Function and regulation of transforming growth factor β1 signalling in antler chondrocyte proliferation and differentiation

Abstract Objectives Chondrocyte 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 methods Immunofluorescence, 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. Results TGFβ1, 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. Conclusions TGFβ1 signalling could induce the proliferation and differentiation of antler chondrocytes through Notch‐Shh‐Foxa pathway.

many biological processes, is expressed abundantly in bone and plays an important role in bone physiology and homeostasis. 3,4 Ablation of TGFβ1 led to visibly decreased longitudinal long bone growth along with reduced hypertrophic chondrocyte numbers. 5 Further analysis shows that TGFβ1 may bind to the TGFβ1 type II receptor (TGFΒR2) and activates the type I transmembrane serine/ threonine kinase receptor (TGFΒR1), leading to the phosphorylation of Smad transcription factors, which serve as the principal facilitators of TGFβ signalling. 6,7 Conditional knockout of TGFΒR2 or mutant of Smad3 exon 8 resulted in the degenerative joint disease and progressive osteoarthritis-like phenotype accompanied by the aberrant chondrocyte proliferation and differentiation. [8][9][10][11] Silencing of Smad3 impeded the TGFβ-induced chondrogenic differentiation. 12 However, few studies have reported the physiological function of TGFβ1 signalling in antler chondrocyte proliferation and differentiation.
Notch signalling is critical for cartilage development and endochondral ossification. [13][14][15][16] Blockage of Notch signalling delayed chondrocyte maturation and suppressed chondrocyte proliferation, whereas activation of Notch signalling promoted chondrocyte hypertrophy. [14][15][16] Further studies demonstrated that Sonic hedgehog (Shh) signalling acted downstream of Notch pathway to mediate the proliferation and differentiation of antler chondrocytes. 17 In rat mesenchymal stem cells (MSCs), TGFβ1 could affect the expression of Shh and Gli1, 18 but the relationship among TGFβ1, Notch and Shh signalling in chondrocyte proliferation and differentiation remains unknown. It has been previously reported that Foxa transcription factors are key regulators of chondrocyte differentiation. Chondrocyte-specific knockout of Foxa2 and Foxa3 led to post-natal dwarfism with profound defects in chondrocyte hypertrophy and mineralization in the sternebrae. 19 However, little is known whether Foxa may mediate the effects of TGFβ1 signalling on chondrocyte proliferation and differentiation.

| Tissue collection
Antler tissues were collected from 3-year-old healthy sika deer as previously described. 20 The distal 5 cm of growing tip was removed and sectioned sagittally along the longitudinal axis. A part of the tip was then cut into 4-6 mm pieces, flash frozen in liquid nitrogen and stored at −80°C for immunofluorescence, and the remaining tip was used for isolation of antler chondrocytes.

| Antler chondrocyte treatment
Antler chondrocytes were isolated by enzymatic digestion as previously described 20

| Western blot
Western blot was performed according to an existing research method, 21 and cells were lysed using protein lysate. The entire protein was then transferred onto PVDF membranes. After blocking with 5% non-fat milk, the membranes were probed with Smad3 (1:5000; Abcam) and p-Smad3 (1:5000; Abcam) antibodies, washed with PBS and then incubated with HRP-linked secondary antibodies. Bands were visualized with an enhanced chemiluminescence substrate (Thermo Fischer Scientific). β-actin was used to normalize the protein levels.

| MTS assay
Cell proliferation was analysed using MTS assay (Promega) in accordance with the manufacturer's protocol. Briefly, antler chondrocytes were treated as described above, at which time 20 µL of MTS reagent was added to each well and incubated for 4 hours. The absorbance was measured at 490 nm using a 96-well plate reader.
Each experiment was performed in triplicate.

| Flow cytometry
After antler chondrocytes were synchronized by serum starvation, they were treated as described above. Cells were harvested by trypsinization and centrifugation, washed with PBS and then fixed overnight at 4°C in 70% ethanol. The fixed cells were washed with PBS and stained with 0.5 mL PI/RNase staining buffer (BD Biosciences) for 15 minutes at room temperature. Then, the stained cells were analysed by flow cytometry.

| RNA interference
Small interfering RNA (siRNA) for targeting Shh, Gli1, Gli2, Gli3, Foxa1, Foxa2 and Foxa3 as well as a scrambled siRNA (negative control) were  Table 1. Transfection for siRNA was performed according to Lipofectamine 2000 protocol (Invitrogen). After transfection with the corresponding siRNA, antler chondrocytes were collected for 24 hours in the absence or presence of rTGFβ1 and rShh, respectively.

| Real-time PCR
Total RNA from cultured chondrocytes was extracted and then reverse-transcribed into cDNA. The expression levels of different genes were determined by real-time PCR analysis using the FS Universal SYBR Green Real Master (Roche) as previously described. 20 The results were analysed using LightCycler 96 Software. After analysis using the 2 −ΔΔCt method, data were normalized to GAPDH expression. Primers were designed according to the conserved regions of white-tailed deer, human, cattle and sheep mRNA sequences and listed in Table 2.

| Statistical analysis
All the experiments were independently repeated at least three times.
The significance of difference was analysed by one-way ANOVA or independent samples t test using the SPSS software program (SPSS Inc). The differences were considered significant at P < 0.05.

| TGFβ1, TGFBR1 and TGFBR2 expression in antler cartilage
To examine the expression of TGFβ1, TGFBR1 and TGFBR2 in antler cartilage, immunofluorescence was performed. The results showed that TGFβ1, TGFBR1 and TGFBR2 were highly expressed in antler chondrocytes ( Figure 1A).

| Effects of TGFβ1 signalling on the proliferation and cell cycle of antler chondrocytes
MTS results showed that rTGFβ1 significantly enhanced the proliferative activity of antler chondrocytes, whereas addition of TGFβ1 receptor inhibitor SB431542 impeded this enhancement (Figure 2A).
Similarly, flow cytometry analysis revealed that rTGFβ1 accelerated the progression of cell cycle from G1 to S phase, whereas SB431542 significantly slowed this progression ( Figure 2B- abolished the rTGFβ1-induced stimulation of these genes ( Figure 2D,E).
Western blot analysis showed that exogenous rTGFβ1 enhanced the expression of Smad3 and p-Smad3, but this enhancement was reversed by SB431542 ( Figure 3A

| Effects of TGFβ1 signalling on antler chondrocyte differentiation
To determine the role of TGFβ1 signalling in antler chondrocyte differentiation, we investigated its influence on the expression of type X collagen (Col X), runt-related transcription factor 2 (Runx2) and alkaline phosphatase (Alpl), the well-known markers for hypertrophic chondrocytes. 22,23 The results indicated that Col X, Runx2 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).

| Notch signalling mediates the effects of TGFβ1 on antler chondrocyte proliferation and differentiation
Notch signalling is important for chondrocyte proliferation and differentiation. [14][15][16] Our previous studies demonstrated that The results showed that DAPT effectively decreased the promotion of Col X, Runx2 and Alpl by rTGFβ1 ( Figure 4G).

| Shh signalling mediates the effects of TGFβ1 on antler chondrocyte proliferation and differentiation
In antler chondrocytes, rTGFβ1 raised the expression of Shh, Smo, Gli1, Gli2 and Gli3, while SB431542 and SIS3 attenuated the effects of rTGFβ1 on these genes ( Figure 5A  Gli2 and Gli3 ( Figure 8A,B).

| TGFβ1 signalling regulates antler chondrocyte differentiation through Foxa
It is well known that Foxa transcription factors are critical for chondrocyte differentiation. 19 In antler chondrocytes, rTGFβ1 enhanced the expression of Foxa1, Foxa2 and Foxa3, but these effects were attenuated by SB431542 and SIS3 ( Figure 8C). Furthermore, silencing

| Notch and Shh signalling mediate the regulation of Foxa by TGFβ1 in antler chondrocytes
As described above, TGFβ1 was upstream of Notch and Shh signal-

| D ISCUSS I ON
The It has previously been shown that TGFβ1 plays a role in inducing chondrocyte proliferation and maintaining phenotypic stability. 5,26 Cell proliferation is dependent on four distinct phases of the cell cycle (G0/G1, S, G2 and M), which are regulated by a complex interplay of cyclins and Cdks. 27  In summary, this study reveals that TGFβ1 signalling may induce the proliferation and differentiation of antler chondrocytes through Notch-Shh-Foxa pathway ( Figure 10).

ACK N OWLED G EM ENTS
This work was financially supported by National Natural Science Foundation of China (31672503 and 31873003) and Natural Science Foundation of Jilin Province (20180101247JC).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.