Figure 7
Thyroid hormone acts through THRβ1 and HH expression to influence epiphyseal chondrocyte differentiation. (A–D) IHC data confirms a higher IHH expression in the SOC vs SHH (green) in P7 hyt/ + mice (A,C), whereas there is an extreme reduction of IHH expression coupled with an increase in SHH expression in hyt/hyt mice (B,D). (E–H) IHC data indicate unchanged THRα1 levels in both hyt/ + and hyt/hyt animals (E,F). Conversely, THRβ1 expression is markedly reduced in hyt/hyt compared to hyt/ + animals (G,H). (I,J) Ihh mRNA expression of P7 epiphyses decreased 7 fold while Shh expression is increased 2.4 fold in hyt/hyt vs hyt/ + animals (I). P7 epiphyses of hyt/hyt animals showed a 2.2 fold decrease in Thrβ1 but no effect in Thrα1 expression compared to hyt/ +animals (J). (K) Thrβ1 knockdown in cultured CCs increased Shh expression 3.4 fold and reduced Ihh expression 2 fold. (L) Adenoviral OE of Shh in epiphyseal CCs increased Sox9, Col2, and Acan mRNA expression (1.5, 3.5, and 3 fold, respectively). Mmp13 and Rankl expression decrease 1.7 and 2.8 fold, respectively. Ptch1 and Gli1 expression is increased 9 and 13.5 fold, respectively. Gli2 and Gli3 expression remain unchanged. All mRNA is tested against GFP adenovirally treated cells. (M, N) shRNA knockdown of Ihh in ATDC5 CCs causes an increase in Gli1, however, Ptch1 and Gli2 are knocked down (2 fold each) (M) It also reduces Mmp13 and Bsp expression (5 and 3.3 fold, respectively) but increases Col2 expression (2.5 fold) (N). Red Circles denote the SOC initiation region. All RNA data are represented as a mean ± SEM, “A” denotes significance. Large Scale bars are 100 μm and small scale bars are 200 μm.
