Background/Purpose:

Enhanced cold sensitivity is an early and consistent phenomenon in scleroderma (SSc). We previously demonstrated increased expression of the transient receptor potential melastatine 8 (TRPM8) in SSc microvascular endothelial cells. TRPM8 protein is a cold-and menthol-sensing calcium (Ca²⁺) ion channel. To date, TRPM8 expression, function and intracellular signaling have not been characterized in human dermal fibroblasts (FB). In this study we evaluated TRPM8 expression in normal and SSc FB and in skin biopsies. We also investigated the effects of TRPM8 activation on selected FB fibrotic gene expression, intracellular calcium concentration ([Ca²⁺] _i), intracellular reactive oxygen species (ROS) generation and SMAD3 phosphorylation.

Methods:

FB were isolated from involved SSc skin and matched control subjects. The expression of TRPM8 was determined by RT-PCR, immunocytochemistry (ICC) and western blot (WB) analysis. TRPM8 activation in FB was triggered by the TRPM8 agonist menthol (MT) or by exposure of cells to cold (18C°). The [Ca²⁺]_i was determined by Ca²⁺ calcium imaging system using Ca²⁺ indicator dye Fura 2 or Fura4. The mRNA and protein expression levels of COL1, aSMA, FN, CTGF and phosphoSMAD3 were determined by q PCR and WB. The production of ROS was detected by fluorescent indicator dye dihydroethidium (DHE). SMAD3 binding to CTGF promoter region was detected by chromatin immunoprecipitation assay (ChIP).

Results:

TRPM8 gene and protein expression in dermal FB was determined by RT-PCR, WB and ICC. TRPM8 mRNA and protein expression were significantly higher in SSc-FB and SSc-skin biopsies. Calcium image studies show that MT increased [Ca²⁺] _i and this response was blocked by capsazepine (CapZ), an antagonist of TRPM8. The activation of the TRPM8 by MT or cold exposure resulted in increased expression of COL1, aSMA, and FN. SSc-FB were more sensitive to MT or cold than normal FB. These effects were blocked by the addition of CapZ or TRPM8 siRNA. Furthermore,  MT evoked production of intracellular ROS, which could be abolished by Ca²⁺ free solution or CapZ. The increased expression of COL1, FN, aSMA and CTGF by MT were inhibited by BAPTA-AM (intracellular calcium chelator) or by antioxidants (a mixture of superoxide dismutase, catalase and N-acetycysteine amide). Additionally, MT induced intracellular SMAD3 phosphorylation and facilitated nuclear accumulation of SMAD3. Finally, Chip assay confirmed that SMAD3 is recruited to the CTGF promoter after MT stimulation in FB. The addition of SB431542 and CTGF antibody partly block the COL1 expression induced by MT.

Conclusion: Functional TRPM8 is expressed in human dermal FB and enhanced expression  was observed in SSc FB and skin. The activation of TRPM8 mediated enhanced expression of the profibrotic genes COL1, aSMA, FN and CTGF via calcium influx, ROS production and SMAD3 transcription activation. SSc-FB were more sensitive to MT or cold exposure than normal FB, implicating potential involvement of TRPM8 in the pathogenesis of scleroderma fibrosis and suggesting that the blockade of TRPM8 may be a novel target for therapeutic intervention in SSc.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/cold-receptor-expression-and-function-in-human-dermal-fibroblast-possible-role-in-the-pathogenesis-of-scleroderma-fibrosis/