Background/Purpose: Hedgehog acyltransferase (Hhat) catalyzes the attachment of the fatty acid palmitate onto Sonic Hedgehog (Shh), a modification essential for Shh signaling activity. Palmitoylation of Shh by Hhat is required for multimerization of Shh proteins to large signaling complexes, which is particularly required for exocrine Shh signaling. Hhat has been identified as a potential target for cancer therapy especially in malignancies characterized by Shh overexpression. Hedgehog signaling activates fibroblasts in vitro and in vivo and plays a prominent role in tissue fibrosis in Systemic sclerosis (SSc). The aim of this study was to evaluate the role of Hhat in the pathogenesis of SSc.

Methods: Expression of Hhat in human and murine skin was analyzed by immunohistochemistry and immunofluorescence. The effects of small interfering RNA (siRNA) induced knockdown of Hhat on TGFβ signaling were analyzed in cultured human fibroblasts in vitro and in the mouse models of bleomycin-induced skin fibrosis and DNA topoisomerase I (topo I) induced fibrosis in vivo. The anti-fibrotic effect on skin was assessed by hydroxyproline assay, alpha smooth muscle cells quantification and measuring the dermal thickness.

Results: Increased expression of Hhat was detected by immunohistochemistry in skin biopsies from SSc patients as compared to healthy volunteers, with a particularly strong expression in fibroblasts. The expression of Hhat was induced in fibroblasts in vitro and in vivo in a TGFβ-dependent manner. Stimulation with TGFβ increased the mRNA and protein levels of Hhat in cultured fibroblasts. Moreover, TGF-β receptor I (TBR)-induced dermal fibrosis upregulated Hhat expression, whereas treatment with a specific inhibitor of TβRI prevented the induction of Hhat in bleomycin-induced skin fibrosis. The TGFβ-induced upregulation of Hhat required Smad3 and knockdown of Smad3 prevented the stimulatory effects of TGFβ on Hhat expression. We also demonstrate that the TGFβ-dependent induction of Hhat is sufficient to promote hedgehog signaling with enhanced activity in reporter assays and increased transcription of hedgehog target genes such as Ptch-1 and Ptch-2. Vice versa, inactivation of Hhat ameliorates the stimulatory effects of TGFβ on fibroblasts. Knockdown of Hhat by siRNA reduced collagen release and myofibroblast differentiation in TGFβ-stimulated fibroblasts. Moreover, inactivation of Hhat also ameliorated experimental dermal fibrosis. Knockdown of Hhat by injection of Hhat siRNA /atelocollagen complexes into the skin effectively ameliorated dermal thickening, reduced myofibroblast counts and decreased collagen content in bleomycin-induced fibrosis. Dermal fibrosis induced by challenge of mice with topoisomerase I was also ameliorated by knockdown of Hhat.

Conclusion: We demonstrate that Hhat is regulated in SSc in a TGFβ-dependent manner. Hhat in turn promotes hedgehog signaling, which directly contributes to the stimulatory effects of TGFβ on fibroblasts. Inactivation of Hhat reduces TGFβ-dependent fibroblast activation and ameliorates experimental fibrosis. These data provide first evidence that targeting Hhat may be a novel target for the treatment of fibrosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/inhibition-of-hedgehog-acyltransferase-alleviates-the-profibrotic-effects-of-transforming-growth-factor-%ce%b2-in-systemic-sclerosis/