TGF-β and BMP signaling in osteoblast, skeletal development, and bone formation, homeostasis and disease

TGFβs ligands

All TGF-β isoforms are expressed by perichondrium and periosteum^21,22 as well as epiphyseal growth plate²³. However, only Tgfb2-null mice displayed severe skeletal abnormalities in both endochondral and intramembranous bone,²⁴ while Tgfb1-deficient mice and Tgfb3-null mice had almost normal skeleton.^25–27 Thus, TGF-β2, but not TGF-β1 or TGF-β3, is essential for embryonic skeleton development.

TGF-β has an important role in maintaining postnatal bone mass by coupling bone resorption and bone formation (Figure 1). TGF-βs are synthesized as large precursor molecules, composed of mature TGF-β and latency-associated protein (LAP).²⁸ LAP remains noncovalently bound to mature TGF-β as it is secreted, rendering it inactive by masking the ECM (extracellular matrix) of many different tissues.²⁸ Cleaving LAP by osteoclastic bone resorption release active TGF-β1 to induce enrichment of osteoprogenitor in the bone resorption lacunae.⁷ Tang et al. crossed Tgfb1-null mice with immunodeficient Rag2−/− mice to overcome the early death of Tgfb1-null mice.^8,25 The Tgfb1−/−Rag2−/− mice showed a significant loss of trabecular bone density and reduced osteoblast number on the bone surface.⁸ A gain-of-function mutant of TGF-β1 causes Camurati–Engelmann disease (CED) in humans,^19,20 which is characterized by diaphyseal thickening and fluctuating bone volume. This mutation results in a similar bone defect in mice (CED mice).⁸ However, TGF-β1 is unable to induce osteogenesis in mesenchymal pluripotent cells, but increases the pool of osteoprogenitors by inducing chemotaxis and proliferation.⁸ Apoptosis of osteoblasts is also blocked by TGF-β1 deletion through maintenance of survival during transdifferentiation into osteocytes.²⁹ In addition, TGF-β treatment blocked osteoblast mineralization in culture,³⁰ indicating its bi-functions in osteoblast differentiation. On the other hand, active TGF-βs regulate bone resorption in a dose-dependent manner. The gradient of TGF-β created during osteoclast bone resorption can limit further osteoclast activity.²⁸ Low concentrations of active TGF-β can induce macrophage migration to the bone resorption pits, whereas high concentrations of active TGF-β inhibit migration of osteoclast precursors.²⁸ TGF-β was also shown to promote osteoclast differentiation at low dose and inhibits osteoclast differentiation at high dose through regulating RANKL/OPG ratio secreted by osteoblasts.^7,31 Furthermore, TGF-β1 via binding to its receptor on osteoclasts activates Smad2/3, which associates directly with TRAF6–TAB1–TAK1 complex and favors osteoclast differentiation.³²

TGF-β is a double-edged sword in the maintenance of articular cartilage metabolic homeostasis and the pathogenesis of arthritis³³. Loss of TGF-β signaling in cartilage induces chondrocyte hypertrophy, ultimately resulting in cartilage degeneration, and pharmacological activation of the TGF-β pathway has therefore been proposed to preserve articular cartilage integrity during osteoarthritis (OA).^19,34,35 However, age-dependent switch from TGF-β-ALK5-Smad2/3 to -ALK1-Smad1/5/8 signaling contributes to osteophyte formation and the pathogenesis of osteoarthritis.^17,36 At the onset of either OA or rheumatoid arthritis (RA), elevated active TGF-β1 resulted from excessive bone resorption recruits MSCs in the subchondral bone marrow and induces the formation of “osteoid islet”, which leads to the degeneration of the overlying articular cartilage.^37,38 Transgenic expression of active TGF-β1 in osteoblastic cells induced OA, whereas inhibition of TGF-β activity in subchondral bone attenuated the degeneration of articular cartilage.³⁷ Similarly, aberrant activation of TGF-β in subchondral bone is involved at the onset of RA joint cartilage degeneration, whereas either systemic or local blockade of TGF-β activity in the subchondral bone attenuated articular cartilage degeneration in RA.³⁸ Furthermore, knockout of the TβRII in nestin-positive MSCs leads to less development of both OA and RA relative to wild-type mice.^37,38

Extracellular regulation of TGF-β signaling

ECM proteins regulate TGF-β signaling through controlling ligand availability (Figure 1). LTBPs covalently bind latent TGF-β and modulate tissue levels of TGF-β. Ablation of LTBP-3 reduces both osteogenesis and bone resorption, and resulted in increased bone mass associated with decreased levels of TGF-β.^39,40 Binding of TGF-β to small leucine-rich proteoglycans (decorin, biglycan, and lumican) restricts the TGF-β in the ECM so as to inhibit its activity.⁴¹ Bgn and Dcn double deficiency results in osteopenia and a striking change in collagen fibril shape.⁴¹ Excessive TGF-β signaling, caused by reduced binding of decorin, is also found to be a common mechanism of osteogenesis imperfecta in mouse models.⁴²

TGF-βs receptors

Similarly to TGF-βs, the expression of TGF-β receptors is also detected in the growth plate and perichondrium.^21,22,43 Mice lacking either TβRI/ALK5 or TβRII in MSC results in short long bone and defects in joint development fusion.^30,44–46 MSC-specific Tgfbr2-deletion and MSC-specific Alk5-deletion both promote chondrocyte hypertrophy and decreased chondrocyte proliferation, which was also observed in DN-TβRII transgenic mice.^{30,34,44–46} Thus, TGF-β signaling favors chondrocyte proliferation but blocks the transition from proliferative chondrocyte into hypertrophic chondrocyte. Mice lacking TβRII/ALK5 in the chondrocyte can reproduce defects in the skull base and vertebrae as those observed in the Tgfb2-null mice,^24,47 but cannot influence normal long bone development.⁴⁷ This phenotype indicates that TGF-β might regulate growth plate development largely through mediating paracrine cytokines secretion by perichondrium cells.

TGF-β signaling favors bone formation by promoting osteoprogenitor enrichment. Consistently, deletion of Alk5 in MSC resulted in reduced bone collars and trabecular bones associated with decreased osteoblast number.³⁰ The study showed that TGF-β, through TβRI, promotes pre-osteoblast commitment and early differentiation.³⁰ However, deletion of Tgfbr2 in osteoblasts resulted in an unexpected increase in bone mass due to enhanced PTHrP signaling.⁴³

Smad-dependent pathway

Smad2 and Smad3 respond to TGF-β and regulate TGF-β-mediated osteoblast and chondrocyte differentiation (Figure 1). Smad2/3 inhibits Runx2 expression, and activated Smad3 also recruits class II histone deacetylases (HDACs) 4 and 5 to repress the function of Runx2.^48–51 TGF-β can no longer inhibit the differentiation of osteoblasts in the absence of Smad3.^52,53 Although TGF-β-Smad3 negatively regulates osteoblastogenesis, it also inhibits osteoblast apoptosis and the differentiation into osteocyte. Thus, Smad3-null mice are osteopenic associated with increased osteocyte number and apoptosis.⁵² Furthermore, TGF-β-induced inhibition of chondrocyte maturation is potentiated by Smad2/3 signaling,⁵⁴ and abolished by disrupting Smad2/3 activation.^54,55 Smad3-null mice also displayed accelerated chondrocyte hypertrophy, at least partially through reduced Noggin level and unopposed BMP signaling.⁵⁶

In addition, TGF-β-Smad3 signaling is required for postnatal maintenance of articular cartilage homeostasis. Smad3-null mice and chondrocyte-specific Smad3 CKO mice exhibit articular cartilage degeneration and automatically develop OA.^57–59 Mutations of Smad3 in human caused aneurysms–osteoarthritis syndrome,^15,60,61 which is presenting with aneurysms, mild craniofacial features, and skeletal and cutaneous anomalies. Mechanistically, TGF-β signals through Smad3 to confer a rapid and dynamic repression of Runx2-induced MMP-13 expression, and signals through p38 to promote Runx2-induced MMP-13 expression in the absence of Smad3.⁵⁸

BMP ligands

Among the 14 BMPs, BMP-2, 4, 5, 6, 7, and 9 exhibit high osteogenic activity.^64,65 Osteogenic capability of BMP-2 and BMP-7 have been vastly studied and the recombinant proteins are currently being investigated in human clinical trials of craniofacial deformities, fracture healing, and spine fusion.^13,14 BMP-2 vastly increases osteocalcin expression⁶⁶ and a short-term expression of the BMP-2 is necessary and sufficient to irreversibly induce bone formation.⁶⁷ BMP-7 induces the expression of osteoblastic differentiation markers, such as ALP and accelerates calcium mineralization.^68–70 BMP-3 is a ‘non-canonical’ BMP molecule that transduces type IIB Activin receptor (AcvrIIB)-Smad2/3 signaling to oppose the osteogenic function of other BMPs.⁷¹ In vivo, BMP-3 is mainly produced by osteoblasts and osteocytes,⁷¹ and negatively regulates bone mass in vivo, as shown by knockout and transgenic mouse models.^72,73 Interestingly, a recent study showed that BMP2 addition to culture media rapidly induced expansion of isolated mouse skeletal stem cell (mSSC).⁷⁴ Delivery of BMP2 induces de novo formation of the mSSC and bone in extraskeletal locations.⁷⁴ In addition, co-delivery of BMP2 and sVEGFR1 induces de novo formation of cartilage in extraskeletal locations.⁷⁴ Expression of the BMP antagonist gremlin 1 defines a population of osteochondroreticular stem cells in the bone marrow, which is self-renewal and is able to generate osteoblasts, chondrocytes, and reticular marrow stromal cells, but not adipocytes.⁷⁵

BMPs have essential roles at many steps in endochondral bone development. Studies showed that BMP signaling promotes chondrocyte proliferation and differentiation,^76–78 by maintaining the expression of Sox9, a transcription factor that is essential for chondrogenic commitment and differentiation.^77,79 BMP-7 null mice died shortly after birth and exhibits skeletal patterning defects restricted to the rib cage, skull, and hindlimbs.⁸⁰ Conditional deletion of BMP-2, BMP-4, or BMP-7 in MSC results in normal skeletongenesis.^70,81,82 Only BMP-2 CKO mice have frequent fractures that fail to heal, which is not observed in either Bmp-4 or Bmp-7 CKO mice.^70,83,84 The studies indicate that BMPs have duplicate functions in skeleton development, while BMP-2 has a unique role in postnatal bone formation. MSC-specific Bmp-2/-4 DKO mice have extremely malformed limbs and a severe impairment of osteogenesis, which is not observed in Bmp-2/-7 DKO mice.^82,85 Chondrogenic condensations in some skeletal elements fail in MSC-specific Bmp-2/-4 DKO mice.⁸² Furthermore, chondrocyte-specific Bmp-2 CKO and Bmp-2/4 DKO mice exhibits severe disorganization of chondrocytes within the growth plate region.⁸⁶ Collectively, these studies revealed that coordinated functions of endogenous BMP-2 and BMP-4 are required for osteoblastogenesis as well as chondrocyte proliferation, differentiation and apoptosis during skeletal development.

BMP7 has a pivotal role in postnatal maintenance of articular cartilage. Intra-articular administration of rhBMP7 significantly inhibited articular cartilage degeneration and blocked production of inflammatory cytokines by the synovial membrane.^18,87,88 Bmp7^f/fPrx1-Cre mice, and Bmpr1a^f/fGdf5-Cre mice exhibit articular cartilage degeneration and automatically develop OA.⁸⁹

BMP receptors

There are three type I receptors for BMPs, type IA BMP receptor (BMPR1A/ALK3), type IB BMP receptor (BMPR1B/ALK6), and type I activin receptor (AcvR1/ ALK2). BMPs exert their osteogenic signaling through those receptors. Activating mutants of Acvr1 cause ectopic ossification in mouse⁹⁰ and in human disease (fibrodysplasia ossificans progressive, FOP).^91,92 Mechanically, active AcvR1 induces epithelial-to-mesenchymal transition and promotes ectopic osteoblastogenesis via canonical Smad1/5 signaling.^90,93 On the other hand, blocking BMP signaling through overexpression of DN-BMPRII or deletion of Bmpr1a in osteoblast results in low bone mass.^94,95 Although deletion of Bmpr1a in preosteoblasts causes an unexpected increase in bone mass due to reduced bone resorption, osteoblast differentiation remains low in this condition.^96–98

The three type I BMP receptors (BMPR1A, BMPR1B, and ALK6) have largely redundant roles in skeletal development. BMPR1A and BMPR1B share similar expression pattern in chondrocyte condensations and developing skeletons,^99–104 and AcvR1 is expressed throughout the growth plate.¹⁰⁵ Activation and dominant-negative experiments proved the BMPRIB is essential for cartilage condensation in chicks.¹⁰³ Activation experiments showed that BMPR1A, BMPR1B, and AcvR1 are all able to promote chondrogenesis.^{77,101,103,106–108} However, deletion of each of them alone only results in mild skeletal defects or defects restricted to certain skeletal elements.^{77,104,105,109,110} In contrast, Bmpr1a/Bmpr1b DKO mice, Acvr1/Bmpr1a DKO mice, and Acvr1/Bmpr1b DKO mice exhibit generalized chondrodysplasia that is much more severe than any of the corresponding mutant strains.^104,105,110 The loss of both BMPR1A and BMPR1B blocks chondrocyte condensation, proliferation, differentiation, survival, and function due to impaired Sox proteins (Sox-5, 6, and 9) expression.^104,110 In conclusion, those mouse models demonstrated that BMP signaling is essential for almost every step during endochondral bone development (Figure 3).

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Figure 3

Crosstalk between BMP, TGF-β, and other signaling during osteoblast differentiation. BMP has dual roles in Wnt signaling. On one hand, BMP inhibits Wnt/β-catenin signaling by increasing Wnt antagonist Dkk1 and Sost expression and by preventing β-catenin nuclei translocation. On the other hand, BMP promotes Wnt/β-catenin signaling by forming co-transcriptional complex with β-catenin/TCF/LEF/Runx2, by increasing Wnt expression, by antagonizing Dvl function and by decreasing b-TrCP expression. BMP signaling promotes FGF, Hh, PTHrP signaling by increasing IHH expression, increasing FGF expression, decreasing Ptch1 expression, respectively. BMP is essential for IHH-induced osteoblast differentiation. FGF, IHH, Wnt, and PTHrP signaling all promotes BMP2 expression so as to enhance BMP signaling. FGF and IHH signaling is essential for BMP-induced osteoblast differentiation. TGF-β antagonizes PTHrP signaling through TGF-β type II receptor complexing and internalizing together with PTHrP receptor (PPR). TGF-β promotes Wnt activity by increasing Wnt ligands expression and decreasing Axin expression. Fibroblast growth factor (FGF) and Wnt all increase TGF-β expression to promote TGF-β signaling. BMP, bone morphogenetic protein; PTHrP, parathyroid hormone-related peptide; TGF-β, transforming growth factor-β.

Smad-depedent pathway

Most BMPs activate Smad1/5/8 as their R-Smad (Figure 2). Smad1/5/8-Smad4 complex transcribed Runx2 expression, as they complex with Runx2 to initiate other osteoblast gene expression.^62,111,112 Smad1 is an important mediator of BMP’s osteogenic function. In osteoblast-specific Smad1-CKO mice, BMP signaling was partially inhibited, osteoblast proliferation and differentiation were impaired and mice developed an osteopenic phenotype.¹¹³ Smad1 and Smad5 together mediate the role of BMP in endochondral bone development. Chondrocyte-specific deletion of Smad1 results in delayed calvarial bone development and shortening of the growth plate.^113,114 The addition of Smad5 haploinsufficiency or insufficiency leads to more severe chondrodysplasia, while addition of Smad8 insufficiency did not worsen the defects.^114,115 Thus, Smad8 contributes much less to skeleton development as compared with Smad1 and Smad5. Only BMP3 also activates Smad2/3, which antagonized osteogenic activity of Smad1/5/8 and opposed the function of other BMPs in osteoblast differentiation.¹¹⁶

Both BMPs and TGF-βs signal through Smad4. Smad4 mutation in humans causes Myhre syndrome, a developmental disorder, characterized by short stature and facial dysmorphism.^117,118 In vitro Smad4 ablation partially suppressed BMP-4-induced osteoblast differentiation.¹¹⁹ In vivo abrogation of Smad4 in chondrocytes results in dwarfism with a severely disorganized growth plate and ectopic bone collars in perichondrium.¹²⁰ In the Smad4-deficient growth plate, resting zone was expanded, while chondrocyte proliferation was reduced and hypertrophic differentiation was accelerated.¹²⁰ Deletion of Smad4 in mature osteoblasts causes lower bone mass and decreased osteoblast proliferation and differentiation up to 6 months of age, while the trabecular bone volume in the mutant mice increased after 7-month-old due to reduced bone resorption.¹²¹ Embryonic deletion of Smad4 in pre-osteoblast causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia, and Wnt-deficiency syndromes.¹²² Postnatal deletion of Smad4 in pre-osteoblasts increases mitosis of cells on trabecular bone surfaces as well as in primary osteoblast cultures, while delays differentiation and matrix mineralization by primary osteoblasts associated with altered β-catenin acvitity.¹²³ Knockout of Smad4 in osteoblast also reveals that BMP/TGFβs coordinate with Wnt signaling to maintain normal bone mass. In summary, Smad4 has multiples roles in growth plate development and postnatal bone homeostasis.

Regulators in the ECM

ECM proteins regulate TGF-β signaling through controlling ligand availability (Figure 1). LTBPs covalently bind latent TGF-β and modulate tissue levels of TGF-β. Ablation of LTBP-3 reduces both osteogenesis and bone resorption, and resulted in increased bone mass associated with decreased levels of TGF-β.^39,40 Binding of TGF-β to small leucine-rich proteoglycans (decorin, biglycan, and lumican) restricts the TGF-β in the ECM so as to inhibit its activity.⁴¹ Bgn and Dcn double deficiency results in osteopenia and a striking change in collagen fibril shape.⁴¹ ExcessiveTGF-β signaling caused by reduced binding of decorin is also found to be a common mechanism of osteogenesis imperfecta in mouse models.⁴²

BMP signaling is regulated by a group of cognate binding proteins (for example, Noggin, Chordin, Gremlin, and Follistatin) that competitively bound BMPs to prevent their binding to receptors^133–135 (Figure 2). Noggin opposes BMP’s osteogenic function in vivo¹³⁶ and in vitro.¹³⁷ Nog-null mice died at birth with severe malformed skeletons due to unopposed BMP signaling,^138,139 and addition of deficiency of other BMP antagonists (Grem1 or Follistatin) aggravated the skeleton malformation of Nog−/− mice.^140,141 Ectopic expression of Noggin in osteoblast impairs osteoblastogenesis and results in osteopenia in mice.^142,143 Ectopic expression of Noggin is also able to block cartilage development, change skeletal morphology, and prevent cranial suture fusion.^144,145 Noggin expression is induced by BMPs, and acts in a negative regulatory loop to inhibit BMP activity.¹³³ Other signals including Sox9 and FGFs also regulate BMP signaling via manipulating Noggin expression.^145–148 Chordin and Chordin-like proteins (CHL) were identified as a factor dorsalizing the Xenopus embryo.^149–152 Chordin and CHL treatment prevented BMP-induced osteoblast differentiation and mineralization,^152,153 as well as BMP-induced chondrocyte maturation.^149,152,154 Furthermore, Chordin−/−Nog+/− mice exhibit defects restricted to the head associated with increased BMP activity.¹⁵⁵

I-Smad

Inhibitory Smads (I-Smad, Smad6, and 7) inhibits BMPs and TGF-βs signal in multiple ways: preventing R-Smad nuclei translocation, competitively binding with type I receptor to prevent R-Smad phosphorylation, and promoting R-smads or receptor degradation by recruiting E3 ubiquitin ligases Smurf1/2.^156,157 Smad6 preferentially inhibits BMP signaling (Figure 2), while Smad7 interferes with both BMP and TGFβ signaling¹⁵⁷ (Figures 1 and ​and2).2). Smad6 and 7 overexpression blocks BMPs-induced osteoblast and chondrocyte differentiation in vitro,^{107,158–161} and vice versa.¹⁶⁰ Chondrocyte-specific Smad6 transgenic mice show postnatal dwarfism, osteopenia, and delayed chondrocyte hypertrophy due to inhibition of Smad1/5/8 signaling.¹⁶² Besides its role in BMP signaling, Smad6 and Smurf1 also induce Runx2 degradation in an ubiquitin-proteasome-dependent manner, which directly inhibits osteoblast differentiation.¹⁶³ Thus, Smad6 expression is also mediated by BMP–Smad1–Runx2 signaling at transcriptional level and regulates BMP and Runx2 activity in a negative feedback loop.^160,164 Conditional-overexpression of Smad7 in MSC or stage-specific chondrocytes results in defective mesenchymal condensation, chondrocyte proliferation and chondrocyte maturation, respectively.¹⁶⁵

Ubiquitin–proteasomal degradation pathway

Stability of protein transducers within TGFβ and BMP signaling is under control of ubiquitin enzymes and de-ubiquitin enzymes (Figures 1 and ​and2).2). Smurf2 is an E3 ubiquitin ligase that targets TGFβ receptor and Smad2/3 for ubiquitin–proteasomal degradation.^166,167 Ectopic overexpression of Smurf2 in condensing chondrogenic mesenchyme of chicken wing bud accelerates chondrocyte maturation and ossification.¹⁶⁶ Smurf1 is also an E3 ubiquitin ligase with a wide range of a targets, including BMP type I receptors, Smad1/5, Runx2, and MKK2.^{162,163,167,168} Smad6/Smurf1 double transgenic pups exhibit delayed endochondral bone formation that is more severe than Smad6 transgenic pups.¹⁶² Smurf1-deficient mice were born normal but exhibited a temporal increase of bone mass as they aged, due to accumulation of phosphorylated MKK2 and activation of the downstream JNK signaling cascade.¹⁶⁷ Arkadia, an E3 ubiquitin ligase that induces ubiquitylation and proteasome-dependent degradation of TGF-β and BMP suppressors, such as Smad6, Smad7, and c-Ski/SnoN, promotes osteoblast mineralization and differentiation induced by BMPs.¹⁶⁹ SUMO (small ubiquitin-related modifier) and Ubc9 (ubiquitin conjugating enzyme 9) target Smad4 for degradation and inhibit osteoblastic differentiation induced by BMP2.^170,171 Furthermore, deubiquitylating enzyme USP15 mediates K48-linked deubiquitylation of ALK3, to promote ALK3-smad1 signal and BMP-induced osteoblast differentiation.¹⁷²

Transcriptional repressors

A group of co-repressors controls transcriptional activity of the R-Smad/co-Smad complex (Figure 2). Ski/SnoN interacts with R-Smad proteins to repress their activity. c-Ski/SnoN, evoked by TGF-βsuppresses the BMP signaling and hypertrophic chondrocyte maturation. SnoN is highly expressed in articular cartilage and may involve in TGFβ-mediated cartilage homeostasis. Tob as a co-repressor is another negative regulator of BMP/Smad signaling in osteoblasts.¹⁷³ BMP2-signal is elevated in the absence of Tob and repressed by overproduction of Tob.^174,175 Mice carrying a targeted deletion of the Tob gene had a greater bone mass resulting from increased numbers of osteoblasts and were protected from OVX-induced osteoporosis.^174,175

MicroRNAs

Recent researches have raised the importance of microRNA (miRNA) in skeleton homeostasis and in TGF-β and BMP signaling pathway (Figures 1 and ​and2).2). BMP treatment regulates multiple miRNA expression during osteoblastogenesis, and a number of those miRNAs feedback to regulate BMP signaling:^176–179 miR-133 targets Runx2 and Smad5 to inhibit BMP-induced osteogenesis;¹⁷⁶ miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2;^177,178 miR-322 targets Tob and enhances BMP response.¹⁷⁹ In addition, some non-BMP-regulated miRNAs also have regulatory roles in BMP signaling: miR-141 and -200a remarkably modulate the BMP-2-induced pre-osteoblast differentiation through the translational repression of Dlx5;¹⁸⁰ miR-542-3p targets BMP7 and represses BMP7-induced osteoblast differentiation and survival;¹⁸¹ miR-20a promotes osteogenic differentiation through upregulation of BMP/Runx2 signaling by targeting PPARγ, Bambi, and Crim1;¹⁸² miR-140 targets a mild inhibitor of BMP Dnpep, and loss of miR-140 in mice causes growth defects of endochondral bones and craniofacial deformities.¹⁸³ Furthermore, several miRNAs are reported to regulate chondrocyte differentiation by targeting TGF-β and BMP signaling. Profiling identified expression of several miRNAs that changed significantly in human OA chondrocyte compared with normal cells targets Smad-signaling, including miR-20b, miR-146a, and miR-345.^184–186 miR-146a, a miRNA increased in OA, targets Smad4 and induces chondrocyte apoptosis and cellular responsiveness to TGF-β.^187,188 miR-199a(*) targets Smad1 and adversely regulates early chondrocyte differentiation.¹⁸⁹

Crosstalk between TGF-β and BMP signaling with Wnt signaling

TGF-β cooperates with Wnt to stimulate both osteoblast and chondrocyte differentiation in a positive regulatory loop. TGF-β upregulates the expression of Wnts (Wnt-2, -4, -5a, -7a, and -10a) as well as Wnt co-receptor LRP5, and suppresses the expression of β-catenin inhibitor Axin1/2, to promote Wnt/β-catenin signaling.^194,195 On the other hand, Wnt signaling induces Runx2-mediated TGFβRI expression in a β-catenin-independent way to promote TGF-β signaling.¹⁹⁶ Bosch et al.²⁰ showed that canonical Wnt signaling skews TGF-β signaling towards signaling via ALK1-Smad1/5/8 to promote chondrocyte hypertrophy.

BMPs have dual functions in regulating Wnt signaling. BMP induces Dkk1 and Sost expression through MAPK and Smad signaling, respectively, via BMPRIA to inhibit Wnt signaling in osteoblast, and negatively regulated bone mass.^96,98 Smad4 competitively binds β-catenin and prevents its binding with Tcf/Lef transcription complex.¹²³ Thus, ablation of Smad4 either in the crest neuron stem cell or in osteoblast results in upregulation of canonical Wnt signaling so as to promote bone formation.^60,123 BMP2-induced formation of Smad1-Dvl1 complex also restricts β-catenin and inhibits its activity.¹⁹⁷ On the other hand, BMPs stimulate osteoblast and chondrocyte differentiation synergistically with Wnt signaling. BMP-2 promotes canonical Wnt signaling by inducing Wnt3a, Wnt1, Lrp5 expression and inhibiting the expression of β-TrCP, the F-box E3 ligase was found responsible for β-catenin degradation in osteoblasts.^198,199 Ablation of Smad4 in the pre-osteoblasts depletes LRP5 expression and impairs both BMP and Wnt signaling.¹²³ BMP-2-induced LRP-5 expression also contributes to chondrocyte hypertrophy and osteoarthritis progress.²⁰⁰ Osteoblastogenesis is maximized in the presence of both BMP and Wnt signaling.^198,201,202 Smad and TCF/LEF/β-catenin cooperatively complex with each other on the promoter to achieve the highest expression of osteoblast genes (Dlx5, Msx2, and Runx2).²⁰² Moreover, activation of Wnt signaling by Wnt3a or overexpression of β-catenin/TCF4 promotes BMP signaling by stimulating BMP2 transcription.²⁰³

Crosstalk between TGF-β and BMP signaling with FGF signaling

FGF and BMP signal synergistically promote osteoblast differentiation. BMP signaling is essential for FGF-induced osteoblast differentiation,²⁰⁴ and the osteogenic effect of BMP2 is also repressed in the absence of FGF2.²⁰⁵ FGF2 is responsible for BMP-induced nuclei translocation and accumulation of Runx2 and P-Smad1/5/8.²⁰⁶ FGF2, FGF9, and FGF18 induce while DN-FGFR reduces BMP2 expression.^204,207,208 Disruption of the FGF2 in mice impairs the expression of BMP2 and reduces bone mass.²⁰⁵ However, FGF2 has a critical role in osteoblast proliferation but inhibits mineralization while BMP2 is instrumental in stimulating mineralization.^209,210 Thus, besides cooperative functions, FGF2 and BMPs have different roles at different stages of osteoblast differentiation.

Antagonistic BMP and FGF pathways control the differentiation and proliferation rate of chondrocytes in the growth plate.^76,110 Although BMP signal promotes chondrogenesis, FGF signal is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia in humans.²¹¹ BMP signaling is required to inhibit activation of FGF signaling (for example, STAT and ERK1/2 activation) at least in part by inhibiting the expression of FGFR1.¹¹⁰ FGF signals are downregulated by ectopic BMP expression or Chordin/Noggin deficiency, and upregulated by CHL1 ectopic expression.^153,212 Chondrocyte-specific activation of Fgfr3 in mice induced premature synchondrosis closure and enhanced osteoblast differentiation around synchondrosis associated with promoted BMP signaling, due to increased BMP ligand and decreased BMP antagonist expression.²¹¹ Chondrocyte-specific deletion of BMPRIA rescued the bone overgrowth phenotype observed in Fgfr3-deficient mice by reducing chondrocyte differentiation.¹⁶⁸

Crosstalk between TGF-β and BMP signaling with Hedgehog signaling

BMPs and Hedgehog signaling cooperatively regulate osteoblastogenesis in long bones.^213,214 IHH is required for BMP-induced osteogenesis in vitro.²¹³ Hedgehog-Gli activators direct osteo-chondrogenic function of BMPs toward osteogenesis in the perichondrium.²¹⁵ SHH-Gli2 stimulates BMP-2 expression at transcription level to enhance osteoblast differentiation.^216,217 BMP2 also induces expression of IHH and downregulates expression of patched 1 (PTC1), a negative regulator of Hedgehog signaling, during osteoblast differentiation in a positive feedback loop.²¹³

During growth plate development, IHH-PTHrP regulatory loop keeps chondrocyte in the proliferation pool and favors elongation of long bones. Previous studies have shown that BMP signaling upregulates IHH–PTHrP signaling in vivo, and maintains a normal chondrocyte proliferation rate in parallel with IHH.^218,219 Expression of IHH, PTC1, and PTHrP receptor (PPR) were decreased in Smad4-deficient growth plates, but were enhanced in CA-ALK2-, CA-BMPRIA-, or CA-BMPRIB-overexpressed chick limb bud.^108,120 In cultured mouse limb explants, BMP2 application induces and Noggin antagonizes IHH expression.²¹⁸ BMP6 also promotes IHH expression to favor chondrocyte hypertrophy.²²⁰ Furthermore, chondrocyte-specific Smad1/5 CKO mice develop chondrodysplasia and exhibit abnormal growth at the end of bones, probably owing to an imbalance in the crosstalk between the BMP, FGF, and IHH/PTHrP pathways.²¹³ On the other hand, IHH also promotes chondrocyte hypertrophy independent of PTHrP via integrating with BMP and Wnt signaling.²²¹ Chondrocyte-specific deletion of PTC1 upregulated BMP expression and BMP–Smad signaling.²²² IHH deficiency downregulates BMP expression and reduces cranial bone size and all markers.²¹⁴

In axial skeleton development, sequential SHH and BMP signals are required for specification of a chondrogenic fate in presomitic tissue.²²³ In this process, SHH promotes BMP expression, and initiates an Nkx3.2/Sox9 autoregulatory loop that is maintained by BMP signals to induce chondrogenesis.²²³ On the other hand, BMP activity negatively regulates SHH transcription and a BMP-SHH negative-feedback loop serves to confine SHH expression during limb development.^153,212 SHH signal was impaired in the defected craniobone in the absence of Chordin and Noggin, the two BMP cognate binding partners.¹⁵⁵

Crosstalk between TGF-β and BMP signaling with Notch signaling

Notch activation favors BMP-induced osteoblast differentiation.^224,225 Notch inhibition represses expression of BMP target genes.²²⁶ BMP-2 and TGF-β regulates expression of signaling proteins in Notch pathway (for example, Lfng, Hey1, and Hes1).²²⁷ Conditional deletion of Jag1, the Notch ligand in CNC, revealed altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification, associated with dysregulation of BMP receptor expression in the Maxillary mesenchymal stem cell.

Fibrodysplasia ossificans progressiva (FOP; OMIM 135100)

FOP is a rare autosomal dominant disease characterized by progressive ectopic ossification of connective tissues and skeletal muscles. FOP is caused by activating mutations in BMP type I receptor Acvr1. Among them, R206H is the first characterized and most prevalent mutation, while other site mutations were also reported and contribute to the clinical variability and diverse severity of the disease (for example, R258S, L196P, and G328E).^{91,92,231–233}

Brachydactyly type A2 (BDA2; OMIM 112600)

BDA2 is characterized by hypoplasia of the second middle phalanx of the index finger and sometimes the little finger.²³⁴ Inactivating mutations of Bmpr1b gene (for example, I200K, R486K, and R486Q)^235,236 or GDF5 gene (for example, L441P and R380Q)^237,238 were reported in BDA2 patients. Dathe et al.²³⁹ reported another genetic cause of BDA2: duplication of a regulatory element downstream of BMP2 repressed BMP2 expression. GDF5 mutations were also found to be the cause of a number of cartilage disorders, including symphalangsism,²³⁸ chondrodysplasia,²⁴⁰ and osteoarthritis.¹⁶

Myhre syndrome (MIM 139210)

Inactivated SMAD4 mutation causes Myhre syndrome, which is a developmental disorder characterized by short stature, short hands and feet, facial dysmorphism, muscular hypertrophy, deafness, and cognitive delay.^117,118 Myhre syndrome patients carry an I500 mutation at Smad4 that results in a stabilized but unfunctional mutant.^118,241

Noggin-mutation-related diseases

As the major BMP antagonist, mutation of Noggin in human causes multiple bone disorders. Missense mutations in BMP antagonist Noggin cause tyrosine kinase-like orphan receptor 2 (ROR2)-negative brachydactyly type B (BDB2; OMIM611377), which is characterized by hypoplasia/aplasia of distal phalanges in combination with distal symphalangism and fusion of carpal/tarsal bones²⁴². Three missense mutations of Noggin were reported to cause Tarsal–carpal coalition syndrome (TCC; OMIM 186570), an autosomal dominant disorder characterized by short first metacarpals causing brachydactyly as well as fusion of the carpals, tarsals, phalanges, and humeroradial.²⁴³ Truncating mutation in the NOG gene in two families caused autosomal dominant stapes ankylosis with broad thumbs and toes, hyperopia, and skeletal anomalies (OMIM 184460).²⁴⁴ Heterozygous noggin mutations were reported to cause segregating proximal symphalangism (SYM1; OMIM 185800), which is characterized by ankylosis of the proximal interphalangeal joints, carpal and tarsal bone fusion or segregating multiple synostoses syndrome (SYNS1; OMIM 186500), which is characterized by multiple joint fusions.¹³⁸

We thank Dr Joel Jules for his extensive reading and discussion of our manuscript. We apologize to the many researchers whose work could not be cited due to space limitations. Work in our laboratory is supported by grants by NIH grant AR-044741 (Y-PL) and R01DE023813 (Y-PL).