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

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.转化生长因子βs（TGFβs）通过经典通路与非经典通路发挥作用，其中经典信号经Smad2与Smad3进行转导。然而，软骨组织中经典与非经典通路的贡献尚不明确，因为Smad2在软骨发生中的体内作用尚未被研究。因此，我们对在软骨中特异性敲除Smad2的小鼠（Smad2CKO）、全身性Smad3敲除（Smad3-/-）突变小鼠，以及这两种品系的杂交小鼠进行了分析。所有突变品系新生小鼠的生长板均表现出柱状区与肥大区扩张，该现象与静息软骨细胞的增殖增强相关。Smad2CKO小鼠与Smad2CKO;Smad3-/-（即Smad2/3）双突变小鼠的软骨发育缺陷较Smad3-/-小鼠更为严重，这证明Smad2在软骨发生中发挥了作用。在新生期三种突变小鼠的前肥大软骨细胞中，关键的软骨细胞增殖与分化调控因子印度豪猪同源物RNA（Ihh RNA）的表达水平升高。与之相符的是，转化生长因子β（TGFβ）处理可降低对照小鼠（Smad2fx/fx）原代软骨细胞中的Ihh RNA水平，但该抑制效应在突变小鼠的软骨细胞中被削弱。与骨骼表型一致，Smad2CKO细胞中TGFβ介导的Ihh RNA表达抑制效应较Smad3-/-细胞更为显著。在Ihh基因的近端启动子区域鉴定出了潜在的Smad2/3结合元件（SBEs）。诱变实验证实其中3个元件具有功能。染色质免疫沉淀（ChIP）分析显示，Smad2与Smad3对这些SBEs的结合亲和力存在差异，且阻遏蛋白SnoN与Ski分别被Smad2与Smad3差异性招募。进一步研究发现，阻遏蛋白组蛋白去乙酰化酶4（Hdac4）的核定位在Smad2CKO小鼠与双突变小鼠的生长板中出现降低。TGFβ可诱导Hdac4与Smad2在Ihh启动子区域结合，但无法使其与Smad3结合。综上，本研究揭示了Smad2在生长板发育中发挥关键作用，证实Smad2与Smad3均可抑制新生小鼠生长板中的Ihh表达，并表明二者通过结合不同的SBEs、介导组装不同的阻遏复合物来实现该调控功能。