Background/Purpose: SOX9, a high mobility group transcription factor is a master regulator of chondrogenesis and plays a crucial role in the regulation of chondrocyte gene expression. However, recent studies on hepatic fibrosis and segmental glomerulosclerosis have suggested that SOX9 may participate in tissue fibrosis. Furthermore, we previously demonstrated that human normal dermal fibroblasts contained high levels of SOX9 phosphorylated at serine residue 181 and that TGFβ1 stimulates Ser181 SOX9 phosphorylation mediated by RhoA kinase and to a lesser extent by PI3K. We explored here the role of Ser181 phosphoSOX9 in the fibrotic process of Systemic Sclerosis (SSc) employing cultured SSc dermal fibroblasts treated in vitrowith specific inhibitors of the kinases that may be responsible for Ser181 SOX9 phosphorylation and examined the effect of these inhibitors on the increased expression of profibrotic genes and exaggerated production of extracellular matrix proteins characteristics of SSc fibroblasts.

Methods: Dermal fibroblasts obtained from normal skin and from clinically affected forearm skin from patients with diffuse SSc of recent onset were studied. Ser181 phosphoSOX9 levels were assessed by Western blot analysis of cell lysates of confluent dermal fibroblast cultures employing a phospho-specific antibody that recognizes a SOX9 epitope containing a phosphorylated Ser 181 residue. Gene expression analyses were performed employing real time PCR. Collagen production was assessed by Western blots of fibroblast culture media. The effects of kinase inhibitor treatment on Ser181 phosphoSOX9 were assessed in confluent cultures in the presence or absence of TGF-ß1 (10ng/mL) for 24h. The potential kinases involved were identified employing Kinexus phosphoproteome databases. The intracellular kinases PIM1, PIM2 and PKCδ were identified as being responsible for Ser181 SOX9 phosphorylation. The role of these kinases was examined by inhibition with specific small molecule kinase inhibitors. For PKCδ inhibition studies two novel specific inhibitors developed by CompleGen (CG1037 and CG1056) were employed.

Results: Previous results showed that dermal fibroblasts from SSc patients displayed marked elevation of Ser181 phosphoSOX9 levels in comparison with normal fibroblasts. Here, we show that TGF-ß caused potent stimulation of Ser181 SOX9 phosphorylation which was abrogated at nM concentrations by the two small molecule inhibitors targeting PKCδ as well as the PIM1 and PIM2 specific kinase inhibitors. The kinase inhibitors did not cause morphological changes or detectable cytotoxicity at the concentrations employed. The levels of type I collagen production were reduced in parallel with the changes in Ser181 phosphoSOX9 levels.

Conclusion: The results indicate that Ser181 phosphoSOX9 participates in the molecular mechanisms responsible for the exaggerated fibrotic process in SSc and demonstrate that PKCδ and PIM1/2 kinases are responsible for Ser181 SOX9 phosphorylation. Thus, the kinases involved in Ser181 SOX9 phosphorylation provide novel therapeutic targets for SSc and other fibrotic disorders.

 

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-sox9-phosphorylation-abrogates-the-increased-expression-of-profibrotic-genes-in-systemic-sclerosis-dermal-fibroblasts/