Human mesenchymal stem cells (hMSCs) present in the bone marrow are the precursors of osteoblasts, chondrocytes and adipocytes, and hold tremendous potential for osteoregenerative therapy. However, achieving directed differentiation into osteoblasts has been a major concern. The use of lithium for enhancing osteogenic differentiation has been documented in animal models but its effect in humans is not clear. We, therefore, performed high throughput transcriptome analysis of lithium-treated hMSCs to identify altered gene expression and its relevance to osteogenic differentiation. Our results show suppression of proliferation and enhancement of alkaline phosphatase (ALP) activity upon lithium treatment of hMSCs under non-osteogenic conditions. Microarray profiling of lithium-stimulated hMSC revealed decreased expression of adipogenic genes (CEBPA, CMKLR1, HSD11B1) and genes involved in lipid biosynthesis. Interestingly, osteoclastogenic factors and immune responsive genes (IL7, IL8, CXCL1, CXCL12, CCL20) were also downregulated. Negative transcriptional regulators of the osteogenic program (TWIST1 and PBX1) were suppressed while genes involved in mineralization like CLEC3B and ATF4 were induced. Gene ontology analysis revealed enrichment of upregulated genes related to mesenchymal cell differentiation and signal transduction. Lithium priming led to enhanced collagen 1 synthesis and osteogenic induction of lithium pretreated MSCs resulted in enhanced expression of Runx2, ALP and bone sialoprotein. However, siRNA-mediated knockdown of RRAD, CLEC3B and ATF4 attenuated lithium-induced osteogenic priming, identifying a role for RRAD, a member of small GTP binding protein family, in osteoblast differentiation. In conclusion, our data highlight the transcriptome reprogramming potential of lithium resulting in higher propensity of lithium “primed” MSCs for osteoblastic differentiation.
References
[1]
Satija NK, Singh VK, Verma YK, Gupta P, Sharma S, et al. (2009) Mesenchymal stem cell-based therapy: a new paradigm in regenerative medicine. J Cell Mol Med 13: 4385–4402.
[2]
Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, et al. (2001) Repair of large bone defects with the use of autologous bone marrow stromal cells. N Eng J Med 344: 385–386.
[3]
Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, et al. (2002) Isolated allogeneic bone marrow-derived mesenchymal stem cells engraft and stimulate growth in children with osteogenesis imperfecta: implications for cell therapy of bone. Proc Natl Acad Sci U S A 99: 8932–8937.
[4]
Satija NK, Gurudutta GU, Sharma S, Afrin F, Gupta P, et al. (2007) Mesenchymal stem cells: Molecular targets for tissue engineering. Stem Cells Dev 16: 7–23.
[5]
Hartmann C (2006) A Wnt canon orchestrating osteoblastogenesis. Trends Cell Biol 16: 151–158.
[6]
Wu D, Pan W (2010) GSK3: a multifaceted kinase in Wnt signaling. Trends Biochem Sci 35: 161–168.
[7]
Stambolic V, Ruel L, Woodgett JR (1996) Lithium inhibits glycogen synthase kinase-3 activity and mimics Wingless signaling in intact cells. Curr Biol 6: 1664–1668.
[8]
Clément-Lacroix P, Ai M, Morvan F, Roman-Roman S, Vayssière B, et al. (2005) Lrp5-independent activation of Wnt signaling by lithium chloride increases bone formation and bone mass in mice. Proc Natl Acad Sci U S A 102: 17406–17411.
[9]
Kugimiya F, Kawaguchi H, Ohba S, Kawamura N, Hirata M, et al. (2007) GSK-3β controls osteogenesis through regulating Runx2 activity. PLoS ONE 2: e837.
[10]
Vestergaard P, Rejnmark L, Mosekilde L (2005) Reduced relative risk of fractures among users of lithium. Calcif Tissue Int 77: 1–8.
[11]
Zamani A, Omarni GR, Nasab MM (2009) Lithium’s effect on bone mineral density. Bone 44: 331–334.
[12]
Wilting I, de Vries F, Thio BM, Cooper C, Heerdink ER, et al. (2007) Lithium use and the risk of fractures. Bone 40: 1252–1258.
[13]
Smith JR, Pochampally R, Perry A, Hsu S-C, Prockop DJ (2004) Isolation of a highly clonogenic and multipotential subfraction of adult stem cells from bone marrow stroma. Stem Cells 22: 823–831.
[14]
Colter DC, Sekiya I, Prockop DJ (2001) Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human bone marrow cells. Proc Natl Acad Sci U S A 98: 7841–7845.
[15]
Oh Y-T, Liu X, Yue P, Kang S, Chen J, et al. (2010) ERK/Ribosomal S6 kinase (RSK) signaling positively regulates death receptor 5 expression through co-activation of CHOP and Elk1. J Biol Chem 285: 41310–41319.
[16]
Baldi P, Long AD (2001) A Bayesian framework for the analysis of microarray expression data: regularized t-test and statistical inferences of gene changes. Bioinformatics 17: 506–519.
[17]
Murie C, Woody O, Lee AY, Nadon R (2009) Comparison of small n statistical tests of differential expression applied to microarrays. BMC Bioinformatics 10: 45.
[18]
Huang DW, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protocols 4: 44–57.
[19]
Pandey R, Guru RK, Mount DW (2004) Pathway miner: extracting gene association networks from molecular pathways for predicting the biological significance of gene expression microarray data. Bioinformatics 20: 2156–2158.
[20]
Curtis RK, Ore？i？ M, Vidal-Puig A (2005) Pathways to the analysis of microarray data. Trends Biotechnol 23: 429–435.
[21]
Sekiya I, Larson BL, Smith JR, Pochampally R, Cui JG, et al. (2001) Expansion of human adult stem cells from bone marrow stroma: Conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells 6: 530–541.
[22]
Neuhuber B, Swanger SA, Howard L, Mackay A, Fischer I (2008) Effects of plating density and culture time on bone marrow stromal cell characteristics. Exp Hematol 36: 1176–1185.
[23]
Berridge MV, Tan AS, McCoy KD, Wang R (1996) The biochemical and cellular basis of cell proliferation assays that use tetrazolium salts. Biochemica 4: 14–19.
[24]
Zhang F, Phiel CJ, Spece L, Gurvich N, Klein PS (2003) Inhibitory phosphorylation of glycogen synthase kinase-3 in response to lithium. J Biol Chem 278: 33067–33077.
[25]
Sinha D, Wang Z, Ruchalski KL, Levine JS, Krishnan S, et al. (2005) Lithium activates the Wnt and phosphatidylinositol 3-kinase Akt signaling pathways to promote cell survival in the absence of soluble survival factors. Am J Physiol Renal Physiol 288: F703–F713.
[26]
Churchill GA (2002) Fundamentals of experimental design for cDNA microarrays. Nat Genet 32 Suppl:490–495
[27]
Rintala J, Seemann R, Chandrasekaran K, Rosenberger TA, Chang L, et al. (1999) 85 kDa cytosolic phospholipase A2 is a target for chronic lithium in rat brain. Neuroreport 10: 3887–3890.
[28]
McEachin RC, Chen H, Sartor MA, Saccone SF, Keller BJ, et al. (2010) A genetic network model of cellular responses to lithium treatment and cocaine abuse in bipolar disorder. BMC System Biol 4: 158.
[29]
Zhang WV, Jullig M, Connolly AR, Stott NS (2005) Early gene response in lithium chloride induced apoptosis. Apoptosis 10: 75–90.
[30]
Kubo H, Shimizu M, Taya Y, Kawamoto T, Michida M, et al. (2009) Identification of mesenchymal stem cell (MSC)-transcription factors by microarray and knockdown analyses, and signature molecule-marked MSC in bone marrow by immunohistochemistry. Genes Cells 14: 407–424.
[31]
Riekstina U, Cakstina I, Parfejevs V, Hoogduijn M, Jankovskis G, et al. (2009) Embryonic stem cell marker expression pattern in human mesenchymal stem cells derived from bone marrow, adipose tissue, heart and dermis. Stem Cell Rev and Rep 5: 378–386.
[32]
Okuda A, Fukushima A, Nishimoto M, Orimo A, Yamagishi T, et al. (1998) UTF1, a novel transcriptional coactivator expressed in pluripotent embryonic stem cells and extra-embryonic cells. EMBO J 17: 2019–2032.
[33]
Motomura H, Niimi H, Sugimori K, Ohtsuka T, Kimura T, et al. (2007) Gas6, a new regulator of chondrogenic differentiation from mesenchymal cells. Biochem Biophys Res Commun 357: 997–1003.
[34]
Tang GH, Xu J, Chen RJ, Qian YF, Shen G (2011) Lithium delivery enhances bone growth during midpalatal expansion. J Dent Res 90: 336–340.
[35]
Warden SJ, Hassett SM, Bond JL, Rydberg J, Grogg JD, et al. (2010) Psychotropic drugs have constrasting skeletal effects that are independent of their effects on physical activity levels. Bone 46: 985–992.
[36]
Bolton JM, Metge C, Lix L, Prior H, Sareen J, et al. (2008) Fracture risk from psychotropic medications: a population-based analysis. J Clin Psychopharmacol 28: 384–391.
[37]
de Boer J, Wang HJ, Blitterswijk CV (2004) Effects of Wnt signaling on proliferation and differentiation of human mesenchymal stem cells. Tissue Eng 10: 393–401.
[38]
Gregory CA, Perry AS, Reyes E, Conley A, Gunn WG, et al. (2005) Dkk-1-derived synthetic peptides and lithium chloride for the control and recovery of adult stem cells from bone marrow. Proc Natl Acad Sci U S A 280: 2309–2323.
[39]
Zaragosi LE, Wdziekonski B, Fontaine C, Villageois P, Peraldi P, et al. (2008) Effects of GSK3 inhibitors on in vitro expansion and differentiation of human adipose-derived stem cells into adipocytes. BMC Cell Biol 9: 11.
[40]
Neth P, Ciccarella M, Egea V, Hoelters J, Jochum M, et al. (2006) Wnt signaling regulates the invasion capacity of human mesenchymal stem cells. Stem Cells 24: 1892–1903.
[41]
Gregory CA, Singh H, Perry AS, Prockop DJ (2003) The Wnt signaling inhibitor Dickkopf-1 is required for reentry into the cell cycle of human adult stem cells from bone marrow. J Biol Chem 278: 28067–28078.
[42]
Krause U, Harris S, Green A, Ylostalo J, Zeitouni S, et al. (2010) Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy. Proc Natl Acad Sci U S A 107: 4147–4152.
[43]
Bain G, Muller T, Wang X, Papkoff J (2003) Activated β-catenin induces osteoblast differentiation of C3H10T1/2 cells and participates in BMP2 mediated signal transduction. Biochem Biophys Res Commun 301: 84–91.
[44]
Eslaminejad MB, Talkhabi M, Zeynali B (2008) Effect of lithium chloride on proliferation and bone differentiation of rat marrow-derived mesenchymal stem cells in culture. Iran J Basic Med Sci 11: 143–151.
[45]
Li J, Khavandgar Z, Lin SH, Murshed M (2011) Lithium chloride attenuates BMP-2 signaling and inhibits osteogenic differentiation through a novel WNT/GSK3-independent mechanism. Bone 48: 321–331.
[46]
Lai WT, Krishnappa V, Phinney DG (2011) Fgf2 inhibits differentiation of mesenchymal stem cells by inducing Twist2 Spry4, blocking extracellular regulated kinase activation and altering Fgfr expression levels. Stem Cells 29: 1102–1111.
[47]
Shahdadfar A, Fronsdal K, Haug T, Reinholt FP, Brinchmann JE (2005) In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression and transcriptome stability. Stem Cells 23: 1357–1366.
[48]
Larson BL, Ylostalo J, Prockop DJ (2008) Human multipotent stem cells undergo sharp transition from division to development in culture. Stem Cells 26: 193–201.
[49]
de Boer J, Siddappa R, Gaspar C, van Apeldoorn A, Fodde R, et al. (2004) Wnt signaling inhibits osteogenic differentiation of human mesenchymal stem cells. Bone 34: 818–826.
[50]
Kortesidis A, Zannettino A, Isenmann S, Shi S, Lapidot T, et al. (2005) Stromal-derived factor-1 promotes the growth, survival, and development of human bone marrow stromal stem cells. Blood 105: 3793–3801.
[51]
Tamura M, Sato MM, Nashimoto M (2011) Regulation of CXCL12 expression by canonical Wnt signaling in bone marrow stromal cells. Int J Biochem Cell Biol 43: 760–767.
[52]
Muruganandan S, Roman AA, Sinal CJ (2010) Role of chemerin/CMKLR1 signaling in adipogenesis and osteoblastogenesis of bone marrow stem cells. J Bone Miner Res 25: 222–234.
[53]
Kang S, Bennett CN, Gerin I, Rapp LA, Hankenson KD, et al. (2007) Wnt signaling stimulates osteoblastogenesis of mesenchymal precursors by suppressing CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. J Biol Chem 282: 14515–14524.
[54]
Shim M, Smart RC (2003) Lithium stabilizes CCAAT/enhancer-binding protein-alpha(C/EBPalpha) through a glycogen synthase kinase 3 (GSK3)-independent pathway involving direct inhibition of proteasomal activity. J Biol Chem 278: 19674–19681.
[55]
Ross SE, Hemati N, Longo KA, Bennett CN, Lucas PC, et al. (2000) Inhibition of adipogenesis by Wnt signaling. Science 289: 950–953.
[56]
Justesen J, Mosekilde L, Holmes M, Stenderup K, Gasser J, et al. (2004) Mice deficient in 11beta-hydroxysteroid dehydrogenase type 1 lack bone marrow adipocytes, but maintain normal bone formation. Endocrinol 145: 1916–1925.
[57]
Bujalska IJ, Gathercole LL, Tomlinson JW, Darimont C, Ermolieff J, et al. (2008) A novel selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis. J Endocrinol 197: 297–307.
[58]
Elabd C, Basillais A, Beaupied H, Breuil V, Wagner N, et al. (2008) Oxytocin controls differentiation of human mesenchymal stem cells and reverses osteoporosis. Stem Cells 26: 2399–2407.
[59]
Tamma R, Colaianni G, Zhu LL, DiBenedetto A, Greco G, et al. (2009) Oxytocin is an anabolic bone hormone. Proc Natl Acad Sci USA 106: 7149–7154.
[60]
Rauch A, Seitz S, Baschant U, Schilling AF, Illing A, et al. (2010) Glucocorticoids suppress bone formation by attenuating osteoblast differentiation via the monomeric glucocorticoid receptor. Cell Metabolism 11: 517–531.
[61]
Lukert BP, Raisz LG (1990) Glucocorticoid-induced osteoporosis: pathogenesis and management. Ann Internal Med 112: 352–364.
[62]
Yang X, Karsenty G (2004) ATF4, the ostoblast accumulation of which is determined post-translationally, can induce osteoblast-specific gene expression in non-osteoblastic cells. J Biol Chem 279: 47109–47114.
[63]
Yang X, Matsuda K, Bialek P, Jacquot S, Masuoka HC, et al. (2004) ATF4 is a substrate of RSK2 and an essential regulator of osteoblast biology: implication for Coffin-Lowry Syndrome. Cell 117: 387–398.
[64]
Qu G, von Schroeder HP (2006) Role of Osterix in endothelin-1-induced downregulation of vascular endothelial growth factor in osteoblastic cells. Bone 38: 21–29.
[65]
Isenmann S, Arthur A, Zannettino AC, Turner JL, Shi S, et al. (2009) TWIST family of basic helix-loop-helix transcription factors mediate human mesenchymal stem cell growth and commitment. Stem Cells 27: 2457–2468.
[66]
Bialek P, Kern B, Yang X, Schrock M, Sosic D, et al. (2004) A twist code determines the onset of osteoblast differentiation. Dev Cell 6: 423–435.
[67]
Zhao Y, Ding S (2007) A high-throughput siRNA library screen identifies osteogenic suppressors in human mesenchymal stem cells. Proc Natl Acad Sci USA 104: 9673–9678.
[68]
Yoshiko Y, Maeda N, Aubin JE (2003) Stanniocalcin 1 stimulates osteoblast differentiation in rat calvaria cell cultures. Endocrinol 144: 4134–4143.
[69]
Johnston J, Ramos-Valdes Y, Stanton LA, Ladhani S, Beier F, et al. (2010) Human stanniocalcin-1 or -2 expressed in mice reduces bone size and severely inhibits cranial intramembranous bone growth. Transgenic Res 19: 1017–1039.
[70]
Choi YA, Lim J, Kim KM, Acharya B, Cho JY, et al. (2010) Secretome analysis of human BMSCs and identification of SMOC1 as an important ECM protein in osteoblast differentiation. J Proteome Res 9: 2946–2956.
[71]
Hong JH, Hwang ES, McManus MT, Amsterdam A, Tian Y, et al. (2005) TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309: 1074–1078.
[72]
Zhang X, Schwarz EM, Young DA, Puzas JE, Rosier RN, et al. (2002) Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair. J Clin Invest 109: 1405–1415.
[73]
Seelan RS, Khalyfa A, Lakshamanan J, Casanova MF, Parthasarathy RN (2008) Deciphering the lithium transcriptome: Microarray profiling of lithium-modulated gene expression in human neuronal cells. Neuroscience 151: 1184–1197.
[74]
Hipskind RA, Bilbe G (1998) MAP kinase signaling cascades and gene expression in osteoblasts. Front Biosci 3: d804–d816.
[75]
Rosaria M, Paterson HF, Marshall CJ (1999) Activation of the Raf/MAP kinase cascade by the Ras-related protein TC21 is required for the TC21-mediated transformation of NIH3T3 cells. EMBO J 18: 1270–1279.
[76]
Ward Y, Gupta S, Jensen P, Wartmann M, Davis RJ, et al. (1994) Control of MAP kinase activation by the mitogen-induced threonine/tyrosine phosphatase PAC1. Nature 367: 651–654.
[77]
Caunt CJ, Rivers CA, Conway-Campbell BL, Norman MR, McArdle CA (2008) Epidermal growth factor receptor and protein kinase C signaling to ERK2: spatiotemporal regulation of ERK2 by dual specificity phosphatases. J Biol Chem 283: 6241–6252.
[78]
Ge C, Xiao G, Jiang D, Franceschi RT (2007) Critical role of the extracellular signal-regulated kinase-MAPK pathway in osteoblast differentiation and skeletal development. J Cell Biol 176: 709–718.
[79]
Yoshida K, Shinohara H, Haneji T, Nagata T (2007) Arachidonic acid inhibits osteoblast differentiation through cytosolic phospholipase A2-dependent pathway. Oral Dis 13: 32–39.
[80]
Chen S, McLean S, Carter DE, Leask A (2007) The gene expression profile induced by Wnt3a in NIH 3T3 fibroblasts. J Cell Commun Signal 1: 175–183.
[81]
Klapholz-Brown Z, Walmsley GG, Nusse YM, Nusse R, Brown PO (2007) Transcriptional program induced by Wnt protein in human fibroblasts suggests mechanisms for cell cooperativity in defining tissue microenvironments. PLoS ONE 2: e945.
[82]
Sachdev S, Bruhn L, Sieber H, Pichler A, Melchior F, et al. (2001) PIASy, a nuclear matrix-associated SUMO E3 ligase, represses LEF1 activity by sequestering into nuclear bodies. Genes Dev 15: 3088–3103.
[83]
Sampson EM, Haque ZK, Ku MC, Tevosian SG, Albanese C, et al. (2001) Negative regulation of the Wnt-β-catenin pathway by the transcriptional repressor HBP1. EMBO J 20: 4500–4511.
[84]
Rawadi G, Vayssiere B, Dunn F, Baron R, Roman-Roman S (2003) BMP-2 controls alkaline phosphatase expression and osteoblast mineralization by a Wnt autocrine loop. J Bone Miner Res 18: 1842–1853.
[85]
Liu G, Vijayakumar S, Grumolato L, Arroyave R, Qiao H, et al. (2009) Canonical Wnt function as potent regulators of osteogenesis by human mesenchymal stem cells. J Cell Biol 185: 67–75.
[86]
Gordon JA, Hassan MQ, Saini S, Montecino M, van Wijnen AJ, et al. (2010) Pbx1 represses osteoblastogenesis by blocking Hoxa10-mediated recruitment of chromatin remodeling factors. Mol Cell Biol 30: 3531–3541.
[87]
Wewer UM, Ibaraki K, Schiorring P, Durkin ME, Young MF, et al. (1994) A potential role for tetranectin in mineralization during osteogenesis. J Cell Biol 127: 1767–1775.
[88]
Larsen KH, Frederiksen CM, Burns JS, Abdallah BM, Kassem M (2010) Identifying a molecular signature for bone marrow stromal cells with in vivo bone-forming capacity. J Bone Miner Res 25: 796–808.
[89]
Ward Y, Yap SF, Ravichandran V, Matsumura F, Ito M, et al. (2002) The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway. J Cell Biol 157: 291–302.
[90]
Kanazawa I, Yamaguchi T, Yano S, Yamauchi M, Sugimoto T (2009) Activation of AMP kinase and inhibition of Rho kinase induce the mineralization of osteoblastic MC3T3-E1 cells through endothelial NOS and BMP-2 expression. Am J Physiol Endocrinol Metab 296: E139–E146.
[91]
Harmey D, Stenbeck G, Nobes CD, Lax AJ, Grigoriadis AE (2004) Regulation of osteoblast differentiation by Pasteurella Multocida toxin (PMT): A role for Rho GTPase in bone formation. J Bone Miner Res 19: 661–670.
[92]
Ohnaka K, Shimoda S, Nawata H, Shimokawa H, Kaibuchi K, et al. (2001) Pitavastatin enhanced BMP-2 and osteocalcin expression by inhibition of Rho-associated kinase in human osteoblasts. Biochem Biophys Res Commun 287: 337–342.
