cAMP Stimulates Osteoblast-like Differentiation of Calcifying Vascular Cells

The role of the cAMP signaling pathway in vascular calcification was investigated using calcifying vascular cells (CVC) derived from primary aortic medial cell cultures. We previously showed that CVC have fibroblastic morphology and express several osteoblastic differentiation markers. After confluency, they aggregate into cellular condensations, which later mature into nodules where mineralization is localized. Here, we investigated the effects of cAMP on CVC differentiation because it plays a role in both osteoblastic differentiation and vascular disease. Dibutyryl-cAMP or forskolin treatment of CVC for 3 days induced osteoblast-like “cuboidal” morphology, inhibited proliferation, and enhanced alkaline phosphatase activity, all early markers of osteoblastic differentiation. Isobutylmethylxanthine and cholera toxin had the same effects. Treatment of CVC with pertussis toxin, however, did not induce the morphological change or increase alkaline phosphatase activity, although it inhibited CVC proliferation to a similar extent. cAMP also increased type I procollagen production and gene expression of matrix γ-carboxyglutamic acid protein, recently shown to play a role in in vivo vascular calcification. cAMP inhibited the expression of osteopontin but did not affect the expression of osteocalcin and core binding factor. Prolonged cAMP treatment enhanced matrix calcium-mineral incorporation but inhibited the condensations resulting in diffuse mineralization throughout the monolayer of cells. Treatment of CVC with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phosphatase activity and mineralization during spontaneous CVC differentiation. These results suggest that the cAMP pathway promotes in vitro vascular calcification by enhancing osteoblast-like differentiation of CVC. The role of the cAMP signaling pathway in vascular calcification was investigated using calcifying vascular cells (CVC) derived from primary aortic medial cell cultures. We previously showed that CVC have fibroblastic morphology and express several osteoblastic differentiation markers. After confluency, they aggregate into cellular condensations, which later mature into nodules where mineralization is localized. Here, we investigated the effects of cAMP on CVC differentiation because it plays a role in both osteoblastic differentiation and vascular disease. Dibutyryl-cAMP or forskolin treatment of CVC for 3 days induced osteoblast-like “cuboidal” morphology, inhibited proliferation, and enhanced alkaline phosphatase activity, all early markers of osteoblastic differentiation. Isobutylmethylxanthine and cholera toxin had the same effects. Treatment of CVC with pertussis toxin, however, did not induce the morphological change or increase alkaline phosphatase activity, although it inhibited CVC proliferation to a similar extent. cAMP also increased type I procollagen production and gene expression of matrix γ-carboxyglutamic acid protein, recently shown to play a role in in vivo vascular calcification. cAMP inhibited the expression of osteopontin but did not affect the expression of osteocalcin and core binding factor. Prolonged cAMP treatment enhanced matrix calcium-mineral incorporation but inhibited the condensations resulting in diffuse mineralization throughout the monolayer of cells. Treatment of CVC with a protein kinase A-specific inhibitor, KT5720, inhibited alkaline phosphatase activity and mineralization during spontaneous CVC differentiation. These results suggest that the cAMP pathway promotes in vitro vascular calcification by enhancing osteoblast-like differentiation of CVC. Arterial calcification is a common and clinically significant complication associated with atherosclerosis (1Honye J. Mahon D.J. Jian A. White C. Ramee S.R. Wallis J.B. Al-Zarka A. Tobis J.M. Circulation. 1992; 85: 1012-1025Crossref PubMed Scopus (300) Google Scholar, 2Farb A. Burke A.P. Tang A.L. Liang Y.H. Mannan P. Smialek J. Virmani R. Circulation. 1996; 93: 1354-1363Crossref PubMed Scopus (1057) Google Scholar). Hoeg and colleagues showed that calcific atherosclerosis is significant in patients with homozygous familial hypercholesterolemia (3Hoeg J.M. Feuerstein I.M. Tucker E.E. Arterioscler. Thromb. 1994; 14: 1066-1074Crossref PubMed Google Scholar). Previously, we found expression of bone morphogenetic protein (BMP-2), a potent bone differentiation factor that drives endochondral bone formation (4Reddi A.H. Cunningham N.S. J. Bone Miner. Res. 1993; 8: 499-502Google Scholar) in human calcified plaque (5Bostrom K. Watson K.E. Horn S. Wortham C. Herman I.M. Demer L.L. J. Clin. Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (891) Google Scholar). Previously we isolated subpopulations of cells from the bovine artery wall that aggregate into mesenchymal condensations that later mature into mineralized multicellular nodules (6Watson K. Bostrom K. Ravindranath R. Lam T. Norton B. Demer L.L. J. Clin. Invest. 1994; 93: 2106-2113Crossref PubMed Scopus (413) Google Scholar). Although nodules occasionally form in primary smooth muscle cells culture, these calcifying vascular cell (CVC) 1The abbreviations used are: CVC, calcifying vascular cell(s); Coll I, type I collagen; GLA, γ-carboxyglutamic acid; MGP, matrix GLA protein; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Cbfa-1, core binding factor 1; db-cAMP, dibutyryl cAMP. 1The abbreviations used are: CVC, calcifying vascular cell(s); Coll I, type I collagen; GLA, γ-carboxyglutamic acid; MGP, matrix GLA protein; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Cbfa-1, core binding factor 1; db-cAMP, dibutyryl cAMP. cultures differ from primary smooth muscle cell cultures in an approximately 10-fold enrichment for nodule formation as well as the expression of molecular markers such as osteopontin, type I collagen, and the epitope for monoclonal antibody 3G5 (6Watson K. Bostrom K. Ravindranath R. Lam T. Norton B. Demer L.L. J. Clin. Invest. 1994; 93: 2106-2113Crossref PubMed Scopus (413) Google Scholar). CVC retain their phenotype through multiple passages, and they exhibit several osteoblastic markers including type I collagen (Coll I), alkaline phosphatase, osteopontin, and osteocalcin (6Watson K. Bostrom K. Ravindranath R. Lam T. Norton B. Demer L.L. J. Clin. Invest. 1994; 93: 2106-2113Crossref PubMed Scopus (413) Google Scholar). Certain agents present in atherosclerotic arteries, such as 25-hydroxycholesterol, transforming growth factor β-1, and lipid oxidation products, such as minimally oxidized low density lipoprotein and 8-isoprostaglandin E2, promote CVC differentiation (6Watson K. Bostrom K. Ravindranath R. Lam T. Norton B. Demer L.L. J. Clin. Invest. 1994; 93: 2106-2113Crossref PubMed Scopus (413) Google Scholar, 7Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D Tintut Y. Berliner J.A. Demer L.L. J. Arterioscler. Thromb. 1997; 17: 680-687Crossref Scopus (544) Google Scholar). Other cloned subpopulations of artery wall cells do not form nodules even in prolonged culture conditions, suggesting that CVC represent a specific subpopulation (6Watson K. Bostrom K. Ravindranath R. Lam T. Norton B. Demer L.L. J. Clin. Invest. 1994; 93: 2106-2113Crossref PubMed Scopus (413) Google Scholar). There are intriguing similarities between CVC and the mesenchymal stem cells present in adult nonhematopoietic tissue (8Prockop D.J. Science. 1997; 276: 71-74Crossref PubMed Scopus (4081) Google Scholar, 9Dennis J.E. Caplan A.I. Connect. Tissue Res. 1996; 35: 93-99Crossref PubMed Scopus (22) Google Scholar, 10Bruder S.P. Fink D.J. Caplan A.I. J. Cell. Biochem. 1994; 56: 283-294Crossref PubMed Scopus (749) Google Scholar) that are capable of differentiating into osteoblasts, chondroblasts, adipocytes, and myoblasts. Such cells may account for pathologic calcification in other mesenchymal tissues. The cAMP signaling pathway plays a role in both osteoblast differentiation and vascular disease. In osteoblasts, parathyroid hormone modulates differentiation via the cAMP-mediated pathway (11Partridge N.C. Bloch S.R. Pearman A.T. J. Cell. Biochem. 1994; 55: 321-327Crossref PubMed Scopus (114) Google Scholar,12Siddhanti S.R. Quarles L.D. J. Cell. Biochem. 1994; 55: 310-320Crossref PubMed Scopus (78) Google Scholar). cAMP functional response elements have been reported in promoter regions of osteoblast associated genes (13Pearman A.T. Chou W.-Y. Bergman K.D. Pulumati M.R. Partridge N.C. J. Biol. Chem. 1996; 271: 25715-25721Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Povinelli C.M. Stewart J.M. Knoll B.J. Biochim. Biophys. Acta. 1992; 1115: 243-251Crossref PubMed Scopus (5) Google Scholar, 15Towler D.A. Rodan G.A. Endocrinology. 1995; 136: 1089-1096Crossref PubMed Scopus (28) Google Scholar). 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Science. 1975; 187: 449-450Crossref PubMed Scopus (13) Google Scholar, 21Langner R.O. Bement C.L. Pepin J.M. Res. Commun. Mol. Pathol. Pharmacol. 1996; 94: 193-202PubMed Google Scholar). During osteoblast development, a series of events occurs as cells undergo differentiation (22Stein G.S. Lian J.B. Stein J.L. Van Wijnen A.J. Montecino M. Physiol. Rev. 1996; 76: 593-629Crossref PubMed Scopus (392) Google Scholar). Proliferation declines before the onset of differentiation, and various osteoblastic marker genes, involved in extracellular matrix development and mineralization, are expressed in waves: Coll I is expressed maximally during proliferation and declines progressively, whereas alkaline phosphatase and matrix GLA protein (MGP) expression start low and peak during the matrix development/maturation stage, and osteopontin and osteocalcin expression increase and reach a maximum during the matrix mineralization stage (22Stein G.S. Lian J.B. Stein J.L. Van Wijnen A.J. Montecino M. Physiol. Rev. 1996; 76: 593-629Crossref PubMed Scopus (392) Google Scholar, 23Barone L.M. Owen T.A. Tassinari M.S. Bortell R. Stein G.S. Lian J.B. J. Cell. Biochem. 1991; 46: 351-365Crossref PubMed Scopus (69) Google Scholar). Because the cAMP pathway plays a role in both osteoblast differentiation and vascular disease, we investigated its regulatory function in CVC differentiation. In this report, we show that the cAMP pathway stimulates the osteoblast-like differentiation of CVC by inducing morphological change, inhibiting proliferation, enhancing osteoblastic markers (alkaline phosphatase, matrix GLA protein, and type I procollagen), and increasing matrix calcium incorporation yet inhibiting CVC condensation resulting in a diffuse pattern of mineralization. 3HThymidine,45CaCl2 and 32PαdCTP were from Amersham Corp. Dibutyryl cAMP, forskolin, cholera toxin, isobutylmethylxanthine, pertussis toxin, and a protein kinase A-specific inhibitor, KT5720, were from Calbiochem (San Diego, CA). The β-glycerophosphate was purchased from Sigma. Human osteopontin (24Young M.F. Kerr J.M. Termine J.D. Wewer U.M. Wang M.G. McBride O.W. Fisher L.W. Genomics. 1990; 7: 491-502Crossref PubMed Scopus (342) Google Scholar) and human type α1(I) collagen cDNA (25Kiefer M.C. Saphire A.C.S. Bauer D.M. Barr P.J. Nucleic Acids Res. 1990; 18: 1909Crossref PubMed Scopus (34) Google Scholar) probes for Northern analysis were from American Tissue Culture Collection, and human 28 S rRNA probe was purchased from CLONTECH (Palo Alto, CA). Type I procollagen polyclonal antibody for Western analysis was from Chemicon International Inc. (Temecula, CA). CVC, the clonal subpopulation of primary bovine aortic smooth muscle cells, were identified as described previously (6Watson K. Bostrom K. Ravindranath R. Lam T. Norton B. Demer L.L. J. Clin. Invest. 1994; 93: 2106-2113Crossref PubMed Scopus (413) Google Scholar). CVC were grown in Dulbecco's modified Eagle's medium (Irvine Scientific, Santa Ana, CA) containing 15% heat-inactivated fetal bovine serum (Hyclone Labs, Logan, UT) and supplemented with sodium pyruvate (1 mm), penicillin (100 units/ml), and streptomycin (100 units/ml), all from Irvine Scientific, CA. The medium was changed every 3–4 days with agents, if applicable. From 5–7 days before von Kossa staining, 5 mm β-glycerophosphate (7Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D Tintut Y. Berliner J.A. Demer L.L. J. Arterioscler. Thromb. 1997; 17: 680-687Crossref Scopus (544) Google Scholar) and 4 mm CaCl2 were added to the media to permit maximal mineralization. CVC seeded in were for with dibutyryl cAMP (1 mm), forskolin or media for and for was added for an and incorporation was as described previously M. D.M. Rodan G.A. Endocrinology. 1990; PubMed Scopus Google Scholar). The were shown as the of grown in were with forskolin for 3 were and in The cell was in containing mm mm mm and The cell was and the protein was using the was isolated on Diego, CA) and to 4 The were with collagen type I polyclonal antibody for The antibody was by enhanced CVC seeded in were with or cAMP for 3 and alkaline phosphatase was as described previously (7Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D Tintut Y. Berliner J.A. Demer L.L. J. Arterioscler. Thromb. 1997; 17: 680-687Crossref Scopus (544) Google Scholar). The alkaline phosphatase activity was to protein using the The were from a of shown as the of spontaneous CVC differentiation, CVC were grown in for the and was isolated CA). During the nodule stage, cells were in and nodules were by The nodules were and from both nodules and monolayer cells were the cAMP CVC were grown in and dibutyryl cAMP (1 mm), or forskolin were added After 3 days of culture, was in were on and to which were with The were with human osteopontin cDNA probe to by The were for with with and for with with before After with with the same were with human type I collagen using the same or with human 28 S rRNA using and with with for 5 and with for The isolated as described was in of containing mm of of reverse and of for using specific for gene (alkaline phosphatase, matrix GLA protein, Cbfa-1, and were in a of with polymerase of 28 of of polymerase of and of was for or (alkaline phosphatase, MGP, and for alkaline phosphatase, MGP, Cbfa-1, and and for were isolated on to and the were with and with incorporation and von Kossa to mineralization was as described previously (7Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D Tintut Y. Berliner J.A. Demer L.L. J. Arterioscler. Thromb. 1997; 17: 680-687Crossref Scopus (544) Google Scholar). The for incorporation were from a of shown as the of During spontaneous differentiation, CVC morphological In cultures days cells into condensations From days these condensations multicellular nodules which days in culture to mineralization, identified by von Kossa Previously we reported that CVC exhibit several osteoblastic differentiation markers (6Watson K. Bostrom K. Ravindranath R. Lam T. Norton B. Demer L.L. J. Clin. Invest. 1994; 93: 2106-2113Crossref PubMed Scopus (413) Google Scholar). Here, we the of their expression during the described was isolated from of (1 days condensation days nodules days and calcification days Type I osteopontin, and 28 S rRNA as an expression were by Northern phosphatase, matrix GLA protein, Cbfa-1, and were by with specific for gene were to the not shown in were and were as the of maximum expression the of days in culture that to the shown in and (1 not Coll I, alkaline phosphatase, matrix GLA protein, and osteocalcin expression increased as CVC morphological whereas osteopontin expression and was expressed during CVC differentiation and the expression of these differentiation markers during the later of CVC was to the cells the nodules and not in the monolayer cells, nodules were from the cells by and and from both were showed that all differentiation markers were expressed similar in both the monolayer and the cells nodules was expressed in the nodules in the monolayer of osteoblastic differentiation markers between nodules monolayer differentiation to GLA in a treatment of CVC with in a and for days induced a morphological change from an to a “cuboidal” which is an of differentiation into osteoblastic cells C. J. A.H. Tissue Res. PubMed Scopus Google Scholar, M. P. J.M. N. PubMed Scopus Google Scholar, J.E. F. 1995; 17: PubMed Scopus Google Scholar). The same morphological change was CVC were with forskolin or cholera toxin not Prolonged treatment of CVC with effects on later differentiation of CVC, including nodule and mineralization. CVC were with mm and every 3–4 days with medium containing mm was of condensation cells days in and nodule formation cells days in von Kossa for mineralization showed that calcification in both and cells days in culture cells In cells, calcification was whereas in cells, calcification was diffuse throughout the monolayer with of increased the of were with or forskolin and for was added to the medium during the and cellular proliferation was to condensation and nodule The results showed that increased cAMP inhibited CVC proliferation Because it been shown in that the of proliferation is to the onset of differentiation (22Stein G.S. Lian J.B. Stein J.L. Van Wijnen A.J. Montecino M. Physiol. Rev. 1996; 76: 593-629Crossref PubMed Scopus (392) Google we investigated cAMP stimulation also expression of osteoblastic differentiation markers. phosphatase activity, a well early marker of osteoblastic differentiation J. Biol. 1995; Google increased during spontaneous CVC differentiation (7Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D Tintut Y. Berliner J.A. Demer L.L. J. Arterioscler. Thromb. 1997; 17: 680-687Crossref Scopus (544) Google Scholar). we its activity in response to cAMP CVC were with various of or forskolin and for 3 phosphatase activity was induced and In addition, cholera toxin and other agents to increase cAMP also had similar effects on alkaline phosphatase activity 4 Because the results showed that cAMP induced early of osteoblast-like differentiation in CVC, we its effects on later markers described of CVC were with mm or forskolin and for 3 and was by Northern analysis or as described Treatment with increased gene expression of alkaline phosphatase and and matrix GLA protein and but a in osteopontin expression 5 cAMP had on the expression of osteocalcin and not Type I collagen production was enhanced as shown by Western analysis cAMP was by 5 the of mineralization in both and cells, calcium incorporation was CVC were with mm and every 3–4 days with medium containing mm or After days in culture, 4 mm CaCl2 and 5 were added to the media to mineralization. After an days in culture, cells were and changed to media containing 5 mm calcium and mm or medium and for an In calcium been shown to represent because similar results were in cultures with which calcium (7Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D Tintut Y. Berliner J.A. Demer L.L. J. Arterioscler. Thromb. 1997; 17: 680-687Crossref Scopus (544) Google Scholar). The results showed that calcium incorporation was enhanced approximately with treatment Because osteoblastic differentiation been to of proliferation, we of CVC proliferation is to promote osteoblast-like differentiation. CVC were with pertussis toxin, which been shown to smooth muscle cell proliferation cell and the of cAMP L.M. M.R. J.D. Endocrinology. 1994; PubMed Scopus Google Scholar). The results showed that pertussis toxin inhibited CVC proliferation inducing morphology or significantly increasing alkaline phosphatase activity increase both of pertussis suggesting that the of proliferation is not to promote osteoblast-like differentiation of CVC. the cAMP pathway osteoblast-like differentiation, CVC were with KT5720, previously used as a protein kinase A-specific R. P. E. P. M. S. A. 1997; 94: PubMed Scopus Google Scholar, S. M.C. S. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). showed that the increase in alkaline phosphatase activity during spontaneous CVC differentiation was not In addition, incorporation showed that mineralization was also inhibited not suggesting that the cAMP pathway a on osteoblast-like differentiation of CVC of its on cAMP osteoblast-like differentiation in a subpopulation of primary smooth muscle cells that do not form were with The results showed that proliferation was inhibited approximately inducing a significant increase in alkaline phosphatase 3 the same of forskolin that a increase in alkaline phosphatase activity in CVC a increase in not with 5 days of forskolin the on alkaline phosphatase activity in did not increase to not These results a role of the cAMP pathway in osteoblast-like differentiation of CVC. These that cAMP treatment the onset of osteoblast-like differentiation of CVC in several of a morphological change, which is of differentiating into C. J. A.H. Tissue Res. PubMed Scopus Google Scholar, M. P. J.M. N. PubMed Scopus Google Scholar, J.E. F. 1995; 17: PubMed Scopus Google of CVC proliferation, which is a for the of differentiation (22Stein G.S. Lian J.B. Stein J.L. Van Wijnen A.J. Montecino M. Physiol. Rev. 1996; 76: 593-629Crossref PubMed Scopus (392) Google of the of osteoblastic differentiation markers including alkaline phosphatase, type I and matrix GLA protein, which were also found to increase during spontaneous CVC and of the in osteopontin which also occurs in spontaneous CVC differentiation. The results showed that cAMP treatment the mineralization pattern in CVC. During spontaneous differentiation, CVC aggregate to form condensations that mature into mineralized Prolonged treatment of CVC with inhibited condensation and nodule in cells, enhanced production of alkaline phosphatase, extracellular matrix and increased matrix calcium incorporation were to the as in the with cells, resulting in a diffuse pattern of mineralization throughout the diffuse pattern was not of increased of pattern intriguing similarities to in vitro mineralization of bone cell (7Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D Tintut Y. Berliner J.A. Demer L.L. J. Arterioscler. Thromb. 1997; 17: 680-687Crossref Scopus (544) Google Scholar). The by which cAMP condensation is not but may to of a M.G. J. 1994; Google Scholar) expression of such as or which have been to T. 1992; PubMed Scopus Google Scholar, A.P. J.A. J. Clin. Invest. 1995; PubMed Scopus Google Scholar). 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E. 1997; PubMed Scopus Google Scholar). a in in with in vitro vascular calcification. are other such as a its factor. of cells in in human cells are increased of in the vascular in human vascular expression is increased may in response to vascular to its extent. In these results the that cAMP vitro vascular calcification. The in atherosclerotic calcification are with the in CVC, both in spontaneous and differentiation, the in vivo of this We J. Berliner for and and and for