Session Type: Abstract Submissions (ACR)
Background/Purpose: SOX9, a high mobility group (HMG) transcription factor is a master regulator of chondrogenesis and is essential for the maintenance of the chondrocyte-specific phenotype regulating the expression of various chondrocyte-specific gene products including type II collagen and COMP. We recently performed an analysis of the kinome of human dermal fibroblasts and demonstrated that these cells contained high levels of SOX9 phosphorylated at serine residue 181 (Ser181 phosphoSOX9). Recent studies on liver fibrosis and segmental glomerulosclerosis have suggested that SOX9 may participate in tissue fibrosis. Therefore, we explored here the involvement of Ser181 phosphoSOX9 in the fibrotic process of Systemic Sclerosis (SSc) employing cultured SSc dermal fibroblasts in vitro.
Methods: Dermal fibroblasts were obtained from skin samples from normal individuals and from clinically affected and non-affected forearm skin from patients with diffuse SSc of recent onset. 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 by real time PCR employing specific primers. Collagen production was assessed by Western blots of fibroblast culture media. The effects of TGF-ß treatment on Ser181 phosphoSOX9 were assessed in confluent cultures in the presence or absence of TGF-ß1 (10ng/mL) for 24h. The intracellular kinases responsible for SOX9 phosphorylation were examined by inhibition with specific small molecule kinase inhibitors.
Results: Dermal fibroblasts from SSc patients displayed marked elevation of Ser181 phosphoSOX9 levels in comparison with normal fibroblasts. Furthermore, fibroblasts cultured from clinically affected SSc skin had significantly greater levels of Ser 181 phosphoSOX9 than fibroblasts cultured from clinically non-affected skin from the same patients. TGF-ß caused a potent stimulation of SOX9 phosphorylation in normal fibroblasts but only minor stimulation in SSc fibroblasts. The stimulation of SOX9 phosphorylation by TGF-ß was inhibited by small molecule inhibitors targeting PKC-delta and Rho Kinase. Inhibitors of PI3 Kinase and other kinases were not effective. The levels of the Type I collagen production changed in parallel with the changes in Ser181 phosphoSOX9 levels.
Conclusion: The results indicate that Ser181 phosphoSOX9 may participate in the molecular mechanisms responsible for the exaggerated fibrotic process in SSc and suggest that PKC-delta and Rho Kinase, the specific kinases responsible for SOX9 phosphorylation, may provide novel therapeutic targets for SSc and other fibrotic disorders involving Ser181 phosphoSOX9.
S. A. Jimenez,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-levels-of-ser-181-phosphorylated-sox9-in-ssc-dermal-fibroblasts-a-novel-participant-in-the-pathogenesis-of-ssc-fibrotic-process/