Fumiaki Shirasaki

Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients.

Abstract:  Human tissue kallikreins are a family of 15 trypsin‐ or chymotrypsin‐like secreted serine proteases (KLK1–KLK15). Many KLKs have been identified in normal stratum corneum (SC) and sweat, and are candidate desquamation‐related proteases.

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).

Connective tissue growth factor causes persistent proα2(I) collagen gene expression induced by transforming growth factor‐β in a mouse fibrosis model

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine‐rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor‐β (TGF‐β). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF‐β transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF‐β caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF‐β and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the proα2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the −17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial β‐galactosidase gene. Serial injections of CTGF after TGF‐β resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF‐β alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF‐β in comparison with the injection of TGF‐β alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF‐β‐induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.

Ets transcription factors cooperate with Sp1 to activate the human Tenascin-C promoter

Tenascin-C (TN-C), an extracellular matrix glycoprotein is expressed during embryonic development, but is present only at low levels in normal adult tissues. TN-C is re-expressed during wound healing, fibrotic diseases and in cancer. To better understand the mechanisms that control TN-C gene expression, we examined the regulation of the human TN-C promoter in human fibroblasts. We demonstrate that a short segment of the TN-C promoter between bp −133 and −27 contains three evolutionarily conserved Ets binding sites (EBS). These three EBSs bind in vitro expressed Fli1 protein and mediate transactivation of the TN-C gene by Fli1. Furthermore, two proximal EBSs contribute significantly to basal activity of the TN-C promoter. GABP, which is present in human fibroblast nuclear extracts, interacts with the two proximal EBSs. In addition, several Sp1 and Sp3 binding sites have been located in close proximity to the EBSs within this promoter region. The studies performed in Drosophila cells demonstrate that either Fli1 or GABPα+β1 functionally interact with Sp1 resulting in a synergistic stimulation of the TN-C promoter activity. In conclusion, this study shows for the first time that the TN-C gene is regulated by Ets proteins, which together with Sp1 act as potent activators of TN-C expression.

Aberrant human tissue kallikrein levels in the stratum corneum and serum of patients with psoriasis: dependence on phenotype, severity and therapy

Background  Human tissue kallikreins (KLKs) are a family of 15 trypsin‐like or chymotrypsin‐like secreted serine proteases (KLK1–KLK15). Multiple KLKs have been quantitatively identified in normal stratum corneum (SC) and sweat as candidate desquamation‐related proteases.

Neutralizing monoclonal antibody to human connective tissue growth factor ameliorates transforming growth factor-β-induced mouse fibrosis

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM) and are understood to develop under the influence of fibrogenic growth factors. To better understand the detailed mechanisms of persistent fibrosis in SSc, we have previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, transforming growth factor‐β (TGF‐β) transiently induced subcutaneous fibrosis and serial injections of connective tissue growth factor (CTGF) after TGF‐β caused persistent fibrosis. These results suggest that CTGF plays an important role in the development of persistent skin fibrosis and that CTGF may be a potential and specific therapeutic target in skin fibrosis. Therefore, the aim of the current study is to develop a neutralizing monoclonal antibody against human CTGF. We also investigated the neutralizing effect of the antibodies in our animal model. Firstly, by using the DNA immunization method, we developed a panel of anti‐CTGF antibodies recognizing the native conformation of human CTGF. Next, to examine the anti‐fibrosing effects of these antibodies, newborn B6 mice received subcutaneous injections of TGF‐β for 3 days with either anti‐CTGF neutralizing antibodies or control purified immunoglobulin. Anti‐CTGF antibodies significantly reduced skin fibrosis and collagen contents compared with the control group. These results suggest that our anti‐CTGF antibodies are capable of blocking the development of skin fibrosis at least partially and these anti‐CTGF neutralizing antibodies may be useful as the feasible strategy to treat skin fibrotic diseases as SSc. J. Cell. Physiol. 216: 680–687, 2008,

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.

Role of connective tissue growth factor and its interaction with basic fibroblast growth factor and macrophage chemoattractant protein-1 in skin fibrosis.

Activation of the immune system and abnormal growth of skin fibroblasts cause systemic sclerosis. Growth factors have various biological activities, including mediation of immune reactions. The growth factor family includes basic fibroblast growth factor (bFGF), transforming growth factor‐beta (TGF‐β), and connective tissue growth factor (CTGF). CTGF, an important downstream mediator of TGF‐β in fibrosis, has been suggested to play a specific role in fibrotic disorders. We have directed our attention to the role of CTGF in sustaining skin fibrosis. To better understand its effects in vivo, we established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, bFGF transiently induced subcutaneous fibrosis. Simultaneous injection of bFGF and CTGF increased skin fibrosis compared with a single injection of bFGF. Serial injections of bFGF for 3 days followed by CTGF for 4 days, or of CTGF followed by bFGF, did not cause skin fibrosis but simultaneous injections increased macrophage chemoattractant protein‐1 (MCP‐1) mRNA expression levels. To further define the mechanisms of skin fibrosis in vivo, bFGF and CTGF were injected simultaneously into MCP‐1 knockout mice, resulting in decreased collagen levels in granulation tissues on day 8. The number of inflammatory cells, such as mast cells, macrophages and lymphocytes, was significantly decreased in MCP‐1 knockout mice compared with wild‐type mice. These results suggest that bFGF induces collagen production by stimulating skin fibroblasts and CTGF cooperates with bFGF. Our results indicate that the induction of MCP‐1 is necessary for infiltration of inflammatory cells. J. Cell. Physiol. 220: 189–195, 2009.

Chemokine receptors CCR2 and CX3CR1 regulate skin fibrosis in the mouse model of cytokine-induced systemic sclerosis

BACKGROUND Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM), and develop under the influence of certain cytokines. We previously established a mouse model of skin fibrosis induced by exogenous application of cytokines. We have revealed that both the number of macrophages and the levels of macrophage chemoattractant protein-1 (MCP-1) mRNA positively correlate with the extent of skin fibrosis. Macrophages can be divided into two subsets, the first expressing CCR2, and the second expressing CX3CR1. OBJECTIVE To elucidate the role of skin infiltrating macrophages based on CCR2 and CX3CR1 in this cytokine-induced murine fibrosis model. METHODS We examined the amounts of collagen deposited in granulation tissues, the numbers of macrophages and the levels of several mRNA in wild type (WT) mice, CCR2(-/-) mice, and CX3CR1(-/-) mice during injections of transforming growth factor-β (TGF-β) followed by injections of connective tissue growth factor (CTGF). RESULTS TGF-β injection increased the expressions of MCP-1, fractalkine, CCR2 and CX3CR1 mRNA in WT mice. The overproduction of collagen induced by TGF-β was significantly reduced by CCR2 deficiency, while collagen contents induced by CTGF were restored to wild-type levels. In contrast, overproduction of collagen in CX3CR1-deficient mice decreased nearly 50% by both TGF-β and CTGF stimulations. CONCLUSION The involvement of CCR2/MCP-1 interaction (CCR2-dependent loop) was during the TGF-β phase. In contrast, the fractalkine/CX3CR1 interaction contributes to the initiation of fibrosis by TGF-β and its maintenance by CTGF. Collectively, two subsets of macrophages both cooperatively and independently play important roles in the development of fibrosis.

Successful Treatment of Livedo Vasculitis with Beraprost Sodium: A Possible Mechanism of Thrombomodulin Upregulation

BACKGROUND Livedo vasculitis is thought to be a thrombogenic disorder. We demonstrated that thrombomodulin (TM) expression on the endothelial cells in livedo vasculitis was markedly reduced, while blood tests of coagulation and fibrinolytic activities were within the normal range. OBJECTIVE Since prostacyclin (PGI2) upregulates the TM expression of endothelial cells, we tried a PGI2 analogue for the treatment of livedo vasculitis. METHOD Four patients with livedo vasculitis were started on a regimen of beraprost sodium (120 micrograms daily). Additional intake of low-dose aspirin was combinated with a maintenance dose of beraprost sodium (60 micrograms daily). RESULT All 4 patients experienced a clinical improvement with a combination therapy with beraprost sodium and low-dose aspirin. CONCLUSION We propose that PGI2 analogue therapy is useful for the treatment of livedo vasculitis, since the drug upregulates TM expression in this disorder.