Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate.
Smad2 and Smad3 Regulate Chondrocyte Proliferation and Differentiation in the Growth Plate.
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DOI：
10.1371/journal.pgen.1006352
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发表时间：
2016-10
期刊：
影响因子：
4.5
通讯作者：
Lyons KM
中科院分区：
文献类型：
--
作者：
Wang W;Song B;Anbarchian T;Shirazyan A;Sadik JE;Lyons KM
TGFβ通过经典和非经典途径起作用，经典信号通过Smad 2和Smad 3转导。然而，软骨中经典途径与非经典途径的贡献是未知的，因为Smad 2在软骨形成中的作用尚未在体内研究。因此，我们分析了在软骨中Smad 2缺失的小鼠（Smad 2CKO）、全局Smad 3-/-突变体以及这些品系的杂交。生长板在出生时从所有突变株表现出扩大柱状和肥大区，与静息软骨细胞增殖增加。Smad 2CKO和Smad 2CKO; Smad 3-/-（Smad 2/3）突变小鼠的缺陷比Smad 3-/-小鼠更严重，表明Smad 2在软骨形成中起作用。增加水平的Ihh RNA，软骨细胞增殖和分化的关键调节因子，在出生时的三个突变株的prehypertrophic软骨细胞。相应地，TGFβ处理降低了来自对照（Smad 2fx/fx）小鼠的原代软骨细胞中的Ihh RNA水平，但抑制作用在来自突变体的细胞中受损。与骨骼表型一致，对TGFβ介导的Ihh RNA表达抑制的影响在Smad 2CKO中比在Smad 3-/-细胞中更严重。在近端Ihh启动子中鉴定了推定的Smad 2/3结合元件（SBE）。其中三种基因的突变作用。ChIP分析表明，Smad 2和Smad 3对这些SBE具有不同的亲和力，并且阻遏物SnoN和Ski分别由Smad 2和Smad 3差异募集。此外，在Smad 2CKO和双突变小鼠的生长板中，阻遏物Hdac 4的核定位降低。TGFβ诱导Hdac 4与Smad 2在Ihh启动子上结合，但不与Smad 3结合。总的来说，这些研究表明，Smad 2在生长板的发育中起着至关重要的作用，Smad 2和3都抑制新生儿生长板中的Ihh表达，并表明它们通过与不同的SBE结合，介导不同的抑制复合物的组装来实现这一点。软骨生长板调节身体中几乎每个骨骼元素的大小和形状。TGFβs是软骨形成的有效诱导剂，但它们在软骨发育过程中传递其信号的机制知之甚少。同样，有强有力的证据表明TGFβ通路的失调增加了人类骨关节炎（OA）的风险，但其潜在机制尚不清楚。TGFβ通过涉及Smad 2和Smad 3的经典途径以及通过几种非经典途径调节其信号。然而，经典信号与非经典信号在软骨中的作用尚不清楚，因为Smad 2和Smad 3的联合作用尚未确定。我们产生了软骨中缺乏Smad 2和Smad 3的小鼠，以确定胚胎发育期间典型TGFβ信号传导的作用。我们确定Smad 2在生长板中的非肥大软骨细胞中具有比Smad 3更突出的作用，并确定了Smad 2和Smad 3突变体中新生软骨中Ihh RNA水平升高。这些发现可能很重要，因为Ihh是软骨发育过程中软骨增殖和分化的重要调节因子。更一般地说，这些研究确定了Smad 2和Smad 3如何通过不同的机制调节共同的靶基因。
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. The cartilage growth plate regulates the size and shape of nearly every skeletal element in the body. TGFβs are potent inducers of cartilage formation, but the mechanisms by which they transduce their signals in cartilage during development are poorly understood. Similarly, there is strong evidence that dysregulation of the TGFβ pathway increases the risk for osteoarthritis (OA) in humans, but the underlying mechanisms are unknown. TGFβs transduce their signals through a canonical pathway involving Smad2 and Smad3, and through several non-canonical pathways. However, the roles of canonical vs. noncanonical signaling are unknown in cartilage because the combined roles of Smad2 and Smad3 have not been determined. We generated mice lacking both Smad2 and Smad3 in cartilage in order to determine the role of canonical TGFβ signaling during embryonic development. We determined that Smad2 has a more prominent role than Smad3 in non-hypertrophic chondrocytes in the growth plate, and identified elevated levels of Ihh RNA in neonatal cartilage in Smad2 and Smad3 mutants. These findings may be important because Ihh is a vital regulator of cartilage proliferation and differentiation during cartilage development. More generally, the studies identify how Smad2 and Smad3 can regulate a common target gene through distinct mechanisms.
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影响因子：
7.5
作者：
Djouad F;Jackson WM;Bobick BE;Janjanin S;Song Y;Huang GT;Tuan RS
通讯作者：
Tuan RS
影响因子：
64.5
作者：
Mullen AC;Orlando DA;Newman JJ;Lovén J;Kumar RM;Bilodeau S;Reddy J;Guenther MG;DeKoter RP;Young RA
通讯作者：
Young RA
影响因子：
27.4
作者：
Davidson, E. N. Blaney;Vitters, E. L.;van den Berg, W. B.
通讯作者：
van den Berg, W. B.
TGFβ 信号的背景
DOI：
10.1038/nrm3434
发表时间：
2012-10
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
通讯作者：
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影响因子：
10.5
作者：
Dunn, NR;Koonce, CH;Robertson, EJ
通讯作者：
Robertson, EJ