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 β 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.

Background: Endoplasmic reticulum (ER) stress is associated with fibrotic diseases, although the mechanisms are not completely understood. Results: The ER stress protein calreticulin regulates TGF-␤ stimulated extracellular matrix through control of intracellular calcium and NFAT signaling. Conclusion: Calreticulin is necessary for TGF-␤ stimulated extracellular matrix production. Significance: These findings identify calreticulin as a mechanistic link between ER stress and fibrosis.

Calreticulin (CRT), a chaperone and Ca2؉ regulator, enhances wound healing, and its expression correlates with fibrosis in animal models, suggesting that CRT regulates production of the extracellular matrix. However, direct regulation of collagen matrix by CRT has not been previously demonstrated. We investigated the role of CRT in the regulation of fibrillar collagen expression, secretion, processing, and deposition in the extracellular matrix by fibroblasts. Mouse embryonic fibroblasts deficient in CRT (CRT ؊/؊ MEFs) have reduced transcript levels of fibrillar collagen I and III and less soluble collagen as compared with wild type MEFs. Correspondingly, fibroblasts engineered to overexpress CRT have increased collagen type I transcript and protein. Collagen expression appears to be regulated by endoplasmic reticulum (ER) calcium levels and intracellular CRT, because thapsigargin treatment reduced collagen expression, whereas addition of exogenous recombinant CRT had no effect. CRT ؊/؊ MEFs exhibited increased ER retention of collagen, and collagen and CRT were co-immunoprecipitated from isolated cell lysates, suggesting that CRT is important for trafficking of collagen through the ER. CRT ؊/؊ MEFs also have reduced type I procollagen processing and deposition into the extracellular matrix. The reduced collagen matrix deposition is partly a consequence of reduced fibronectin matrix formation in the CRT-deficient cells. Together, these data show that CRT complexes with collagen in cells and that CRT plays critical roles at multiple stages of collagen expression and processing. These data identify CRT as an important regulator of collagen and suggest that intracellular CRT signaling plays an important role in tissue remodeling and fibrosis.Calreticulin (CRT), 3 also known as the C1q receptor, is an endoplasmic reticulum (ER) chaperone, and a modulator of intracellular calcium signaling. CRT also is a multifunctional protein that is present in numerous cellular compartments, including the ER, cytoplasm, nucleus, at the cell surface, and as a released protein (1-5). There is evidence to suggest the involvement of CRT in tissue remodeling and wound healing. In a porcine dermal wound healing model, topical application of purified CRT increases the rate of wound healing and wound tensile strength (6). Proteomic data show up-regulation of CRT expression with fibrosis in a rat model of unilateral ureteric obstruction kidney fibrosis and in a mouse model of bleomycininduced lung fibrosis (7). The mechanisms by which CRT regulates tissue remodeling are not well understood, although fibronectin matrix deposition and modulation of cell adhesion, motility, proliferation, and matrix metalloproteinase expression have been implicated (6, 8 -13).CRT has effects on the extracellular matrix (ECM) and cellular responses to the ECM. Fibroblasts overexpressing CRT have increased fibronectin mRNA, protein, and matrix deposition, and cells lacking CRT express less fibronectin than wild type cells (9,14). CRT in the ER is thought to reg...

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BackgroundThe ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)-mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc). Because recent studies implicate endogenous ligand-driven TLR signaling in SSc pathogenesis, we sought to investigate the regulation, role and mechanism of action of A20 in skin fibroblasts.MethodA20 expression and the effects of forced A20 expression or siRNA-mediated A20 knockdown on fibrotic responses induced by transforming growth factor-ß (TGF-ß) were evaluated was evaluated in explanted human skin fibroblasts. Additionally, A20 regulation by TGF-ß, and by adiponectin, a pleiotropic adipokine with anti-fibrotic activity, was evaluated.ResultsIn normal fibroblasts, TGF-ß induced sustained downregulation of A20, and abrogated its TLR4-dependent induction. Forced expression of A20 aborted the stimulation of collagen gene expression and myofibroblast transformation induced by TGF-ß, and disrupted canonical Smad signaling and Smad-dependent transcriptional responses. Conversely, siRNA-mediated knockdown of A20 enhanced the amplitude of fibrotic responses elicited by TGF-ß. Adiponectin, previously shown to block TLR-dependent fibrotic responses, elicited rapid and sustained increase in A20 accumulation in fibroblasts.ConclusionThese results identify the ubiquitin-editing enzyme A20 as a novel endogenous mechanism for negative regulation of fibrotic response intensity. Systemic sclerosis-associated genetic variants of A20 that cause impaired A20 expression or function, combined with direct suppression of A20 by TGF-ß within the fibrotic milieu, might play a significant functional role in persistence of fibrotic responses, while pharmacological augmentation of A20 inhibitory pathway activity might represent a novel therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1118-7) contains supplementary material, which is available to authorized users.