kinase A analysis was performed according to Yang et al. (Yang et al., 1999). The kinase reaction mixture consists of 10 μl of substrate cocktail (500 μM camptide and 10 μM cAMP), 10 μl of inhibitor cocktail (20 μM PKC inhibitor peptide and 20 μM compound R24571), 10 μl of tissue grind and 0.5 mM ATP 10 μl of a mixed solution containing 75 mM MgCl ₂ and 10 μCi of [γ- ³² P] ATP (3000 Ci / mmol). The mixture was allowed to react at 30 ° C. for 10 minutes and then 25 μl of the mixture was blotted onto P81 paper square. The paper was washed once with 0.75% phosphoric acid and the radioactivity bound to the camptide was quantified in a scintillation counter (Model Tri-Carb 1600 CA, Packard Instrument Company). Samples were analyzed three times for each condition. PKI-inhibitory kinase activity was calculated and then expressed as a percentage of total PKA activity.

Cell extracts were treated with 50 mM Tris, pH 7.5, 10 mM MgCl ₂ , 100 μM ATP, 4 nnmol [γ- ³² P] ATP, 0.25 mg / ml BSA and 50 μM of camptide (Leu-Arg-Arg-Ala-Ser -Leu-Gly), either without additives (control) or with 1 μM PKI peptide (background), with 10 μM cAMP (for total activity) or with kinase reaction buffer containing PKI and cAMP (for total background activity) The reaction was carried out at 5 ° C. for 5 minutes. Samples were analyzed three times for each condition and the camptide bound radioactivity was quantified in a scintillation counter. PKI-inhibitory kinase activity was calculated and its value expressed as a percentage of total PKA activity.

Semi-quantitative RT-PCR

Total RNA was extracted from pre-aging and nearly aged cells using the acid guanidium thiocyanate phenol-chloroform extraction method (Chomczynski and Sacchi, 1987). To compare the relative amounts of mRNA in pre-aging and nearly aged cells, a semi-quantitative reverse transcription / polymerization chain reaction (RT-PCR) was performed according to Nicotti and Sassy-Prigent (1996). It was. Stepwise mixtures were prepared by mixing RNA isolated from pre-aging and nearly aged cells, whereby each mixture had the same total RNA amount (2 μg) in the same volume (12 μl). The RT reaction was carried out in a final volume of 20 μl using SuperScript II reverse transcriptase according to the manufacturer's policy, and PCR amplified with 4 μl of the final RT product mixture. The primer set used was 5'-ATG ATC GAC CGC AAC CTC-3 'and 5'-CTT CAA CCT CCC CAT AGC C-3' to amplify G _i1 , 5'-GAT CGA CTT to amplify G _i2 TGC CGA CCC-3 'and 5'-TCG TTC AGG TAG TAG GCA GC-3', G to amplify _{i3 5'-TGG CAG TGC TGA AGA} AGG-3 ' and 5'-GGT CTT CAC TCT CGT CCG- 3 'was used (Takano et al., 1997). Primers for amplifying Gqα include 5'-ATG ACT TGG ACC GTG TAG CCG ACC-3 '(sense) and 5'-CCA TGC GGT TCT CAT TGT CTG ACT as described in Shah et al. (Shah (1999)). -3 '(antisense) was used. In addition, the DNA fragments corresponding to the tertiary cytoplasmic loop (i3) of EDG-1 include 5'-AGA ATC TAC TCC TTG GTC AGG ACT-3 '(sense) and 5'-TAC CCG GGT TAC TTG AGC GCC AGC GAC TTC TC- Amplification via PCR using 3 ′ (antisense) primers (Lee et al., 1996). PCR primers for EDG-2, -4, and -7 were designed based on unconserved amino acid sequences including other membrane transfer sites (Bandoh et al., 1999). EDG-2 primers comprise a site comprising TM 3 through TM 1 (360 bp); EDG-4 primers comprise a region comprising 5 to 5 (582 bp); And EDG-7 primers amplify a region (750 bp) containing 6 to TM 1. Oligonucleotides used were 5'-GGA AAG CAT CTT GCC ACA GAA-3 '(sense) and 5'-GCC GGT TGC TCA TCC GTG TGT-3' (antisense) for EDG-2, 5 for EDG-4 '-GGC AAA GAG CTC AGC TCC CAC-3' (sense) and 5'-CAT GCG CTG CAC TCG CCG CCG-3 '(antisense) and 5'-AAC ACT GAT ACT GTC GAT GAC-3 for EDG-7 (Sense) and 5'GAC AGT CAT CAC CGT CTT CAT-3 '(antisense). Primers for adenylyl cyclase variants, phosphodiesterases and protein kinase A were described in Jan et al. (2001), Cho et al. (2000) and Jang et al. Prepared according to the literature of Jang and Juhnn (2001).

Real-time PCR or concentration gradient PCR was used to select PCR conditions appropriately such that there was a linear relationship between the amount of template mRNA and the amount of amplified product mixture. PCR products were electrophoresed on 1.2% agarose gels containing ethidium bromide. The amount of mRNA was compared using the results of the concentration measurement of the band obtained by Bio-Rad GS 710 Calibrated Imaging Densitometry.

DNA synthesis

Stabilized cells were labeled using [ ³ H] thymidine (0.5 μCi / ml) for 4 hours, and unlabeled radioactive thymidine was removed by washing with PBS. DNA was precipitated with 10% TCA and dissolved in 0.5N NaOH. Radioactivity in the neutralized digest was counted in the liquid scintillation cocktail.

Measurement of the Action of Adenylyl Cylase Inhibitors in Aging Cells

Young or aged HDF (passage 8-11) was mixed at 37 ° C. with a mixture of LPA (30 μM), dbcAMP (cAMP analog, 1 mM, Sigma) and adenylyl cyclase inhibitor, SQ22536 (300 μM, Calbiochem) Treated for 30-60 minutes. Treated cells were stained with Trypan Blue (GiBco / BRL) at 37 ° C. for 10 minutes and then only cells that were not blue were counted with a hemacytometer.

In addition, the treated cells were analyzed by flow cytometry. The treated cells were harvested and treated with 1 ml of 0.1% trypsin for 30 seconds, and then centrifuged at 1000 rpm for 10 minutes at 4 ° C. The cell concentration was then adjusted to a concentration of 1-3 × 10 ⁶ cells / ml in the sample buffer. Sample buffers were prepared by placing 1 g of glucose in 1 l PBS without Ca ²⁺ and Mg ²⁺ and filtering with a 0.22 μm filter. The solution containing the cells was centrifuged at 1000 rpm at 4 ° C. for 10 minutes and ice-cooled 70% ethanol was added dropwise to the pellet. The sample tube was closed and left in ethanol at 4 ° C. overnight to allow fixation, followed by brief vortexing and centrifugation at 3000 rpm for 5 minutes. Then, ethanol was discarded and PI (propidium iodide) staining solution (0.5 mL 20 × propidium iodide concentrated solution (final 50 μg / mL), 1000 kunits RNase A (final 100 U / mL) and 10 ML of sample buffer) was added to the sample tube. Finally, samples were analyzed with a slow flow cytometer (FACS Calibur, Beckton-Dikinson) within 24 hours.

result

1. Intracellular Ca in Aging Human Diploid Fibroblasts ²⁺  Differential change in level

Primary human diploid fibroblasts isolated from the penile lids of newborns were used to study the mechanism of decreasing response to growth factor accumulation with age. These cells were maintained and passaged as described in Yeo et al ., 2000a. These senescent cells showed characteristic morphological changes, increased beta-galactosidase activity, and verified to decrease the rate of proliferation. As observed in other cellular systems, aging and nearly aging cells grew larger and at lower density compared to their pre-aging equivalents. Here we report differential changes in signaling phenomena caused by platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA). In human diploid fibroblasts, PDGF induced signaling at a concentration range of 10-100 ng / ml, while LPA induced phospholipid hydrolysis, Ca ²⁺ migration, and actin at a concentration range of 0.1-10 ㎍ / ml. Promote the polymerization. To compare these cellular responses, we treated pre-aging and aging (or nearly aged) cells with PDGF and LPA, each with 50 ng / ml and 1 μg / ml maximum response doses. .

Changes in intracellular Ca ²⁺ were confirmed by staining pre-aging and aging fibroblasts with fluorine-3-AM and observing with confocal microscopy. Since senescent cells are much larger and flatter, the number of pre-aging cells was much higher than aging cells within the same scanned range. When we repeated this experiment, the cells showed similar results. We provide one of the representative experiments in FIG. 1. PDGF and LPA showed a sharp increase in cytosolic Ca ²⁺ levels in presenescent fibroblasts (passage 7) and the oscillating pattern of Ca ²⁺ increased agonist-specifically. However, this phenomenon changed in senescent cells (passage 27). Ca ²⁺ response was significantly weakened in PDGF-stimulated senescent cells (FIG. 1B) but not significantly weakened in LPA-stimulated senescent cells (FIG. 1D).

2. Differential Changes in Actin Polymerization in Aging Fibroblasts

Since LPA and PDGF are Ca ²⁺ -migrating agonists, actin polymerization was also observed in fibroblasts. As indicated in FIG. 2, the reference level of F-actin was much higher in senescent cells (passage 27) than in pre-aging cells (passage 10). Both PDGF and LPA increased the formation of stress fibers in fibroblasts before aging, and the changes induced by LPA were much greater. PDGF-induced actin polymerization was almost blocked in senescent cells, but LPA-induced actin polymerization was partially reduced.

3. Differential Changes of Agonist-Stimulated Phospholipid Hydrolysis by Phospholipases C and D in Nearly Aged Fibroblasts

Phospholipase C (PLC) is activated depending on its isoform via receptor tyrosine kinase or G-protein. One of the PLC activation products is inositol 1,4,5-triphosphate, which increases intracellular Ca ²⁺ secretion. In addition, since PIP ₂ , a major substrate of PLC, is involved in actin polymerization, we validate PLC activity by measuring the production of IP ₃ in pre-aging cells (passage 6) and nearly aged cells (passage 23). It was. As shown in FIG. 3, PDGF-stimulated inositol phosphate (IPs) production significantly decreased in nearly aged cells but nevertheless its level increased in LPA-stimulated senescent cells.

Since a deficiency of protein kinase C-dependent phospholipase D (PLD) activity has been shown in cell aging and PDGF has been shown to activate PLD through protein kinase C activity, we have found that The effect of aging on activity was verified. PLD activity was confirmed by measuring phosphatidylbutanol formation in the presence of 0.5% 1-butanol. As expected, the effect of PDGF and LPA on PLD activity was reduced in nearly aged cells at passage 24 (FIG. 4); An 80% reduction in PLD activity was observed in PDGF-stimulated cells. In contrast, LPA treatment showed only a 20% reduction in nearly aged cells.

4. Differential Changes in Growth Factor-Induced DNA Synthesis in Nearly Aged Fibroblasts

Age-dependent decrease in DNA synthesis following growth factor promotion may be a cause for the rate of proliferation (Peacocke and Campisi, 1991, Smith and Pereira-Smith, 1996). DNA synthesis rates were verified by measuring [ ³ H] thymidine binding to DNA. Reference levels of [ ³ H] thymidine binding to DNA were significantly reduced in nearly aged fibroblasts of passage 24. PDGF is a very strong agonist for [ ³ H] thymidine that binds to DNA of fibroblasts before aging in a concentration range of 10-100 ng / ml (FIG. 5A). When the initial passaged pre-aging cells (passage 8) stabilized to about 70% cell density, [ ³ H] thymidine binding increased 7-9 fold with 50-100 ng / ml of PDGF. However, treatment with 50-100 ng / ml of PDGF showed a significant decrease in PDGF-induced DNA synthesis in nearly aged cells, but only a 1.3-fold increase in [ ³ H] thymidine binding (FIG. 5B). Incubation of fibroblasts prior to aging with LPA increased the level of [ ³ H] thymidine binding, which began to increase at 10 μg / ml and peaked at 50-70 μg / ml. There was also a decrease in response to LPA but less than PDGF (FIG. 5A). When the fold increase was calculated, the response to 50-70 μg / ml LPA was very high in nearly aged cells (FIG. 5B). This observation was closely related to the effect on agonist-stimulated signaling phenomena involving the pattern of variation in [Ca ²⁺ ] _i , where PDGF-induced intracellular Ca ²⁺ secretion was much more than LPA-induced. Strongly suppressed.

5. Molecular changes associated with PDGF-signaling phenomena

In order to examine the reactivity of Ca ²⁺ response to PDGF promotion and reduction of IP ₃ production with age, we further validated the molecular changes associated with PDGF-signaling phenomena. PLC-γ1 is activated by binding to a growth factor receptor that is autophosphorylated, after which tyrosine-phosphorylation by activated receptor kinase is promoted by binding of the corresponding growth factor. Tyrosine phosphorylation of many proteins, including the PDGF receptor and PLC- [gamma] 1, gradually declined with increasing age of the cells (Figure 6). The first and second bands were the PDGF receptor itself and PLC-γ1, which was confirmed by a clearance experiment with protein A beads to which anti-PDGF receptor and anti-PLC-γ1 antibody were attached (Yeo et al. , 1997). In addition, when verified by Western blot analysis, the expression level of PLC-γ1 was not changed by increasing passage (FIG. 7A). Thus, it is believed that the reduced response of aged (or nearly aged) cells to PDGF promotion is not due to an increase in PLC-γ1 protein. In order to determine whether the significant reduction in PDGF-induced signaling is due to a decrease in the PDGF receptor itself, we further used polyclonal anti-human PDGF type A / B receptor antibodies to level the PDGF receptor. Was verified. Although the antibody recognizes both PDGF type A (170 kDa) and B (190 kDa) receptors, fibroblasts mainly comprise B-type receptors. Expression of PDGF receptor type B was found to be progressively decreased in regulation over the age of the cells (FIG. 7B).

Because PLD activity decreases in PDGF-stimulated nearly aged cells, in addition to the PDGF receptor, some proteins related to PLD activity, such as PKC and PLD variants, have changed with age. Western blot analysis revealed no change in the levels of PKCs (FIG. 8A), but gradually decreased levels of PLD1 protein with age (FIG. 8B).

6. LPA-induced cAMP signaling is altered in nearly aged human diploid fibroblasts

To study the differential changes in growth factor-stimulated signaling systems in senescent cells, we investigated the effects of aging on cAMP signaling in response to physiological lipid mitogen lysophosphatidic acid (LPA). I looked at it. Under basic conditions, the cAMP of the cells appeared to be 37.0 +/- 2.6 and 36 +/- 3.1 pmol / mg proteins, respectively, in pre-aging (passage 8) and almost aged fibroblasts (passage 27), respectively. In fibroblasts before aging, the cAMP of the cells decreased by 33-43% after 30 minutes of reaction with LPA in a concentration range of 1-70 μg / ml (FIG. 9A). In contrast, the LPA response increased cellular cAMP 31-77% in nearly aged fibroblasts. A 30 μg / ml dose of LPA effectively induced a decrease in cAMP in pre-aging cells (FIG. 9A). The profile of the time-dependent cAMP response also changed in near aging LPA-stimulated cells. The cAMP content of the cells increased by 70-80% while the nearly aged fibroblasts were reacted with 30 μg / ml LPA for 60 minutes (FIG. 9B).                 

7. Changes in Protein Kinase A Activity and CREB Phosphorylation by LPA Treatment

PKA is activated as cAMP accumulates. As expected, PKA activity is closely related to the changed profile of cAMP content. PKA activity decreased with LPA treatment in pre-aging cells but increased in nearly aged cells (FIG. 10A). CREB phosphorylation is one of the cAMP-induced responses for the regulation of gene expression. CREB phosphorylation was closely related to cAMP accumulation and subsequent PKA activity induced in LPA-stimulated nearly aged fibroblasts (FIG. 10B).

8. Changes in Adenylyl Cylase and Phosphodiesterase Activity Induced in Aging Cells

cAMP is produced by activated adenylyl cyclases (ACs) and degraded by activated phosphodiesterases (PDEs). The ability to break the PKA activity threshold depends on the activity of AC and the inhibition of each or all of the particular PDE variants. To verify whether the activity or protein content of ACs or PDEs changes with age, we first measured total AC and PDE activity. As shown in FIG. 11A, total AC activity decreased time-dependently in pre-aging cells by LPA, but increased in nearly aged cells. In contrast, as LPA accumulated, PDE activity increased in pre-aging cells and decreased in nearly aged cells (FIG. 11B).                 

9. Differential Changes in LPA Receptors

To investigate the mechanism of increased cAMP accumulation by LPA in senescent human diploid fibroblasts, we identified changes in LPA receptors and G-proteins involved in cAMP-signaling phenomena. In addition to the major members of the EDG family that work with LPA, EDG-2, EDG-4, and EDG-7, experiments were made with low-affinity LPA receptor EDG1. Specific mRNA transcripts for human LPA-receptors were detected by semi-quantitative RT-PCR using specific primers and each product was quantitatively analyzed. We detected mRNA transcripts for EDG-1, EDG-2, EDG-4 and EDG-7 (EDG1>EDG2>EDG4> EDG7) in human diploid fibroblasts before aging (FIG. 12). The inventors found that the levels of EDG-1 and -4 mRNA did not change with age in nearly aged fibroblasts, but the levels of EDG-2 and -7 decreased significantly.

10. Differential Changes in G Proteins

The cAMP system is one of the important G-protein signaling systems. Certain extracellular signals bind to and activate seven transmembrane receptors and bind to promoter G protein (Gsα) or inhibitory G protein (Giα) to regulate adenylyl cylase (AC). The LPA receptor EDG-2 binds to pertussis toxin-sensitive Gi, but EDG4 binds to both Gi and Gq (An et al., 1998), and EDG7 is a pertussis toxin-sensitive G-protein, possibly Gq. (Bandoh et al., 1999; Im et al., 2000). Among the subunits of G protein involved in the regulation of adenylyl cyclase, Giα directly inhibits some of the AC isoforms and Gq indirectly promotes some of the AC isoforms through Ca ⁺⁺ and / or calmodulin. Suppress Thus, mRNA levels of G protein were analyzed by semi-quantitative RT-PCR. As shown in FIG. 13, the mRNA level of Gq did not change even with age. In addition, mRNA levels of Gi1, Gi2 and Gi3 decreased in nearly aged cells.

11. Changes in the expression of adenylyl cyclase, phosphodiesterase and protein kinase A variants induced in senescent cells

Because the pluralism of this form of AC, PDE and PKA plays an important role in the regulation of cAMP signaling (Houslay and Milligan, 1997), we use semi-quantitative RT-PCR with specific primers to achieve this at the transcription level. Heterologous expression of the enzyme was verified. Each RT-PCR product was electrophoretically separated on an agarose gel containing ethidium bromide and photographed for quantitative analysis. Semi-quantitative RT-PCR results were confirmed by real time PCR.

At least nine genes for mammalian AC have been cloned (Sunahara et al., 1996). AC variants have common values and exhibit essentially different characteristics. All this forms are activated by both forskolin and GPT-binding Gsα and are inhibited by specific adenosine analogs such as 2'-deoxy-3'-AMP. All of these AC variants are further regulated in type-specific patterns by input of other signals including calcium ions, other G protein subunits (Gi / oα, Gqα and βγ) and protein kinase C (Taussig and Gilman, 1995; Sunahara et al., 1996). In pre-aging human diploid fibroblasts, ACII, ACIV, ACVI and ACIX mRNAs were detected by the experimental method of the present invention. In nearly aged fibroblasts the expression of ACII, ACIV and ACVI was increased but the expression of ACIX was decreased (FIG. 14A).

The cAMP of the cell can be hydrolyzed and inactivated by cyclic nucleotide phosphodiesterase (PDE). Mammalian PDEs are encoded by at least 19 different genes. The use of different transcriptional units and splicing of the exon of a single GDE gene results in the formation of proteins with different regulatory domains that bind to common catalytic domains, resulting in minor differences in properties and sensitivity to other signals. Has been extended to a heterogeneous array (Conti and Jin, 1999). Of the 19 PDE genes, types 4B and 4C were mainly expressed in human diploid fibroblasts before aging (FIG. 14B). MRNA levels of type 1A / B, 1C and 3A increased in nearly aged condition. Nearly aged, the mRNA levels of 4B were significantly reduced but the levels of 4C were not changed.

In addition, mRNA expression of PKA catalytic and regulatory subunits was compared before and between aging fibroblasts (FIG. 6). PKA catalytic subunits Cα, regulatory subunits RIα and RIβ increased with age, but regulatory subunit RIIα decreased.                 

12. Changes in PKC and ERK Activity According to LPA

ACII is a PKC-stimulated form. Increasing ACII in nearly aged fibroblasts will indicate PKC-dependence in AC activity by LPA. We detected cAMP content in LPA-stimulated nearly aged HDF in the presence of PKC inhibitor bis-indolylmaleiamide. As expected, we observed inhibition of cAMP accumulation in the presence of protein kinase C inhibitor (FIG. 15).

Since LPA promotes phospholipase C through Gq and results in calcium fluctuation, calcium-dependent PKC can be activated. Since calcium and PKC are modulators of AC activity, we identified whether LPA regulates PKC activity before and in aging HDF (FIG. 16). Baseline levels of PKC activity were much higher in nearly aged HDF compared to pre-aging HDF. Although the magnitude of increase in PKC activity was low in nearly aged cells, PKC activity by LPA was additionally increased.

ERKs may be accumulated through the βγ subunit of Gi or activated PKC / PLD pathways. Since Raf, an upregulator of ERK, is inhibited by cAMP-dependent protein kinase A, we verified the ERK activity induced by LPA. Levels of ERK activity were confirmed by comparing the levels of phospho-ERK via Western blot analysis. As shown in FIG. 17, both ERK1 and ERK2 in HDF before aging were promoted by treatment of LPA for 10-30 minutes and returned to baseline after treatment for 60 minutes. The basal level of ERK phosphorylation increased but did not additionally increase the level of phospho-ERK.

13. Changes in DNA Synthesis and Cell Proliferation Induced by LPA

Age-dependent decrease in DNA synthesis following LPA promotion was confirmed by measuring [ ³ H] thymidine binding to DNA. Baseline levels of [ ³ H] thymidine binding to DNA were significantly reduced in nearly aged fibroblasts. Culture of pre-aging fibroblasts with LPA increased the binding level of [ ³ H] thymidine, which began to increase at 10 μg / ml and reached a maximum at 50-70 μg / ml. In addition, the response to LPA decreased in nearly aged cells (FIG. 18A). When the fold increase was calculated, the response to 50-70 μg / ml LPA was very high in senescent cells (FIG. 18B).

To study the action of LPA-induced cAMP signaling, we compared LPA-stimulated cell proliferation prior to aging and between nearly aged cells. After 4 days of treatment with vehicle or LPA, the level of viable cells was measured by counting viable cells or by MTT assay. LPA stimulated proliferation of pre-aging cells was dose-dependent, but the rate of proliferation was partially reduced in nearly aged cells (Fig. 19).

14. Restoration of DNA Synthesis and Cell Proliferation in Aging Cells by Adenylyl Cylase Inhibitor

Recovery of DNA synthesis and cell proliferation in senescent cells by treatment with adenylyl cyclase inhibitor was verified by flow cytometry and cell count. The number of viable cells in senescent cell culture increased significantly with treatment of adenylyl cyclase inhibitor with LPA, and the pattern appeared very similar to young cells. In other words, cell proliferation in senescent cells changed in an increasing pattern (FIG. 20).

As shown in FIG. 21, in the flow cytometry assay, the number of cells in S phase in senescent cells increased significantly with treatment of adenylyl cyclase inhibitor. This increase was further enhanced by simultaneous treatment of adenylyl cyclase inhibitor with LPA.

In conclusion, in view of various physiological phenotypes, senescent cells can be transformed into young cells by treating adenylyl cyclase inhibitors and the response to LPA in senescent cells can be restored by treatment with adenylyl cyclase. It can be seen that there is.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.                 

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Claims (27)

Measuring a relative change in the young cell of the signal or molecular species involved in signal transduction, wherein the change in the signal or molecular species is (b) reduced expression of F-actin; (c) reduced activity of phospholipase C; (d) decreased activity of phospholipase D; (e) decreased expression or phosphorylation of platelet-derived growth factor receptors; (f) reduced phosphorylation of phospholipase C-γ1; (g) decreased expression of phospholipase D1; (h) decreased expression of EDG-2; (i) decreased expression of EDG-7; (j) decreased expression of Gi1; (k) decreased expression of Gi2; (l) decreased expression of Gi3; (m) increased adenylyl cyclase activity or expression; (n) decreased activity and expression of phosphodiesterases; (o) increased activity of protein kinase C; (p) increased activity and expression of protein kinase A; (q) increased phosphorylation of CREB; And (r) A method for detecting senescent cells, characterized in that at least one selected from the group consisting of increased cAMP content. The method of claim 1, wherein the signal or molecular species of (b)-(g) are involved in signal transduction caused by platelet-derived growth factors. The method of claim 1, wherein the (h)-(r) signal or molecular species is involved in signal transduction caused by lysophosphatidic acid. The method of claim 1, wherein the senescent cells are derived from human cells. The method of claim 4, wherein the human cells are fibroblasts. 4. The detection of senescent cells according to claim 3, wherein said adenylyl cyclase with increased expression in senescent cells is adenylyl cyclase II, adenylyl cyclase IV and adenylyl cyclase VI. Way. 4. The method of claim 3, wherein said phosphodiesterase having reduced expression in senescent cells is phosphodiesterase 4B. 4. The method of claim 3, wherein said protein kinase A with increased expression in senescent cells is a Cα, RIα or RIβ subunit. delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete

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