Different Roles of GNAS and cAMP Signaling During Early and Late Stages of Osteogenic Differentiation

Abstract

Progressive osseous heteroplasia (POH) and fibrous dysplasia (FD) are genetic diseases of bone formation at opposite ends of the osteogenic spectrum: imperfect osteogenesis of the skeleton occurs in FD, while heterotopic ossification in skin, subcutaneous fat, and skeletal muscle forms in POH. POH is caused by heterozygous inactivating germline mutations in GNAS, which encodes G-protein subunits regulating the cAMP pathway, while FD is caused by GNAS somatic activating mutations. We used pluripotent mouse ES cells to examine the effects of Gnas dysregulation on osteoblast differentiation. At the earliest stages of osteogenesis, Gnas transcripts G_sα, XLαs and 1A are expressed at low levels and cAMP levels are also low. Inhibition of cAMP signaling (as in POH) by 2′,5′-dideoxyadenosine enhanced osteoblast differentiation while conversely, increased cAMP signaling (as in FD), induced by forskolin, inhibited osteoblast differentiation. Notably, increased cAMP was inhibitory for osteogenesis only at early stages after osteogenic induction. Expression of osteogenic and adipogenic markers showed that increased cAMP enhanced adipogenesis and impaired osteoblast differentiation even in the presence of osteogenic factors, supporting cAMP as a critical regulator of osteoblast and adipocyte lineage commitment. Furthermore, increased cAMP signaling decreased BMP pathway signaling, indicating that G protein-cAMP pathway activation (as in FD) inhibits osteoblast differentiation, at least in part by blocking the BMP-Smad pathway, and suggesting that GNAS inactivation as occurs in POH enhances osteoblast differentiation, at least in part by stimulating BMP signaling. These data support that differences in cAMP levels during early stages of cell differentiation regulate cell fate decisions.

Supporting information available online at http:/www.thieme-connect.de/ejournals/toc/hmr

Key words

progressive osseous heteroplasia - POH - fibrous dysplasia - FD - BMP - SMAD

• References

• 1 Shore EM, Ahn J, Jan de Beur S, Li M, Xu M, Gardner RJ, Zasloff MA, Whyte MP, Levine MA, Kaplan FS. Paternally inherited inactivating mutations of the GNAS1 gene in progressive osseous heteroplasia. N Engl J Med 2002; 346: 99-106
• 2 Shore EM, Kaplan FS. Inherited human diseases of heterotopic bone formation. Nat Rev Rheumatol 2010; 6: 518-527
• 3 Kaplan FS, Shore EM. Progressive osseous heteroplasia. J Bone Miner Res 2000; 15: 2084-2094
• 4 Dumitrescu CE, Collins MT. McCune-Albright syndrome. Orphanet J Rare Dis 2008; 3: 12
• 5 Riminucci M, Robey PG, Saggio I, Bianco P. Skeletal progenitors and the GNAS gene: fibrous dysplasia of bone read through stem cells. J Mol Endocrinol 2010; 45: 355-364
• 6 Weinstein LS, Liu J, Sakamoto A, Xie T, Chen M. Minireview: GNAS: normal and abnormal functions. Endocrinology 2004; 145: 5459-5464
• 7 Weinstein LS, Chen M, Xie T, Liu J. Genetic diseases associated with heterotrimeric G proteins. Trends Pharmacol Sci 2006; 27: 260-266
• 8 Aydin C, Aytan N, Mahon MJ, Tawfeek HA, Kowall NW, Dedeoglu A, Bastepe M. Extralarge XL(alpha)s (XXL(alpha)s), a variant of stimulatory G protein alpha-subunit (Gs(alpha)), is a distinct, membrane-anchored GNAS product that can mimic Gs(alpha). Endocrinology 2009; 150: 3567-3575
• 9 Bastepe M. The GNAS Locus: Quintessential Complex Gene Encoding Gsalpha, XLalphas, and other Imprinted Transcripts. Curr Genomics 2007; 8: 398-414
• 10 Bastepe M, Gunes Y, Perez-Villamil B, Hunzelman J, Weinstein LS, Juppner H. Receptor-mediated adenylyl cyclase activation through XLalpha(s), the extra-large variant of the stimulatory G protein alpha-subunit. Mol Endocrinol 2002; 16: 1912-1919
• 11 Plagge A, Gordon E, Dean W, Boiani R, Cinti S, Peters J, Kelsey G. The imprinted signaling protein XL alpha s is required for postnatal adaptation to feeding. Nat Genet 2004; 36: 818-826
• 12 Spiegel AM, Weinstein LS. Inherited diseases involving g proteins and g protein-coupled receptors. Annu Rev Med 2004; 55: 27-39
• 13 Kronenberg HM. Gs signaling in osteoblasts and hematopoietic stem cells. Ann NY Acad Sci 1192: 327-329
• 14 Bowler WB, Gallagher JA, Bilbe G. G-protein coupled receptors in bone. Front Biosci 1998; 3: d769-d780
• 15 Wang K, Wong YH. G protein signaling controls the differentiation of multiple cell lineages. Biofactors 2009; 35: 232-238
• 16 Hong SH, Lu X, Nanes MS, Mitchell J. Regulation of osterix (Osx, Sp7) and the Osx promoter by parathyroid hormone in osteoblasts. J Mol Endocrinol 2009; 43: 197-207
• 17 Hollnagel A, Ahrens M, Gross G. Parathyroid hormone enhances early and suppresses late stages of osteogenic and chondrogenic development in a BMP-dependent mesenchymal differentiation system (C3H10T1/2). J Bone Miner Res 1997; 12: 1993-2004
• 18 Ishizuya T, Yokose S, Hori M, Noda T, Suda T, Yoshiki S, Yamaguchi A. Parathyroid hormone exerts disparate effects on osteoblast differentiation depending on exposure time in rat osteoblastic cells. J Clin Invest 1997; 99: 2961-2970
• 19 Isogai Y, Akatsu T, Ishizuya T, Yamaguchi A, Hori M, Takahashi N, Suda T. Parathyroid hormone regulates osteoblast differentiation positively or negatively depending on the differentiation stages. J Bone Miner Res 1996; 11: 1384-1393
• 20 Koh AJ, Beecher CA, Rosol TJ, McCauley LK. 3′,5′-Cyclic adenosine monophosphate activation in osteoblastic cells: effects on parathyroid hormone-1 receptors and osteoblastic differentiation in vitro. Endocrinology 1999; 140: 3154-3162
• 21 Lanske B, Amling M, Neff L, Guiducci J, Baron R, Kronenberg HM. Ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J Clin Invest 1999; 104: 399-407
• 22 Datta NS, Abou-Samra AB. PTH and PTHrP signaling in osteoblasts. Cell Signal 2009; 21: 1245-1254
• 23 Ghayor C, Ehrbar M, San Miguel B, Gratz KW, Weber FE. cAMP enhances BMP2-signaling through PKA and MKP1-dependent mechanisms. Biochem Biophys Res Commun 2009; 381: 247-252
• 24 Ohta Y, Nakagawa K, Imai Y, Katagiri T, Koike T, Takaoka K. Cyclic AMP enhances Smad-mediated BMP signaling through PKA-CREB pathway. J Bone Miner Metab 2008; 26: 478-484
• 25 Nagata A, Tanaka T, Minezawa A, Poyurovsky M, Mayama T, Suzuki S, Hashimoto N, Yoshida T, Suyama K, Miyata A, Hosokawa H, Nakayama T, Tatsuno I. cAMP activation by PACAP/VIP stimulates IL-6 release and inhibits osteoblastic differentiation through VPAC2 receptor in osteoblastic MC3T3 cells. J Cell Physiol 2009; 221: 75-83
• 26 Siddappa R, Mulder W, Steeghs I, van de Klundert C, Fernandes H, Liu J, Arends R, van Blitterswijk C, de Boer J. cAMP/PKA signaling inhibits osteogenic differentiation and bone formation in rodent models. Tissue Eng Part A 2009; 15: 2135-2143
• 27 Sakamoto A, Chen M, Nakamura T, Xie T, Karsenty G, Weinstein LS. Deficiency of the G-protein alpha-subunit G(s)alpha in osteoblasts leads to differential effects on trabecular and cortical bone. J Biol Chem 2005; 280: 21369-21375
• 28 Yamaguchi A, Komori T, Suda T. Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 2000; 21: 393-411
• 29 Pera MF, Tam PP. Extrinsic regulation of pluripotent stem cells. Nature 465: 713-720
• 30 Yu J, Thomson JA. Pluripotent stem cell lines. Genes Dev 2008; 22: 1987-1997
• 31 Nishikawa S, Jakt LM, Era T. Embryonic stem-cell culture as a tool for developmental cell biology. Nat Rev Mol Cell Biol 2007; 8: 502-507
• 32 Duplomb L, Dagouassat M, Jourdon P, Heymann D. Concise review: embryonic stem cells: a new tool to study osteoblast and osteoclast differentiation. Stem Cells 2007; 25: 544-552
• 33 Nagy A, Rossant J, Nagy R, Abramow-Newerly W, Roder JC. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 1993; 90: 8424-8428
• 34 Kurosawa H. Methods for inducing embryoid body formation: in vitro differentiation system of embryonic stem cells. J Biosci Bioeng 2007; 103: 389-398
• 35 Dani C, Smith AG, Dessolin S, Leroy P, Staccini L, Villageois P, Darimont C, Ailhaud G. Differentiation of embryonic stem cells into adipocytes in vitro. J Cell Sci 1997; 110 (Pt 11) 1279-1285
• 36 Wardle FC, Papaioannou VE. Teasing out T-box targets in early mesoderm. Curr Opin Genet Dev 2008; 18: 418-425
• 37 Kelsey G. Imprinting on chromosome 20: tissue-specific imprinting and imprinting mutations in the GNAS locus. Am J Med Genet C Semin Med Genet 2010; 154C: 377-386
• 38 Nuttall ME, Gimble JM. Controlling the balance between osteoblastogenesis and adipogenesis and the consequent therapeutic implications. Curr Opin Pharmacol 2004; 4: 290-294
• 39 Muruganandan S, Roman AA, Sinal CJ. Adipocyte differentiation of bone marrow-derived mesenchymal stem cells: cross talk with the osteoblastogenic program. Cell Mol Life Sci 2009; 66: 236-253
• 40 Ichida F, Nishimura R, Hata K, Matsubara T, Ikeda F, Hisada K, Yatani H, Cao X, Komori T, Yamaguchi A, Yoneda T. Reciprocal roles of MSX2 in regulation of osteoblast and adipocyte differentiation. J Biol Chem 2004; 279: 34015-34022
• 41 Ryoo HM, Lee MH, Kim YJ. Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells. Gene 2006; 366: 51-57
• 42 Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA. MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem 2003; 278: 45969-45977
• 43 Madsen L, Kristiansen K. The importance of dietary modulation of cAMP and insulin signaling in adipose tissue and the development of obesity. Ann NY Acad Sci 1190: 1-14
• 44 Lian JB, Stein GS, Javed A, van Wijnen AJ, Stein JL, Montecino M, Hassan MQ, Gaur T, Lengner CJ, Young DW. Networks and hubs for the transcriptional control of osteoblastogenesis. Rev Endocr Metab Disord 2006; 7: 1-16
• 45 Matsubara T, Kida K, Yamaguchi A, Hata K, Ichida F, Meguro H, Aburatani H, Nishimura R, Yoneda T. BMP2 regulates Osterix through Msx2 and Runx2 during osteoblast differentiation. J Biol Chem 2008; 283: 29119-29125
• 46 Xu M, Kaplan FS, Shore EM. GNAS mRNAs from the Paternally-Inherited allele are Decreased in Progressive Osseous Heteroplasia (POH), a Human Disorder of Ectopic Ossification. J Bone Min Res 2005; 20: S417
• 47 Luo J, Zhou W, Zhou X, Li D, Weng J, Yi Z, Cho SG, Li C, Yi T, Wu X, Li XY, de Crombrugghe B, Hook M, Liu M. Regulation of bone formation and remodeling by G-protein-coupled receptor 48. Development 2009; 136: 2747-2756
• 48 Zhao Y, Ding S. A high-throughput siRNA library screen identifies osteogenic suppressors in human mesenchymal stem cells. Proc Natl Acad Sci USA 2007; 104: 9673-9678
• 49 Yamashita A, Takada T, Nemoto K, Yamamoto G, Torii R. Transient suppression of PPARgamma directed ES cells into an osteoblastic lineage. FEBS Lett 2006; 580: 4121-4125
• 50 Akune T, Ohba S, Kamekura S, Yamaguchi M, Chung UI, Kubota N, Terauchi Y, Harada Y, Azuma Y, Nakamura K, Kadowaki T, Kawaguchi H. PPARgamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors. J Clin Invest 2004; 113: 846-855
• 51 Shockley KR, Lazarenko OP, Czernik PJ, Rosen CJ, Churchill GA, Lecka-Czernik B. PPARgamma2 nuclear receptor controls multiple regulatory pathways of osteoblast differentiation from marrow mesenchymal stem cells. J Cell Biochem 2009; 106: 232-246
• 52 Weinstein LS, Xie T, Qasem A, Wang J, Chen M. The role of GNAS and other imprinted genes in the development of obesity. Int J Obes (Lond) 2010; 34: 6-17
• 53 Plagge A, Kelsey G, Germain-Lee EL. Physiological functions of the imprinted Gnas locus and its protein variants Galpha(s) and XLalpha(s) in human and mouse. J Endocrinol 2008; 196: 193-214
• 54 Li H, Fong C, Chen Y, Cai G, Yang M. beta2- and beta3-, but not beta1-adrenergic receptors are involved in osteogenesis of mouse mesenchymal stem cells via cAMP/PKA signaling. Arch Biochem Biophys 2010; 496: 77-83
• 55 Tintut Y, Parhami F, Bostrom K, Jackson SM, Demer LL. cAMP stimulates osteoblast-like differentiation of calcifying vascular cells. Potential signaling pathway for vascular calcification. J Biol Chem 1998; 273: 7547-7553
• 56 Doorn J, Siddappa R, van Blitterswijk CA, de Boer J. Forskolin enhances in vivo bone formation by human mesenchymal stromal cells. Tissue Eng Part A 2012; 18: 558-567
• 57 Siddappa R, Martens A, Doorn J, Leusink A, Olivo C, Licht R, van Rijn L, Gaspar C, Fodde R, Janssen F, van Blitterswijk C, de Boer J. cAMP/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo. Proc Natl Acad Sci U S A 2008; 105: 7281-7286
• 58 Lo KW, Kan HM, Ashe KM, Laurencin CT. The small molecule PKA-specific cyclic AMP analogue as an inducer of osteoblast-like cells differentiation and mineralization. J Tissue Eng Regen Med 2012; 6: 40-48
• 59 Hakeda Y, Ikeda E, Kurihara N, Nakatani Y, Maeda N, Kumegawa M. Induction of osteoblastic cell differentiation by forskolin. Stimulation of cyclic AMP production and alkaline phosphatase activity. Biochim Biophys Acta 1985; 838: 49-53
• 60 Nakao Y, Koike T, Ohta Y, Manaka T, Imai Y, Takaoka K. Parathyroid hormone enhances bone morphogenetic protein activity by increasing intracellular 3′,5′-cyclic adenosine monophosphate accumulation in osteoblastic MC3T3-E1 cells. Bone 2009; 44: 872-877
• 61 Yang DC, Tsay HJ, Lin SY, Chiou SH, Li MJ, Chang TJ, Hung SC. cAMP/PKA regulates osteogenesis, adipogenesis and ratio of RANKL/OPG mRNA expression in mesenchymal stem cells by suppressing leptin. PLoS One 2008; 3: e1540
• 62 Huang H, Song TJ, Li X, Hu L, He Q, Liu M, Lane MD, Tang QQ. BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci U S A 2009; 106: 12670-12675