Kiyoshi Higashi

Interferon-γ Interferes with Transforming Growth Factor-β Signaling through Direct Interaction of YB-1 with Smad3

Transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ) exert antagonistic effects on collagen synthesis in human dermal fibroblasts. We have recently shown that Y box-binding protein YB-1 mediates the inhibitory effects of IFN-γ on α2(I) procollagen gene (COL1A2) transcription through the IFN-γ response element located between –161 and –150. Here we report that YB-1 counter-represses TGF-β-stimulated COL1A2 transcription by interfering with Smad3 bound to the upstream sequence around –265 and subsequently by interrupting the Smad3-p300 interaction. Western blot and immunofluorescence analyses using inhibitors for Janus kinases or casein kinase II suggested that the casein kinase II-dependent signaling pathway mediates IFN-γ-induced nuclear translocation of YB-1. Down-regulation of endogenous YB-1 expression by double-stranded YB-1-specific RNA abrogated the transcriptional repression of COL1A2 by IFN-γ in the absence and presence of TGF-β. In transient transfection assays, overexpression of YB-1 in human dermal fibroblasts exhibited antagonistic actions against TGF-β and Smad3. Physical interaction between Smad3 and YB-1 was demonstrated by immunoprecipitation-Western blot analyses, and electrophoretic mobility shift assays using the recombinant Smad3 and YB-1 proteins indicated that YB-1 forms a complex with Smad3 bound to the Smad-binding element. Glutathione S-transferase pull-down assays showed that YB-1 binds to the MH1 domain of Smad3, whereas the central and carboxyl-terminal regions of YB-1 were required for its interaction with Smad3. YB-1 also interferes with the Smad3-p300 interaction by its preferential binding to p300. Altogether, the results provide a novel insight into the mechanism by which IFN-γ/YB-1 counteracts TGF-β/Smad3. They also indicate that IFN-γ/YB-1 inhibits COL1A2 transcription by dual actions: via the IFN-γ response element and through a cross-talk with the TGF-β/Smad signaling pathway.

Interferon alfa down-regulates collagen gene transcription and suppresses experimental hepatic fibrosis in mice.

The equilibrium between the production and degradation of collagen is rigorously controlled by a number of growth factors and cytokines. Interferon alfa (IFN‐α) is now widely used for the treatment of chronic hepatitis C, which can improve serum levels of fibrotic markers and the degree of hepatic fibrosis, not only in patients who responded to therapy but also in those in whom it is ineffective. These findings may suggest that IFN‐α possesses direct antifibrotic effects in addition to its antiviral activity. However, in contrast to IFN‐γ, which has been shown to suppress collagen gene transcription, little is known about the mechanisms responsible for the antifibrotic effects of IFN‐α. Here, we report that IFN‐α, when administered into transgenic mice harboring the α2(I) collagen gene (COL1A2) promoter sequence, significantly repressed promoter activation and prevented the progression of hepatic fibrosis induced by carbon tetrachloride injection. Transient transfection assays indicated that IFN‐α decreased the steady‐state levels of COL1A2 messenger RNA (mRNA) and inhibited basal and TGF‐β/Smad3‐stimulated COL1A2 transcription in activated hepatic stellate cells (HSC). These inhibitory effects of IFN‐α on COL1A2 transcription were exerted through the interaction between phosphorylated Stat1 and p300. Blocking of the IFN‐α signal by overexpressing the intracellular domain‐deleted IFN receptor increased basal COL1A2 transcription and abolished the inhibitory effects of IFN‐α. In conclusion, our results indicate that IFN‐α antagonizes the TGF‐β/Smad3‐stimulated COL1A2 transcription in vitro and suppresses COL1A2 promoter activation in vivo, providing a molecular basis for antifibrotic effects of IFN‐α. (Hepatology 2003;38:890–899).

Hepatocyte Growth Factor Suppresses Profibrogenic Signal Transduction via Nuclear Export of Smad3 With Galectin-7

BACKGROUND & AIMS Hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) regulate diversified cellular functions and often act antagonistically against each other. For example, TGF-beta is the most potent factor accelerating liver fibrosis, whereas HGF treatment prevents its progression. Here, we propose a novel molecular mechanism by which HGF counter represses TGF-beta-stimulated profibrogenic signal transduction. METHODS Effects of HGF on TGF-beta-responsive gene transcription of type I collagen, the major matrix component of fibrotic liver, were examined by using cultured hepatic stellate cells (HSC) and transgenic mice harboring alpha2(I) collagen gene (COL1A2) promoter. Expression and subcellular localization of Smad3 were determined by Western blot analyses and immunofluorescence staining, respectively. A mass spectrometric analysis was employed to identify immunoprecipitated proteins with antiphospho-Smad2/3 antibodies. RESULTS Over expression of HGF inhibited COL1A2 transcription in cultured HSC and suppressed activation of COL1A2 promoter in liver tissue induced by carbon tetrachloride administration. A mass spectrometric analysis identified galectin-7 as one of the immunoprecipitated proteins with antiphospho-Smad2/3 antibodies following HGF treatment. HGF accelerated nuclear export of Smad3 by enhancing its interaction with galectin-7. Transfection of cells with galectin-7 small interfering RNA inhibited nuclear export of Smad3 and abolished suppressive effect of HGF on expression of TGF-beta-responsive genes such as COL1A2 and plasminogen activator inhibitor-1. On the other hand, over expression of galectin-7 suppressed TGF-beta-stimulated expression of those target genes. CONCLUSIONS These results reveal a novel function of intracellular galectin-7 as a transcriptional regulator via its interaction with Smad3 and provide a molecular basis for the antifibrotic effect of HGF.

A novel inhibitor of Smad‐dependent transcriptional activation suppresses tissue fibrosis in mouse models of systemic sclerosis

OBJECTIVE Tissue fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc), and an increasing number of promising molecular targets for antifibrotic therapies have been described recently. Transforming growth factor beta (TGFbeta) is well known to be the principal factor that leads to tissue fibrosis. The present study was undertaken to investigate the ability of HSc025, a novel small compound that antagonizes TGFbeta/Smad signaling through the activation of nuclear translocation of Y-box binding protein 1, to prevent tissue fibrosis in vitro or in mouse models of SSc. METHODS Human dermal fibroblasts were exposed to HSc025 at various concentrations in the presence of TGFbeta, and levels of collagen or fibronectin expression were determined. HSc025 (15 mg/kg/day for 14 days) was administered orally to tight skin mice and to mice with bleomycin-induced pulmonary fibrosis. Improvement of tissue fibrosis was evaluated by histologic or biochemical examination in each model. RESULTS Pretreatment with HSc025 prevented Smad-dependent promoter activation, in a dose-dependent manner; however, HSc025 had no effect on TGFbeta-induced phosphorylation of Smad3. The inhibitory effects of HSc025 on TGFbeta-induced collagen or fibronectin expression were also confirmed in vitro. Orally administered HSc025 significantly reduced hypodermal thickness and hydroxyproline content in tight skin mice, and markedly decreased the histologic score and hydroxyproline content in the lungs of bleomycin-treated mice. CONCLUSION These results demonstrate that HSc025 is a novel inhibitor of TGFbeta/Smad signaling, resulting in the improvement of skin and pulmonary fibrosis. Orally available HSc025 might therefore be useful in the treatment of SSc.

A Novel Small Compound That Promotes Nuclear Translocation of YB-1 Ameliorates Experimental Hepatic Fibrosis in Mice

Transforming growth factor-β (TGF-β) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-β/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-β-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.

Novel anti-fibrotic modalities for liver fibrosis: molecular targeting and regenerative medicine in fibrosis therapy.

Based on the cellular and molecular mechanisms underlying hepatic fibrogenesis, several kinds of approaches have been proposed to treat liver fibrosis. Among a number of growth factors and cytokines that regulate collagen metabolism, transforming growth factor (TGF)‐β is the most potent factor to accelerate liver fibrosis by activating hepatic stellate cells, stimulating collagen gene transcription, and suppressing matrix metalloproteinases expression. Thus, TGF‐β as well as its intracellular mediators, Smad proteins, can be potential therapeutic targets for liver fibrosis. Constitutive phosphorylation and nuclear accumulation of Smad3 is the common feature of activated stellate cells. We have synthesized a novel small compound that inhibits Smad3‐dependent collagen gene transcription by promoting nuclear import of a transcriptional repressor, YB‐1. Another insight into anti‐fibrotic strategies is the contribution of bone marrow‐derived cells to the regression of liver fibrosis. Administration of granulocyte‐colony stimulating factor enhanced the migration of bone marrow‐derived cells into fibrotic liver tissue and accelerated the regression of experimental liver fibrosis. We have recently identified novel unknown factors expressed by bone marrow‐derived cells that not only ameliorate liver fibrosis but also accelerate regeneration of fibrotic liver.

Dermokine inhibits ELR(+)CXC chemokine expression and delays early skin wound healing.

BACKGROUND Dermokine-β is abundant in stratified epithelia and in differentiating keratinocytes in culture. We have recently shown that treatment of keratinocytes with dermokine-β attenuates phosphorylation of extracellular signal-regulated kinase, however, the roles of dermokine-β in vivo remain unknown. OBJECTIVE Dermokine-β is overexpressed in marginal keratinocytes during wound healing. This study was conducted to investigate the roles of dermokine-β in the wound healing process. METHODS Recombinant human dermokine-β or its active peptide was topically applied to excisional wounds in mice and the relative wound area was calculated. Histological and chemokine expression analyses in wounds were also performed. The chemokine expression levels as well as the chemotactic activity of dermokine-β in cultured keratinocytes were determined. RESULTS Topical application of recombinant dermokine-β as well as its carboxy-terminal domain peptide inhibited mouse wound healing at an early phase, reduced infiltration of neutrophils and macrophages into the wounds, inhibited angiogenesis, and decreased the number of myofibroblasts in the wounds. Treatment with dermokine-β augmented IL-10 expression, but attenuated expression of transforming growth factor-β and tumor necrosis factor-α. In addition, application of dermokine-β to skin wounds reduced the expression of CXCL1 and CXCL5, both of which are chemoattractant for neutrophils into wounds. Both dermokine-β and its active peptide decreased the expression of CXCL1, CXCL6, and CXCL8 in cultured human keratinocytes. Treatment of human keratinocytes with dermokine-β inhibited neutrophil chemotaxis. CONCLUSION These results suggest that dermokine-β delays early cutaneous wound healing in part by inhibiting expression of CXC chemokines containing the ERL-sequence motif.

Altered expression of dermokine in skin disorders

Background  Although dermokine‐β, a glycoprotein expressed in epithelial cells, does not have significant homology to other proteins, its carboxyl‐terminal domain shares a high pI value with many cytokines, suggesting similar functions.

Dermokine-β impairs ERK signaling through direct binding to GRP78

dermokine‐Beta binds to GRP78 by anti bait coimmunoprecipitation (View interaction)

A novel small compound accelerates dermal wound healing by modifying infiltration, proliferation and migration of distinct cellular components in mice

BACKGROUND Impaired wound healing in skin ulcer is one of the major medical issues in the aged society. Wound healing is a complex process orchestrated by a number of humoral factors and cellular components. TGF-β is known to stimulate collagen production in dermal fibroblasts while inhibiting proliferation of epidermal keratinocyte. A screening of small compounds that suppress type I collagen production in fibroblasts has identified HSc025 that antagonizes the TGF-β/Smad signal. OBJECTIVE We examined the effects of HSc025 on dermal wound healing and elucidated the underlying mechanisms. METHODS Effects of HSc025 on the wound closure process were evaluated in a murine full-thickness excisional wound healing model. Cell proliferation and migration were estimated using primary cultures of human keratinocytes and fibroblasts. Comprehensive analyses of gene expression profiles were performed using untreated and HSc025-treated fibroblasts. RESULTS Oral HSc025 administration suppressed macrophage infiltration and accelerated wound closure as early as at day 2 after the dermal excision. Treatment of cultured keratinocytes with HSc025 counteracted the inhibitory effects of TGF-β on cell proliferation and migration. On the other hand, HSc025 stimulated migration, but not proliferation, of dermal fibroblasts independently of TGF-β. Experiments using an artificial dermis graft revealed that HSc025 stimulated migration of collagen-producing cells into the graft tissue. A cDNA microarray analysis of untreated and HSc025-treated fibroblasts identified pirin as a critical mediator accelerating fibroblast migration. CONCLUSION HSc025 accelerates wound healing by modifying infiltration, proliferation and migration of distinct cellular components, which provides a novel insight into the therapy for intractable skin ulcer.