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

Weiguang Wang,Buer Song,Teni Anbarchian,Joshua E Sadik,Anna Shirazyan,Karen M Lyons,Vicki Rosen

doi:10.1371/journal.pgen.1006352

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.

Highlights

The cartilage growth plate is the primary driver of endochondral bone growth
There is strong evidence that dysregulation of the Transforming growth factor βs (TGFβs) pathway increases the risk for osteoarthritis (OA) in humans, but the underlying mechanisms are unknown
We generated mice lacking both Smad2 and Smad3 in cartilage in order to determine the role of canonical TGFβ signaling during embryonic development

Summary

Introduction

The cartilage growth plate is the primary driver of endochondral bone growth. In the growth plate, resting, columnar, prehypertrophic and hypertrophic chondrocytes are arrayed in discrete zones. Upon stimulation by extracellular signals, cells near the bottom of the resting zone transition to columnar chondrocytes, which exhibit a higher rate of proliferation and a flatter morphology. These cells form stacks along the long axis of the developing skeletal element. Once cells escape the zone of influence of Pthrp, they exit the cell cycle, become prehypertrophic, and upregulate Ihh expression, which promotes hypertrophy and matrix mineralization. This feedback loop controls the transition of chondrocytes through each zone of the growth plate

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Conclusion