Vitamin D receptor regulates TGF-ß signalling in systemic sclerosis.

BACKGROUND: Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily. Its ligand, 1,25-(OH)2D, is a metabolically active hormone derived from vitamin D3. The levels of vitamin D3 are decreased in patients with systemic sclerosis (SSc). Here, we aimed to analyse the role of VDR signalling in fibrosis. METHODS: VDR expression was analysed in SSc skin, experimental fibrosis and human fibroblasts. VDR signalling was modulated by siRNA and with the selective agonist paricalcitol. The effects of VDR on Smad signalling were analysed by reporter assays, target gene analyses and coimmunoprecipitation. The effects of paricalcitol were evaluated in the models of bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active transforming growth factor-ß (TGF-ß) receptor I (TBRI(CA)). RESULTS: VDR expression was decreased in fibroblasts of SSc patients and murine models of SSc in a TGF-ß-dependent manner. Knockdown of VDR enhanced the sensitivity of fibroblasts towards TGF-ß. In contrast, activation of VDR by paricalcitol reduced the stimulatory effects of TGF-ß on fibroblasts and inhibited collagen release and myofibroblast differentiation. Paricalcitol stimulated the formation of complexes between VDR and phosphorylated Smad3 in fibroblasts to inhibit Smad-dependent transcription. Preventive and therapeutic treatment with paricalcitol exerted potent antifibrotic effects and ameliorated bleomycin- as well as TBRI(CA)-induced fibrosis. CONCLUSIONS: We characterise VDR as a negative regulator of TGF-ß/Smad signalling. Impaired VDR signalling with reduced expression of VDR and decreased levels of its ligand may thus contribute to hyperactive TGF-ß signalling and aberrant fibroblast activation in SSc.

Inhibition of hedgehog signaling for the treatment of murine sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a prognosis limiting complication of allogeneic stem cell transplantation. The molecular mechanisms underlying cGVHD are incompletely understood, and targeted therapies are not yet established for clinical use. Here we examined the role of the hedgehog pathway in sclerodermatous cGVHD. Hedgehog signaling was activated in human and murine cGVHD with increased expression of sonic hedgehog and accumulation of the transcription factors Gli-1 and Gli-2. Treatment with LDE223, a highly selective small-molecule antagonist of the hedgehog coreceptor Smoothened (Smo), abrogated the activation of hedgehog signaling and protected against experimental cGVHD. Preventive therapy with LDE223 almost completely impeded the development of clinical and histologic features of sclerodermatous cGVHD. Treatment with LDE223 was also effective, when initiated after the onset of clinical manifestations of cGVHD. Hedgehog signaling stimulated the release of collagen from cultured fibroblasts but did not affect leukocyte influx in murine cGVHD, suggesting direct, leukocyte-independent stimulatory effects on fibroblasts as the pathomechanism of hedgehog signaling in cGVHD. Considering the high morbidity of cGVHD, the current lack of efficient molecular therapies for clinical use, and the availability of well-tolerated inhibitors of Smo, targeting hedgehog signaling might be a novel strategy for clinical trials in cGVHD.

Combined inhibition of c-Abl and PDGF receptors for prevention and treatment of murine sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGvHD) is a common complication of allogeneic bone marrow transplantation, and has a major effect on the long-term prognosis. The molecular mechanisms underlying cGvHD have been only partially revealed, and molecular targeted therapies have not yet been established for clinical use. We examined the effects of the combined inhibition of the Abelson kinase (c-Abl) and platelet-derived growth factor receptors (PDGFR) in experimental sclerodermatous cGvHD. Treatment using imatinib or nilotinib abolished the aberrant activation of c-Abl and PDGFR and protected against experimental cGvHD. Preventive therapy using imatinib or nilotinib inhibited the development of sclerodermatous cGvHD. Clinical features such as weight loss, alopecia, and skin ulcers, and histologic features with dermal thickening and accumulation of collagen were significantly reduced in mice that received imatinib or nilotinib therapy, but not in mice that received prednisone therapy. Of note, imatinib and nilotinib were also effective for treatment of experimental cGvHD that had already been clinically manifested. In summary, the combined inhibition of c-Abl and PDGFR is effective for prevention and treatment of experimental sclerodermatous cGvHD. Considering the high morbidity associated with cGvHD, the lack of efficient molecular therapies for clinical use, and first positive signals from uncontrolled studies of imatinib, combined inhibition of c-Abl and PDGFR might be a promising future strategy for treatment of sclerodermatous cGvHD.

The transcription factor JunD mediates transforming growth factor {beta}-induced fibroblast activation and fibrosis in systemic sclerosis.

OBJECTIVES: Transforming growth factor ß (TGFß) has been identified as a key player in fibrotic diseases. However, the molecular mechanisms by which TGFß activates fibroblasts are incompletely understood. Here, the role of JunD, a member of the activator protein 1 (AP-1) family of transcription factors, as a downstream mediator of TGFß signalling in systemic sclerosis (SSc), was investigated. METHODS: The expression of JunD was analysed by real-time PCR, immunofluorescence, western blotting and immunohistochemistry. The canonical Smad pathway was specifically targeted by small interfering (si)RNA. The expression of extracellular matrix proteins in JunD deficient (JunD(-/-)) fibroblasts was analysed by real-time PCR and hydroxyproline assays. The mouse model of bleomycin-induced dermal fibrosis was used to assess the role of JunD in experimental fibrosis. RESULTS: JunD was overexpressed in SSc skin and in cultured fibroblasts in a TGFß dependent manner. The expression of JunD colocalised with pSmad 3 in fibrotic skin and silencing of Smad 3 or Smad 4 by siRNA prevented the induction of JunD by TGFß. JunD(-/-) fibroblasts were less responsive to TGFß and released less collagen upon stimulation with TGFß. Moreover, JunD(-/-) mice were protected from bleomycin-induced fibrosis with reduced dermal thickening, decreased myofibroblast counts and lower collagen content of lesional skin. CONCLUSIONS: These data demonstrate that JunD is overexpressed in SSc and that JunD is a mediator of the profibrotic effects of TGFß. Considering that inhibitors of AP-1 signalling have recently been developed and are available for clinical trials in SSc, these findings may have translational implications.