Abstract
ObjectivesChondrocyte proliferation and differentiation are crucial for endochondral ossification, but their regulatory mechanism remains unclear. The present study aimed to determine the physiological function of TGFβ1 signalling in the proliferation and differentiation of antler chondrocytes and explore its relationship with Notch, Shh signalling and Foxa.Materials and methodsImmunofluorescence, Western blot, MTS assay, flow cytometry, RNA interference and real‐time PCR were used to analyse the function and regulatory mechanisms of TGFβ1 signalling in antler chondrocyte proliferation and differentiation.ResultsTGFβ1, TGFBR1 and TGFBR2 were highly expressed in antler cartilage. TGFβ1 promoted chondrocyte proliferation, increased the proportion of S‐phase cells and induced the expression of hypertrophic chondrocyte markers Col X, Runx2 and Alpl. However, this induction was weakened by TGFβ receptor inhibitor SB431542 and Smad3 inhibitor SIS3. Simultaneously, TGFβ1 activated Notch and Shh signalling whose blockage attenuated the above effects of rTGFβ1, whereas addition of rShh rescued the defects in chondrocyte proliferation and differentiation elicited by SB431542 and SIS3. Further analysis revealed that inhibition of Notch signalling impeded TGFβ1 activation of the Shh pathway. Knockdown of Foxa1, Foxa2 and Foxa3 abrogated the effects of TGFβ1 on chondrocyte differentiation. Notch and Shh signalling mediated the regulation of Foxa transcription factors by TGFβ1.ConclusionsTGFβ1 signalling could induce the proliferation and differentiation of antler chondrocytes through Notch‐Shh‐Foxa pathway.
Highlights
Chondrocyte proliferation and differentiation are two key processes in cartilage ossification.[1]
The results showed that rTGFβ1 increased the expression of Ccnd[1], Ccnd[2], Ccnd[3], FIGURE 4 Notch signalling mediates the effects of TGFβ1 on antler chondrocyte proliferation and differentiation
The results indicated that Col X, Runx[2] and Alpl mRNA levels were increased in a time‐dependent manner after rTGFβ1 treatment, but this increase was abrogated by SB431542 and SIS3 (Figure 3C‐F)
Summary
Chondrocyte proliferation and differentiation are two key processes in cartilage ossification.[1]. Cal function of TGFβ1 signalling in antler chondrocyte proliferation and differentiation. Further studies demonstrated that Sonic hedgehog (Shh) signalling acted downstream of Notch pathway to mediate the prolif‐ eration and differentiation of antler chondrocytes.[17]. In rat mesenchy‐ mal stem cells (MSCs), TGFβ1 could affect the expression of Shh and Gli[1,18] but the relationship among TGFβ1, Notch and Shh signalling in chondrocyte proliferation and differentiation remains unknown. We investigated the function of TGFβ1 signalling in the proliferation and differentiation of antler chondrocytes and explored its relationship with Notch, Shh signalling and Foxa. The results evidenced that TGFβ1 signalling might induce the prolifer‐ ation and differentiation of antler chondrocytes through activating Notch‐Shh‐Foxa pathway
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