Human bone marrow stromal/stem cells (hBMSCs) have an inherent tendency to undergo hypertrophy when induced into the chondrogenic lineage using transforming growth factor-beta 1 (TGFβ) in vitro, reminiscent of what occurs during endochondral ossification. Surprisingly, Indian Hedgehog (IHH) has received little attention for its role during hBMSC chondrogenesis despite being considered a master regulator of endochondral ossification. In this study, we investigated the role that endogenously produced IHH plays during hBMSC chondrogenesis. We began by analyzing the expression of IHH throughout differentiation using quantitative polymerase chain reaction and found that IHH expression was upregulated dramatically upon chondrogenic induction and peaked from days 9 to 12 of differentiation, which coincided with a concomitant increase in the expression of chondrogenesis- and hypertrophy-related markers, suggesting a potential role for endogenously produced IHH in driving hBMSC chondrogenesis. More importantly, pharmacological inhibition of Hedgehog signaling with cyclopamine or knockdown of IHH almost completely blocked TGFβ1-induced chondrogenesis in hBMSCs, demonstrating that endogenously produced IHH is necessary for hBMSC chondrogenesis. Furthermore, overexpression of IHH was sufficient to drive chondrogenic differentiation, even when TGFβ signaling was inhibited. Finally, stimulation with TGFβ1 induced a significant and sustained upregulation of IHH expression within 3 h that preceded an upregulation in all cartilage-related genes analyzed, and knockdown of IHH blocked the effects of TGFβ1 entirely, suggesting that the effects of TGFβ1 are being mediated through endogenously produced IHH. Together, our findings demonstrate that endogenously produced IHH is playing a critical role in regulating hBMSC chondrogenesis.
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References
1.
JohnstoneB, HeringTM, CaplanAI, GoldbergVM and YooJU. (1998). In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res, 238:265–272.
2.
BaiX, XiaoZ, PanY, HuJ, PohlJ, WenJ and LiL. (2004). Cartilage-derived morphogenetic protein-1 promotes the differentiation of mesenchymal stem cells into chondrocytes. Biochem Biophys Res Commun, 325:453–460.
3.
SteinertAF, WeissenbergerM, KunzM, GilbertF, GhivizzaniSC, GobelS, JakobF, NothU and RudertM. (2012). Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells. Arthritis Res Ther, 14:R168
4.
SteinertAF, ProffenB, KunzM, HendrichC, GhivizzaniSC, NothU, RethwilmA, EulertJ and EvansCH. (2009). Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer. Arthritis Res Ther, 11:R148
5.
PalmerGD, SteinertA, PascherA, GouzeE, GouzeJN, BetzO, JohnstoneB, EvansCH and GhivizzaniSC. (2005). Gene-induced chondrogenesis of primary mesenchymal stem cells in vitro. Mol Ther, 12:219–228.
6.
ShenB, WeiA, WhittakerS, WilliamsLA, TaoH, MaDD and DiwanAD. (2010). The role of BMP-7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro. J Cell Biochem, 109:406–416.
7.
WeissS, HennigT, BockR, SteckE and RichterW. (2010). Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells. J Cell Physiol, 223:84–93.
8.
KimYJ, KimHJ and ImGI. (2008). PTHrP promotes chondrogenesis and suppresses hypertrophy from both bone marrow-derived and adipose tissue-derived MSCs. Biochem Biophys Res Commun, 373:104–108.
9.
WangL and DetamoreMS. (2009). Insulin-like growth factor-I improves chondrogenesis of predifferentiated human umbilical cord mesenchymal stromal cells. J Orthop Res, 27:1109–1115.
10.
ByersBA, MauckRL, ChiangIE and TuanRS. (2008). Transient exposure to transforming growth factor beta 3 under serum-free conditions enhances the biomechanical and biochemical maturation of tissue-engineered cartilage. Tissue Eng Part A, 14:1821–1834.
11.
HuangAH, SteinA, TuanRS and MauckRL. (2009). Transient exposure to transforming growth factor beta 3 improves the mechanical properties of mesenchymal stem cell-laden cartilage constructs in a density-dependent manner. Tissue Eng Part A, 15:3461–3472.
12.
MuellerMB, FischerM, ZellnerJ, BernerA, DienstknechtT, PrantlL, KujatR, NerlichM, TuanRS and AngeleP. (2010). Hypertrophy in mesenchymal stem cell chondrogenesis: effect of TGF-beta isoforms and chondrogenic conditioning. Cells Tissues Organs, 192:158–166.
13.
ScottiC, TonnarelliB, PapadimitropoulosA, ScherberichA, SchaerenS, SchauerteA, Lopez-RiosJ, ZellerR, BarberoA and MartinI. (2010). Recapitulation of endochondral bone formation using human adult mesenchymal stem cells as a paradigm for developmental engineering. Proc Natl Acad Sci U S A, 107:7251–7256.
14.
DreierR. (2010). Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther, 12:216
15.
PooleAR. (1999). An introduction to the pathophysiology of osteoarthritis. Front Biosci, 4:D662–D670.
16.
LaiLP and MitchellJ. (2005). Indian hedgehog: its roles and regulation in endochondral bone development. J Cell Biochem, 96:1163–1173.
17.
OlsenBR, ReginatoAM and WangW. (2000). Bone development. Annu Rev Cell Dev Biol, 16:191–220.
18.
VortkampA, LeeK, LanskeB, SegreGV, KronenbergHM and TabinCJ. (1996). Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science, 273:613–622.
19.
St-JacquesB, HammerschmidtM and McMahonAP. (1999). Indian hedgehog signaling regulates proliferation and differentiation of chondrocytes and is essential for bone formation. Genes Dev, 13:2072–2086.
20.
LanskeB, KaraplisAC, LeeK, LuzA, VortkampA, PirroA, KarperienM, DefizeLH, HoC, et al. (1996). PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science, 273:663–666.
21.
AlvarezJ, SohnP, ZengX, DoetschmanT, RobbinsDJ and SerraR. (2002). TGFbeta2 mediates the effects of hedgehog on hypertrophic differentiation and PTHrP expression. Development, 129:1913–1924.
22.
PathiS, RutenbergJB, JohnsonRL and VortkampA. (1999). Interaction of Ihh and BMP/Noggin signaling during cartilage differentiation. Dev Biol, 209:239–253.
23.
MakKK, KronenbergHM, ChuangPT, MackemS and YangY. (2008). Indian hedgehog signals independently of PTHrP to promote chondrocyte hypertrophy. Development, 135:1947–1956.
24.
KarpSJ, SchipaniE, St-JacquesB, HunzelmanJ, KronenbergH and McMahonAP. (2000). Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein-dependent and -independent pathways. Development, 127:543–548.
25.
LongF, ChungUI, OhbaS, McMahonJ, KronenbergHM and McMahonAP. (2004). Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton. Development, 131:1309–1318.
26.
PittengerMF, MackayAM, BeckSC, JaiswalRK, DouglasR, MoscaJD, MoormanMA, SimonettiDW, CraigS and MarshakDR. (1999). Multilineage potential of adult human mesenchymal stem cells. Science, 284:143–147.
27.
SotiropoulouPA, PerezSA, SalagianniM, BaxevanisCN and PapamichailM. (2006). Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells, 24:462–471.
28.
HandorfAM and LiWJ. (2014). Induction of mesenchymal stem cell chondrogenesis through sequential administration of growth factors within specific temporal windows. J Cell Physiol, 229:162–171.
29.
HandorfAM and LiWJ. (2011). Fibroblast growth factor-2 primes human mesenchymal stem cells for enhanced chondrogenesis. PLoS One, 6:e22887
30.
KarlssonC, BrantsingC, SvenssonT, BrisbyH, AspJ, TallhedenT and LindahlA. (2007). Differentiation of human mesenchymal stem cells and articular chondrocytes: analysis of chondrogenic potential and expression pattern of differentiation-related transcription factors. J Orthop Res, 25:152–163.
31.
FreyriaAM and Mallein-GerinF. (2012). Chondrocytes or adult stem cells for cartilage repair: the indisputable role of growth factors. Injury, 43:259–265.
32.
WuX, CaiZD, LouLM and ChenZR. (2013). The effects of inhibiting hedgehog signaling pathways by using specific antagonist cyclopamine on the chondrogenic differentiation of mesenchymal stem cells. Int J Mol Sci, 14:5966–5977.
33.
PepinskyRB, ZengC, WenD, RayhornP, BakerDP, WilliamsKP, BixlerSA, AmbroseCM, GarberEA, et al. (1998). Identification of a palmitic acid-modified form of human Sonic hedgehog. J Biol Chem, 273:14037–14045.
34.
MaG, YuJ, XiaoY, ChanD, GaoB, HuJ, HeY, GuoS, ZhouJ, et al. (2011). Indian hedgehog mutations causing brachydactyly type A1 impair Hedgehog signal transduction at multiple levels. Cell Res, 21:1343–1357.
35.
LeeJM and ImGI. (2012). PTHrP isoforms have differing effect on chondrogenic differentiation and hypertrophy of mesenchymal stem cells. Biochem Biophys Res Commun, 421:819–824.
36.
SekiyaI, VuoristoJT, LarsonBL and ProckopDJ. (2002). In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A, 99:4397–4402.
37.
van GoolSA, EmonsJA, LeijtenJC, DeckerE, StichtC, van HouwelingenJC, GoemanJJ, KleijburgC, ScherjonSA, et al. (2012). Fetal mesenchymal stromal cells differentiating towards chondrocytes acquire a gene expression profile resembling human growth plate cartilage. PLoS One, 7:e44561
38.
SekiK and HataA. (2004). Indian hedgehog gene is a target of the bone morphogenetic protein signaling pathway. J Biol Chem, 279:18544–18549.
39.
DennlerS, ItohS, VivienD, ten DijkeP, HuetS and GauthierJM. (1998). Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene. EMBO J, 17:3091–3100.
40.
ZawelL, DaiJL, BuckhaultsP, ZhouS, KinzlerKW, VogelsteinB and KernSE. (1998). Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell, 1:611–617.
41.
FurumatsuT, TsudaM, TaniguchiN, TajimaY and AsaharaH. (2005). Smad3 induces chondrogenesis through the activation of SOX9 via CREB-binding protein/p300 recruitment. J Biol Chem, 280:8343–8350.
42.
WatanabeH, de CaesteckerMP and YamadaY. (2001). Transcriptional cross-talk between Smad, ERK1/2, and p38 mitogen-activated protein kinase pathways regulates transforming growth factor-beta-induced aggrecan gene expression in chondrogenic ATDC5 cells. J Biol Chem, 276:14466–14473.
43.
ZengL, KempfH, MurtaughLC, SatoME and LassarAB. (2002). Shh establishes an Nkx3.2/Sox9 autoregulatory loop that is maintained by BMP signals to induce somitic chondrogenesis. Genes Dev, 16:1990–2005.
44.
ParkJ, ZhangJJ, MoroA, KushidaM, WegnerM and KimPC. (2010). Regulation of Sox9 by Sonic Hedgehog (Shh) is essential for patterning and formation of tracheal cartilage. Dev Dyn, 239:514–526.
45.
Bien-WillnerGA, StankiewiczP and LupskiJR. (2007). SOX9cre1, a cis-acting regulatory element located 1.1 Mb upstream of SOX9, mediates its enhancement through the SHH pathway. Hum Mol Genet, 16:1143–1156.
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