Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate

Abstract

TGFβs act through canonical and non-canonical pathways, and canonical signals are transduced via Smad2 and Smad3. However, the contribution of canonical vs. non-canonical pathways in cartilage is unknown because the role of Smad2 in chondrogenesis has not been investigated in vivo. Therefore, we analyzed mice in which Smad2 is deleted in cartilage (Smad2CKO), global Smad3-/- mutants, and crosses of these strains. Growth plates at birth from all mutant strains exhibited expanded columnar and hypertrophic zones, linked to increased proliferation in resting chondrocytes. Defects were more severe in Smad2CKO and Smad2CKO;Smad3-/- (Smad2/3) mutant mice than in Smad3-/- mice, demonstrating that Smad2 plays a role in chondrogenesis. Increased levels of Ihh RNA, a key regulator of chondrocyte proliferation and differentiation, were seen in prehypertrophic chondrocytes in the three mutant strains at birth. In accordance, TGFβ treatment decreased Ihh RNA levels in primary chondrocytes from control (Smad2fx/fx) mice, but inhibition was impaired in cells from mutants. Consistent with the skeletal phenotype, the impact on TGFβ-mediated inhibition of Ihh RNA expression was more severe in Smad2CKO than in Smad3-/- cells. Putative Smad2/3 binding elements (SBEs) were identified in the proximal Ihh promoter. Mutagenesis demonstrated a role for three of them. ChIP analysis suggested that Smad2 and Smad3 have different affinities for these SBEs, and that the repressors SnoN and Ski were differentially recruited by Smad2 and Smad3, respectively. Furthermore, nuclear localization of the repressor Hdac4 was decreased in growth plates of Smad2CKO and double mutant mice. TGFβ induced association of Hdac4 with Smad2, but not with Smad3, on the Ihh promoter. Overall, these studies revealed that Smad2 plays an essential role in the development of the growth plate, that both Smads 2 and 3 inhibit Ihh expression in the neonatal growth plate, and suggested they accomplish this by binding to distinct SBEs, mediating assembly of distinct repressive complexes.

miR‑486‑5p is upregulated in osteoarthritis and inhibits chondrocyte proliferation and migration by suppressing SMAD2

“…A previous study indicated that TGF-β signaling contributed to the development of OA through the activin receptor-like kinase (ALK) 5/SMAD2/SMAD3 pathway or the ALK1/SMAD2/SMAD5 pathway (24). SMAD2 appeared to exhibit a stronger effect on chondrogenesis compared with SMAD3 in vivo (25). In addition, a lower level of SMAD2 protein expression was detected in patients with OA compared with in patients without OA (13).…”

The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-β1 concentrations during primary fibroblast proliferation

“…In addition, previous studies have reported that low TGF-β1 concentrations promote the proliferation of muscle, bone (or chondrocytes) and endothelial cells [4,17]. In addition, c-Ski can promote muscle proliferation [18] and participates in endothelial, chondrocyte proliferation [19,20]; c-Ski knockout mice and c-Ski-deficient individuals have defective bone and muscle development [21]. Therefore, these results indicate that proliferation of these cells induced by low TGF-β1 concentrations may also be mediated through the induction of c-Ski.…”

Exosomes produced from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor show improved osteochondral regeneration activity

“…Our preliminary data indicate that 3D-Exos might exert its effects through the TGF-β1-dependent Smad2/3 signaling pathway. Several studies have demonstrated that TGF-βs are potent inducer of chondrogenesis (Wang et al 2016;Fig. 2 High-yield exosomes production from hollow-fiber bioreactor.…”