SMAD4 contributes to chondrocyte and osteocyte development

Abstract Different cellular and molecular mechanisms contribute to chondrocyte and osteocyte development. Although vital roles of the mothers against decapentaplegic homolog 4 (also called ‘SMAD4’) have been discussed in different cancers and stem cell‐related studies, there are a few reviews summarizing the roles of this protein in the skeletal development and bone homeostasis. In order to fill this gap, we discuss the critical roles of SMAD4 in the skeletal development. To this end, we review the different signalling pathways and also how SMAD4 defines stem cell features. We also elaborate how the epigenetic factors—ie DNA methylation, histone modifications and noncoding RNAs—make a contribution to the chondrocyte and osteocyte development. To better grasp the important roles of SMAD4 in the cartilage and bone development, we also review the genotype‐phenotype correlation in animal models. This review helps us to understand the importance of the SMAD4 in the chondrocyte and bone development and the potential applications for therapeutic goals.


| INTRODUC TI ON
The formation of skeletal elements often follows a basic path that involves different chondrogenic and osteogenic programmes. 1 Among these, a significant number of signalling pathways, eg SMAD family, play roles. The term 'SMAD' was coined from a combination of a gene name from Caenorhabditis elegans SMA ("small" worm phenotype) and MAD family ("mothers against decapentaplegic") of genes in Drosophila melanogaster. 2 By considering their functions, eight members of SMAD proteins are categorized into three main classes including (i) receptor-regulated or regulatory SMADs (also known as R-SMADs), ie SMADs 1, 2, 3, 5 and 8, (ii) common SMAD or co-SMAD that only involves SMAD4 and (iii) inhibitory SMADs (I-SMADs) that contain SMAD6 and SMAD7 (reviewed in Ref. [3]) ( Figure 1). SMADs play the fundamental roles in cell signalling during the skeletal development (from chondrocyte precursors to mature osteocytes). 3 However, the underlying molecular mechanisms whereby the SMADs (especially SMAD4) exert their functions in these procedures are yet unclear. SMAD4 was first investigated in the context of transforming growth factorβ (TGFβ) family signal transduction. 4 In general, by the time TGFβ upstream signals stimulate cell signalling, SMAD4 interacts with R-SMADs (ie SMAD2/3) and subsequently forms an oligomer complex that modulates the expression of target genes. 4 By interacting with SMAD1, 2, 3 and 5, SMAD4 is actively involved in the intracellular signalling pathways of all three types of TGF ligands. 5 Aberrant expression of SMAD4 affects the normal TGFβ signalling and leads to the uncontrolled cell growth and tumour induction in different tissues. [5][6][7][8][9] This, therefore, shows the paramount importance of SMAD4 in cell growth and development.
SMAD family has similar structures in general. They are composed of two important conserved domains including N-terminal Mad homology domain-1 (MH1 domain) and the C-terminal Mad homology domain 2 (MH2 domain). 10 While DNA-binding activity is often attributed to the MH1 domain, the MH2 domain fulfills some transcriptional activities. 11 These two domains are connected each other by a linker region (a regulatory region in which the different phosphorylation signatures are located) that in turn changes the SMAD4 functions 12 (Figure 1). R-SMADs have a short conserved pattern of two serines separated by another amino acid (Ser-X-Ser). After phosphorylation, 13 this sequence activates R-SMADs, but this section is absent in SMAD4 (Figure 1).
During the development, SMADs contribute to primitive streak formation, 14 neural crest migration, 15 gastrulation, 16 left-right asymmetry, 17 self-renewal of hematopoietic stem cells 18,19 and morphogenesis of different tissues. 20,21 These processes cover some aspects of the chondrocyte and osteocyte development. Axiomatically, the SMAD family-especially SMAD4-may play the important roles in the skeletal development and tissue homeostasis. In order to show how SMAD4 plays a role during the cartilage-to-bone development, herein, we discuss the molecular mechanisms mediated by this protein. Furthermore, we put forth some information about the SMAD4 epigenetic regulations (eg noncoding RNAs, DNA methylation and histone modifications) during the skeletal development. We also discuss how the genotype-phenotype correlations of SMAD4 frame our understanding about the complexities of concepts of the skeletal development and also discuss the possible therapeutic applications using this protein.

| S MAD 4 MODUL ATE S S TEM CELL FE ATU R E S
Migratory neural crest cells follow different pathways to differentiate into neurons, chondrocytes, bones and mesodermal cells. 22 Therefore, every disrupted signalling pathway may change these cell fates. As the most important precursor for cartilage and bone cells, The structure of some important members of the SMAD family. The comparison between the three main classes of SMADs including R-SMADs, co-SMAD and I-SMADs is depicted. The MH1 domain is shown by red and is absent in the I-SMADs including SMAD6/7. This section is linked to the MH2 region (Purple) by a linker region that can be subjected to phosphorylation. This state can change the functions of putative SMADs. SMAD4 embraces nucleus export signal (NES) in its linker region. Additionally, SMAD2, SMAD3 and SMAD4 contain a nucleus localization signal (NLS) in their MH1 domain. C-terminal SS-X-S motifs are present in R-SMADs. The figure is redrawn from Ref. [10,140] to promote their differentiation towards chondrogenesis. 28 In sum, SMAD4 is vital for the commitment and the proper differentiation for the chondrocyte and osteocyte lineage formation.
SMAD4 plays the dual roles regarding 'self-renewal.' The SMAD4 inhibition affects neither human embryonic stem cell selfrenewal nor the neuroectoderm formation, 14 although this protein may play roles in the self-renewal of hematopoietic stem cells. 18,19 SMAD4 may exert its different roles in self-renewal of cells in a celland tissue-specific manner.
SMAD4 also functions in 'stem cell migration' that is undertaken in two different levels: in intracellular and in the cell population. In the former, TGF-β1 not only promotes the formation of gap junctions in chondrocytes via SMAD3/4 signalling pathways but also increases the cartilage precursor cell differentiation and chondrocyte proliferation, migration and metabolism. 29 In cell population levels, the SMAD4/ TGFβ pathways promote cell migration, adhesion and cytoskeletal organization in different cells. 30 In fact, these pathways involved in cell polarity are highly conserved whereby cells regulate the cytoskeletal organization above and beyond the subcellular organelle localization to facilitate cell proliferation and migration. SMAD4 regulates cell polarity in chondrocytes 31 that is a process changing theshape, size, migration and orientation of the chondrocytes.
SMAD4 also plays in 'stem cell maintenance'. The targeted ablation of SMAD4 in the epidermis increases the β-catenin nuclear localization and c-Myc activation that can deplete follicle stem cells.
These suggest a critical role of SMAD4 in the normal maintenance of follicle stem cells. 32 In osteoblasts, SMAD4 regulates hematopoietic stem cell fate and maintenance in a stage-dependent manner. 33 There is a snippet of information about how SMAD4 plays in the stem cell differentiation, maintenance and self-renewal in bone biology; however, many aspects still need to be cleared in future studies by considering the chondrocyte and osteocyte as the target cells.

| S MAD 4 FUN C TI ON S IN ANIMAL S KELE TAL DE VELOPMENT
Knockdown of SMAD4 in mice causes early embryonic death, 34,35 ie SMAD4 / mouse die before E7.5 mainly due to extensive gastrulation defects 34 (Figure 2A). To assess postnatal complications, tissuespecific SMAD4 knockout (KO) mice have been generated. The SMAD4 dysregulation is correlated with different embryonic developmental disorders, 36 impairment in the skeletal muscle differentiation and regeneration, 37 deficiency in stem cell pluripotency 38 and impaired nervous system development. 39 Herein, we only discuss those complications that are related to the skeletal development.  Figure 2G).
The expression of Col10a1, Panx3 and RUNX2 was decreased in SMAD4 −/− mice. 44 In animal models, SMAD4-inactivating mutations cause dwarfism and spontaneous fractures ( Figure 2I). SMAD4 controls the maturation of skeletal collagen and osteoblast survival. This protein is also necessary for matrix-forming responses. Although affected bones in SMAD4 Δ/Δ mice show fully differentiated osteoblast markers, they did not have multiple collagen-processing enzymes, particularly lysyl oxidase that is regulated by SMAD4 and RUNX2.
In addition to impaired chondrocytes and osteocytes, the SMAD4 depletion impresses the scleroaxis and coordinated tendon elongation, eg SMAD4ScxCre mice develop a joint contracture that is stochastic in the direction and is exacerbated with age 45 ( Figure 2J-L). This can substantiate the vital roles of SMAD4 in the initiation and fixation of the chondrocyte and bone development.
The SMAD4 morphant zebrafish manifests severely impaired growth and notochord defects in comparison with SMAD2/3a/3b morphants. These severe phenotypes were imputed to the pleiotropic permissive functions of SMAD4. 46 This study also showed that SMAD4 morphant caused a more severe phenotype in the spinal cord (compared with other SMAD genes), verifying its important roles in neurogenesis, chondrogenesis and osteogenesis F I G U R E 2 SMAD4 mutations cause some important manifestations in animal models. (A) The entire deletion of SMAD4 (SMAD4 Δ/Δ ) causes failure in gastrulation in putative mice. The wild-type (WT) and the mutated models of mouse embryos at E6.5 are shown. The mutation changes the epiblast (epi) epithelium and visceral endoderm (ve) section in affected mouse models. The figure is from Ref. [141]. (B) Bright-field images of WT and SMAD4 Δ/ΔM embryos at E14.5 reveal the short paddle-like limb morphology in affected individuals. This morphology is extendable to both forelimb and hindlimb. The figure is from Ref. [28]. (C) The Skeletal phenotype of SMAD4 F/F ; Osx1-Cre mice confirms the dwarfism and impaired extended osteogenesis. The figure is from Ref. [142]. (D) Cyp26b1 marks the phalanx-forming regions of all developing digit primordia in WT, while almost no differential expression is detectable in SMAD4 Δ/Δ forelimb buds at E12.25. (E) The similar pattern was detected regarding the SOX9 expression in limb region at E12.25. The figures are from Ref. [28]. (F) The impaired and short skeletal development of the SMAD4 mutant mouse model. (G) Skeleton staining affirms such a hypothesis. In this figure, arrows, arrowheads and un-notched arrows delineate humerus, radius and ulna respectively. The figure is from Ref. [44]. (H) The collagen type II distribution (green fluorescence) at E16.5 is shown in WT and mutant mouse models. In WT, collagen type II shows all developing limb skeletal elements, while in SMAD4 Δ/Δ forelimb buds, a thin signal of collagen type II antibodies was detected (arrowheads). No boundary elements for fingers or other advanced structures were detected in the mutant model. (I) The three-dimensional mouse computerized tomography scan showed the skeletal structures of 4-week-old mouse models and highlighted the underdeveloped and hypomineralized sections as well as severe hypomineralization of the craniofacial and axial skeleton in a mutant mouse model. The figure is from Ref. [45]. (J) SMAD4 mutant mouse displays forelimb abduction (black arrowhead) in comparison with the WT littermates. The figure is from Ref. [45]. (K) GFP-labelled tendons in SMAD4 ScxCre mutant showed thinner tendons than control at P5. This figure is from Ref. [45]. (L) SMAD4 ScxCre limb skeletons show some abnormality in chondrogenesis and osteogenesis in addition to the tendons. The figure is from Ref. [45]. (M, N) The SMAD4 morphants manifest a shortened body due to the BMP inhibition, as the most severe form of manifestation. The SMAD4 mutants showed anterior truncation along with the crooked shortened body axis and absence of floorplate. The figure is from Ref. [46]. All figures were used with registered permission.

| S MAD REG UL ATE S AP OP TOS IS
The importance of the cell-programmed death or apoptosis in skeletal tissues and its functional relationship with the chondrocyte and osteocyte development has been thoroughly investigated. 48,49 Osteogenic lineage cells (eg osteoblasts and osteocytes) in addition to the osteoclastic cells are influenced by apoptosis. 49,50 There is a controversy regarding the roles of SMAD4 in apoptosis; although some studies attribute the apoptotic roles to this protein, recent investigations ascribe an apoptotic inhibitory function to this protein.
For instance, SMAD4 contributes to follicular atresia by suppressing granulosa cell apoptosis. 51 TGFβ induces the SMAD4-dependent epithelial-to-mesenchymal transition followed by apoptosis in colorectal cancer cells. 52 Regarding the skeletal development, the protective roles of SMAD4 against apoptosis have been identified, 53 eg the depletion of SMAD4 in chondrocytes resulted in a higher rate of apoptosis and ectopic bone collars in perichondrium that disorganizes growth plate cartilage. 41 In other words, the SMAD4-mediated TGFβ signalling pathway suppresses the chondrocyte hypertrophic differentiation and maintains the normal organization of chondrocytes in growth plate. 41 Likewise, osteoblasts overexpressing SMAD4 show increased apoptosis. SMAD4 plays an important function by regulating osteoblast/ osteocyte viability and bone homeostasis. 54 Apoptosis is essential for the differentiation and bone homeostasis, 55 ie osteoblast apoptosis promotes the osteoclastogenesis and bone resorption, which is a vital process for bone homeostasis. Enhancing osteoblast or osteocyte viability sets the stage for protection against osteoporosis, ie SMAD4 is essential for recovery from pathological bone conditions. 54,56 Osteoclast-specific SMAD4-cKO mice exhibited reduced bone mass with increased osteoclast formation. 57 Yang et al. showed that chondrocyte-specific SMAD4-cKO increased apoptosis. 58 The SMAD4 inhibition in osteoarthritis has also been documented, 59 revealing the protective roles of SMAD4 against apoptosis and its pivotal roles in inducing the chondrocyte differentiation and proliferation.

| TGFβ signalling axis
TGFβ needs the SMAD family to function normally, and it stimulates a variety of physiological processes such as tissue repair, cell growth, cell differentiation and cell proliferation. 60,61 TGFβ activates two types of receptors including the activin receptor-like kinase 1 (ALK1) and ALK5. TGFβ stimulates (using ALK1) or inhibits (via ALK5) the migration and proliferation of endothelial cells. 62 Seven TGFβ superfamily type I receptors (are also known as ALKs) have been identified so far. ALK1, 2, 3 and 6 contribute to signalling by activating the SMAD1, 5 or 8. 62 On the other hand,

| Bone morphogenic protein
As important growth factors, BMPs contribute to the chondrocyteto-osteocyte and craniofacial development. BMP ligands bind to type II BMP receptors (BMPRII), subsequently followed by recruiting type I BMP receptors (BMPRI) to form a heterogeneous tetramer. BMPRII phosphorylates itself in addition to the BMPRI in the tetramer. These events give rise to transduce signals into the cytoplasm by activating the SMAD-dependent pathway (as canonical BMP signalling) or the SMAD-independent pathway (as noncanonical BMP signalling). 70 In the former, BMPRI phosphorylates SMAD1, 5 or 8, which forms transactivator with SMAD4 to activate the transcription of downstream target genes (Figure 3). In the SMAD-independent pathway, the signal is transferred through the phosphorylation of p38, Erk or JNK. so can in turn impress the stem cell commitment. 86 Therefore, inhibiting RUNX2 and SMAD4, eg using RNA interference, is a powerful approach to prevent or treat heterotopic ossification. 87

| Notch signalling
Notch signalling is vital for the normal embryonic development, coordinated tissue homeostasis and stem cell maintenance. 88 Notch functions on chondrogenesis using both CSL (CBF1, suppressor of hairless, Lag-1)-dependent and CSL-independent mechanisms. 89,90 Upon activation, Notch signalling controls a balance between the chondrogenic proliferation and differentiation at the early stages of somite compartmentalization and the long bone development. 89 Notch promotes the final differentiation of the osteoblast progenitors but seems to have no obvious effects on mature osteoblasts. 91 It also inhibits the chondrogenic differentiation by suppressing the activity of COL2A1 promoter and the expression of SOX9. 92 Notch regulates cartilage link protein 1 (Crtl1) as a target of SOX9. 93 In osteoblasts, Notch signalling plays a dual role to either suppress or induce the osteoblastic differentiation. Notch signalling has been demonstrated in vivo to inhibit the osteoblastic differentiation by suppressing both early and late differentiation markers as in collagen type 1, RUNX2, alkaline phosphatase and osteocalcin. 94,95 SMAD4 helps to keep the cerebrovascular endothelial cellpericyte interactions stable by modulating the transcription of Ncadherin by affecting its promoter. This mechanism links TGFβ/ SMAD4 and Notch signalling and maintains cerebral vascular integrity. 96 The thrust of these results is that SMADs form a complex

| Noncoding RNAs
Noncoding RNAs take part in the skeletal development. however, this signalling pathway remains to be discovered in the skeletal development. 104 MiR-224 directly regulates SMAD4 and results in the inhibition of osteoblast differentiation. 105 Besides, TGF-β1/SMAD4 signalling affects the osteoclast differentiation by regulating miR-155 expression, and using miR-155 as a therapeutic target for osteoclast-related disorders has shown a lot of promise. 106   functions as a positive regulator of the osteogenic differentiation of human aortic valve interstitial cells. 115 Thirdly, using osteosarcoma cell models, MALAT1 has been identified to promote metastasis and proliferation by inducing miR-144-3p, which in turn targets SMAD4. 116 lncRNA AWPPH stimulates cell proliferation, autophagy and migration, and inhibits apoptosis in bladder cancer by suppressing SMAD4. 117 This lncRNA upregulates RUNX2 and in turn contributes to the development of nontraumatic osteonecrosis of the femoral head. 118 While AWPPH is downregulated in osteoporosis, in normal tissues, it regulates the balance between type I collagen α1 and α2 ratio, 119   Moreover, SMAD4 has been recently employed as a therapeutic target in a variety of diseases such as pancreatic adenocarcinoma cells, 137 colorectal cancer 138 and fibrosis 139 ; however, we do not know whether this protein can be used as a therapeutic agent/ target for cartilage-and bone-related disorders or not. We believe that the future investigations can answer these kinds of questions.
Additionally, there are plenty of reports demonstrating that SMAD4 bestows some features to the putative stem cells, proposing that SMAD4 and other members of this family may potentially be used to reprogram the stem cells. This can in turn provide valuable information about the biological aspects of bone biology, which will pave the way to utilize this for therapeutic purposes.

ACK N OWLED G M ENTS
This work was supported by Tarbiat Modares University.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B L E S TAT E M E N T
The paper is exempt from data sharing.