Myofibroblasts in murine cutaneous fibrosis originate from adiponectin-positive intradermal progenitors.

OBJECTIVE: Accumulation of myofibroblasts in fibrotic skin is a hallmark of systemic sclerosis (SSc; scleroderma), but the origins of these cells remain unknown. Because loss of intradermal adipose tissue is a consistent feature of cutaneous fibrosis, we sought to examine the hypothesis that myofibroblasts populating fibrotic dermis derive from adipocytic progenitors. METHODS: We performed genetic fate mapping studies to investigate the loss of intradermal adipose tissue and its potential role in fibrosis in mice with bleomycin-induced scleroderma. Modulation of adipocytic phenotypes ex vivo was investigated in adipose tissue-derived cells in culture. RESULTS: A striking loss of intradermal adipose tissue and its replacement with fibrous tissue were consistently observed in mice with bleomycin-induced fibrosis. Loss of adipose tissue and a decline in the expression of canonical adipogenic markers in lesional skin preceded the onset of dermal fibrosis and expression of fibrogenic markers. Ex vivo, subcutaneous adipocytes were driven by transforming growth factor ß to preferentially undergo fibrogenic differentiation. Cell fate mapping studies in mice with the adiponectin promoter-driven Cre recombinase transgenic construct indicated that adiponectin-positive progenitors that are normally confined to the intradermal adipose tissue compartment were distributed throughout the lesional dermis over time, lost their adipocytic markers, and expressed myofibroblast markers in bleomycin-treated mice. CONCLUSION: These observations establish a novel link between intradermal adipose tissue loss and dermal fibrosis and demonstrate that adiponectin-positive intradermal progenitors give rise to dermal myofibroblasts. Adipose tissue loss and adipocyte-myofibroblast transition might be primary events in the pathogenesis of cutaneous fibrosis that represent novel potential targets for therapeutic intervention.

Calreticulin regulates transforming growth factor-ß-stimulated extracellular matrix production.

Endoplasmic reticulum (ER) stress is an emerging factor in fibrotic disease, although precise mechanisms are not clear. Calreticulin (CRT) is an ER chaperone and regulator of Ca(2+) signaling up-regulated by ER stress and in fibrotic tissues. Previously, we showed that ER CRT regulates type I collagen transcript, trafficking, secretion, and processing into the extracellular matrix (ECM). To determine the role of CRT in ECM regulation under fibrotic conditions, we asked whether CRT modified cellular responses to the pro-fibrotic cytokine, TGF-ß. These studies show that CRT-/- mouse embryonic fibroblasts (MEFs) and rat and human idiopathic pulmonary fibrosis lung fibroblasts with siRNA CRT knockdown had impaired TGF-ß stimulation of type I collagen and fibronectin. In contrast, fibroblasts with increased CRT expression had enhanced responses to TGF-ß. The lack of CRT does not impact canonical TGF-ß signaling as TGF-ß was able to stimulate Smad reporter activity in CRT-/- MEFs. CRT regulation of TGF-ß-stimulated Ca(2+) signaling is important for induction of ECM. CRT-/- MEFs failed to increase intracellular Ca(2+) levels in response to TGF-ß. NFAT activity is required for ECM stimulation by TGF-ß. In CRT-/- MEFs, TGF-ß stimulation of NFAT nuclear translocation and reporter activity is impaired. Importantly, CRT is required for TGF-ß stimulation of ECM under conditions of ER stress, as tunicamycin-induced ER stress was insufficient to induce ECM production in TGF-ß stimulated CRT-/- MEFs. Together, these data identify CRT-regulated Ca(2+)-dependent pathways as a critical molecular link between ER stress and TGF-ß fibrotic signaling.