Activation of hepatic stellate cells (HSC), a key event in liver fibrosis, is caused by diminished adipogenic transcription. This study investigated whether Wnt signaling contributes to "antiadipogenic" activation of HSC and liver fibrogenesis. Culture-activated HSC from normal rats and HSC from cholestatic rat livers were examined for expression of Wnt, Frizzled (Fz) receptors, and coreceptors by quantitative PCR. Wnt signaling was assessed by nuclear beta-catenin and T cell factor (TCF) promoter activity. Dickkopf-1 (Dkk-1), a Wnt coreceptor antagonist, was transduced by an adenoviral vector to assess the effects of Wnt antagonism on culture activation of HSC and cholestatic liver fibrosis in mice. Messenger RNA for canonical (Wnt3a and 10b) and noncanonical (Wnt4 and 5a) Wnt genes, Fz-1 and 2, and coreceptors [low-density lipoprotein-receptor-related protein (LRP)6 and Ryk] are increased approximately 3-12-fold in culture-activated HSC compared with quiescent HSC. The nuclear beta-catenin level and TCF DNA binding are markedly increased in activated HSC. TCF promoter activity is stimulated with Wnt1 but inhibited by Chibby, a protein that blocks beta-catenin interaction with TCF, and by Dkk-1. Dkk-1 enhances peroxisome proliferator-activated receptor-gamma (PPARgamma)-driven PPAR response element (PPRE) promoter activity, a key adipogenic transcriptional parameter, abrogates agonist-stimulated contraction, and restores HSC quiescence in culture. High expression of Dkk-1 increases apoptosis of cultured HSC. Expression of Wnt and Fz genes is also induced in HSC isolated from experimental cholestatic liver fibrosis, and Dkk-1 expression ameliorates this form of liver fibrosis in mice. These results demonstrate antiadipogenic Wnt signaling in HSC activation and therapeutic potential of Wnt antagonism for liver fibrosis.

Jump-starting and subsequently maintaining epidermal and dermal cell migration are essential processes for skin wound healing. These events are often disrupted in nonhealing wounds, causing patient morbidity and even fatality. Currently available treatments are unsatisfactory. To identify novel wound-healing targets, we investigated secreted molecules from transforming growth factor ␣ (TGF␣)-stimulated human keratinoytes, which contained strong motogenic, but not mitogenic, activity. Protein purification allowed us to identify the heat shock protein 90␣ (hsp90␣) as the factor fully responsible for the motogenic activity in keratinocyte secretion. TGF␣ causes rapid membrane translocation and subsequent secretion of hsp90␣ via the unconventional exosome pathway in the cells. Secreted hsp90␣ promotes both epidermal and dermal cell migration through the surface receptor LRP-1 (LDL receptor-related protein 1)/CD91. The promotility activity resides in the middle domain plus the charged sequence of hsp90␣ but is independent of the ATPase activity. Neutralizing the extracellular function of hsp90␣ blocks TGF␣-induced keratinicyte migration. Most intriguingly, unlike the effects of canonical growth factors, the hsp90␣ signaling overrides the inhibition of TGF␤, an abundant inhibitor of dermal cell migration in skin wounds. This finding provides a long-sought answer to the question of how dermal cells migrate into the wound environment to build new connective tissues and blood vessels. Thus, secreted hsp90␣ is potentially a new agent for wound healing.

Epithelial-mesenchymal transition (EMT), characterized by loss of epithelial adhesion and gain of mesenchymal features, is an important mechanism to empower epithelial cells into the motility that occurs during embryonic development and recurs in cancer and fibrosis. Whether and how EMT occurs in wound healing and fibrosis in human skin remains unknown. In this study we found that migrating epithelial cells in wound margins and deep epithelial ridges had gained mesenchymal features such as vimentin and FSP1 expression. In hypertrophic scars, EMTrelated genes were elevated along with inflammatory cytokines, indicating a causal relationship. To reconstitute EMT in vitro, normal human skin and primary keratinocytes were exposed to cytokines such as tumor necrosis factor-␣ (TNF-␣), resulting in expression of vimentin, FSP1, and matrix metalloproteinases. Moreover, TNF-␣-induced EMT was impaired by antagonists against bone morphogen proteins (BMP) 2/4, suggesting that BMP mediates the TNF-␣-induced EMT in human skin. Indeed, TNF-␣ could induce BMP-2 and its receptor (BMPR1A) in human skin and primary keratinocytes , and BMP2 could induce EMT features in skin explants and primary keratinocytes. In summary , we uncovered EMT features in both acute and fibrotic cutaneous wound healing of human skin. Moreover , we propose that the mesenchymal induction in wound healing is motivated by TNF-␣ , in part , through induction of BMP. (Am J

Abnormal wound healing encompasses a wide spectrum, from chronic wounds to hypertrophic scars. Both conditions are associated with an abnormal cytokine profile in the wound bed. In this study, we sought to understand the dynamic relationships between myofibroblast differentiation and mechanical performance of the collagen matrix under tissue growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) stimulation. We found TGF-beta increased alpha-smooth muscle actin (alpha-SMA) and TNF-alpha alone decreased the basal alpha-SMA expression. When TGF-beta1 and TNF-alpha were both added, the alpha-SMA expression was suppressed below the baseline. Real-time PCR showed that TNF-alpha suppresses TGF-beta1-induced myofibroblast (fibroproliferative) phenotypic genes, for example, alpha-SMA, collagen type 1A, and fibronectin at the mRNA level. TNF-alpha suppresses TGF-beta1-induced gene expression by affecting its mRNA stability. Our results further showed that TNF-alpha inhibits TGF-beta1-induced Smad-3 phosphorylation via Jun N-terminal kinase signaling. Mechanical testing showed that TNF-alpha decreases the stiffness and contraction of the lattices after 5 days in culture. We proposed that changes in alpha-SMA, collagen, and fibronectin expression result in decreased contraction and stiffness of collagen matrices. Therefore, the balance of cytokines in a wound defines the mechanical properties of the extracellular matrix and optimal wound healing.

Metabolic syndrome (MetS), characterized as obesity, insulin resistance, and non-alcoholic fatty liver diseases (NAFLD), is associated with vitamin D insufficiency/deficiency in epidemiological studies, while the underlying mechanism is poorly addressed. On the other hand, disorder of gut microbiota, namely dysbiosis, is known to cause MetS and NAFLD. It is also known that systemic inflammation blocks insulin signaling pathways, leading to insulin resistance and glucose intolerance, which are the driving force for hepatic steatosis. Vitamin D receptor (VDR) is highly expressed in the ileum of the small intestine, which prompted us to test a hypothesis that vitamin D signaling may determine the enterotype of gut microbiota through regulating the intestinal interface. Here, we demonstrate that high-fat-diet feeding (HFD) is necessary but not sufficient, while additional vitamin D deficiency (VDD) as a second hit is needed, to induce robust insulin resistance and fatty liver. Under the two hits (HFD+VDD), the Paneth cell-specific alpha-defensins including α-defensin 5 (DEFA5), MMP7 which activates the pro-defensins, as well as tight junction genes, and MUC2 are all suppressed in the ileum, resulting in mucosal collapse, increased gut permeability, dysbiosis, endotoxemia, systemic inflammation which underlie insulin resistance and hepatic steatosis. Moreover, under the vitamin D deficient high fat feeding (HFD+VDD), Helicobacter hepaticus, a known murine hepatic-pathogen, is substantially amplified in the ileum, while Akkermansia muciniphila, a beneficial symbiotic, is diminished. Likewise, the VD receptor (VDR) knockout mice exhibit similar phenotypes, showing down regulation of alpha-defensins and MMP7 in the ileum, increased Helicobacter hepaticus and suppressed Akkermansia muciniphila. Remarkably, oral administration of DEFA5 restored eubiosys, showing suppression of Helicobacter hepaticus and increase of Akkermansia muciniphila in association with resolving metabolic disorders and fatty liver in the HFD+VDD mice. An in vitro analysis showed that DEFA5 peptide could directly suppress Helicobacter hepaticus. Thus, the results of this study reveal critical roles of a vitamin D/VDR axis in optimal expression of defensins and tight junction genes in support of intestinal integrity and eubiosis to suppress NAFLD and metabolic disorders.