Cancer-associated fibroblasts (CAFs) regulate cancer progression through the modulation of extracellular matrix (ECM) and cancer cell adhesion. While undergoing a series of phenotypic changes, CAFs control cancer-stroma interactions through integrin receptor signaling. Here, we isolated CAFs from patients with non-small-cell lung cancer (NSCLC) and examined their gene expression profiles. We identified collagen type XI α1 (COL11A1), integrin α11 (ITGA11), and the ITGA11 major ligand collagen type I α1 (COL1A1) among the 390 genes that were significantly enriched in NSCLC-associated CAFs. Increased ITGA11 expression in cancer stroma was correlated with a poor clinical outcome in patients with NSCLC. Increased expression of fibronectin and collagen type I induced ITGA11 expression in CAFs. The cellular migration of CAFs toward collagen type I and fibronectin was promoted via ERK1/2 signaling, independently of the fibronectin receptor integrin α5β1. Additionally, ERK1/2 signaling induced ITGA11 and COL11A1 expression in cancer stroma. We, therefore, propose that targeting ITGA11 and COL11A1 expressing CAFs to block cancer-stroma interactions may serve as a novel, promising anti-tumor strategy.

PURPOSE: The proinflammatory cytokine interleukin (IL)-1 is implicated in corneal ulceration and promotes collagen degradation by corneal fibroblasts cultured in a three-dimensional (3D) collagen gel. Epigallocatechin-3-gallate (EGCG), the principal polyphenol in extracts of green tea, has various beneficial health effects, some of which appear to be mediated through direct or indirect inhibition of protease activity. We therefore examined the effect of EGCG on IL-1β-induced collagen degradation by corneal fibroblasts embedded in a collagen gel. METHODS: Human corneal fibroblasts were cultured in a type I collagen gel. Collagen degradation was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. The expression of urokinase-type plasminogen activator (uPA) was examined by real-time and RT-PCR analysis and by fibrin zymography, and that of the collagenase matrix metalloproteinase 1 (MMP1) was detected by immunoblot analysis. RESULTS: EGCG inhibited IL-1β-induced, plasminogen-dependent collagen degradation by corneal fibroblasts in a concentration-dependent manner. It also attenuated the IL-1β-induced expression of uPA at both mRNA and protein levels. EGCG inhibited the IL-1β-induced conversion of exogenous plasminogen to plasmin as well as the plasminogen-dependent activation of pro-MMP1 in the 3D cultures without a substantial effect on pro-MMP1 abundance. CONCLUSIONS: EGCG inhibits IL-1β-induced collagen degradation by corneal fibroblasts, with this effect likely being mediated by suppression of the upregulation of uPA, the uPA-mediated conversion of plasminogen to plasmin, and the plasmin-mediated activation of pro-MMP1. EGCG thus warrants further investigation as a potential treatment for corneal ulcer.

PURPOSE. Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1 beta (IL-1 beta). METHODS. Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. RESULTS. Collagen and IL-1 beta synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1 beta, were all abolished by a selective uPA inhibitor. CONCLUSIONS. Collagen and IL-1 beta cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1 beta-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

Background: We encountered a rare case of Waldenstrom macroglobulinemia with temporary appearance of 7S IgM half molecule and with monoclonal proteins binding to agarose gel. Methods: The patient's serum and urine were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The N-terminal amino acid sequences of the IgM with abnormal mass (68 kDa) were determined and compared with those of known immunoglobulin. Results: The 68 kDa IgM consisted of a defective Et chain (36 kDa) and an intact kappa chain. N-terminal amino acid sequence analysis demonstrated that the defective It chain had the variable region of IgM. The agarose gel-binding ability of the IgM-w M-protein was lost after reduction or alkaline treatment of serum. Conclusions: The 7S half molecule IgM in the present case may miss a large part of the constant region of the mu chain.

Background: Expression of heat shock protein A4 (HSPA4, also called Apg-2), a member of the HSP110 family, is induced by several forms of stress. The physiological and pathological functions of HSPA4 in the intestine remain to be elucidated. Methods: We assessed HSPA4 expression and function by generating HSPA4-deficient mice and using 214 human intestinal mucosa samples from patients with inflammatory bowel disease (IBD). Results: In the colonic mucosa of patients with IBD, a significant correlation was observed between the expression of HSPA4 and antiapoptotic protein Bcl-2, a T-cell-derived cytokine IL-17 or stem cell markers, such as Sox2. In refractory ulcerative colitis, a condition associated with increased cancer risk, expression of HSPA4 and Bcl-2 was increased in inflammatory cells of colonic mucosae. HSPA4 was overexpressed both in cancer cells and immune cells of human colorectal cancers. Patients with high expression of HSPA4 or Bmi1 showed significantly lower response rates upon subsequent steroid therapy as compared with patients with low expression of each gene. HSPA4-deficient mice exhibit more apoptosis and less expression of IL-17/IL-23 in inflammatory cells and less number of Sox(2+) cells after administration of dextran sodium sulfate than control mice. Transduction of HspaA4(+/-) bone marrow into wild-type mice reduced the immune response. Conclusions: Upregulation of Bcl-2 and IL-17 by HSPA4 would control apoptosis of inflammatory cells and immune response in the gut, which might develop treatment resistance in IBD. HSPA4 and Bmi1 would be a useful biomarker for refractory clinical course and a promising approach for a therapeutic strategy in patients with IBD.

PURPOSE. Urokinase-type plasminogen activator (u-PA) plays an important role in corneal wound healing, yet its role in corneal inflammation remains poorly understood. We investigated the role of u-PA in a murine model of lipopolysaccharide (LPS)-induced corneal inflammation. METHODS. The corneal epithelium was scraped and LPS was applied to u-PA wild-type (u-PA(+/+)) and u-PA-deficient (u-PA(-/-)) mice. Corneal re-epithelialization and opacity were measured by stereomicroscopy. Fibrin zymography was performed to detect plasminogen activators in corneas from u-PA(+/+) and u-PA(-/-) mice. Neutrophil, macrophage, and u-PA receptor (u-PAR) expression were determined by immunohistochemistry. Gene expression of corneal macrophage chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 was assessed with reverse transcription-polymerase chain reaction. The in vitro effects of endogenous u-PA on MCP-1, MIP-2, matrix metalloproteinase (MMP)-2, and MMP-9 expression, and macrophage migration activity in mouse ocular fibroblasts stimulated by LPS, were examined. RESULTS. The u-PA(+/+) mice showed enhanced corneal inflammation as compared with u-PA(-/-) mice. The u-PA expression was increased by LPS stimulation. Immunohistochemical analyses indicated that more neutrophils and macrophages were present in corneas from u-PA(-/-) mice than u-PA(-/-)mice. The u-PAR expression was detected in inflammatory cells and in the leading edges of the epithelial migrating cells. Enhanced mRNA expression of MCP-1 and MIP-2 was observed in corneas from u-PA(+/+) mice compared to u-PA(-/-) mice. Macrophage chemoattractant protein-1, MIP-2, and MMP-9, but not MMP-2, significantly increased in corneal fibroblasts from u-PA(+/+) mice compared with u-PA(-/-) mice. CONCLUSIONS. These data indicate that u-PA promotes LPS-induced leukocyte infiltration in cornea and that u-PA is an important component in LPS-induced corneal inflammatory responses.

Transforming growth factor-beta (TGF-beta) is one of the main epithelial mesenchymal transition (EMT)-inducing factors. In general, TGF-beta-induced EMT promotes cell migration and invasion. TGF-beta also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPAR's interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-beta and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-beta 2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-beta 2 and/or the inhibitor of uPA (u-PA-STOP (R)) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-beta 2 or u-PA-STOP (R) suppressed cell proliferation. Combination treatment of TGF-beta 2 and u-PA-STOP (R) enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-beta 2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-beta inhibitor SB431542 suppressed TGF-beta 2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-beta 2 alone or with TGF-beta 2 and u-PA-STOP (R). TGF-beta 2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-beta 2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with TGF-beta 2 alone. Furthermore, the interaction between uPA and ARPE-19 cells was analyzed using a surface plasmon biosensor system. The binding of uPA to ARPE-19 cells was observed. In addition, TGF-beta 2 significantly promoted the binding activity of uPA to ARPE-19 cells in a time-dependent or cell-number-dependent fashion. These results indicate that TGF-beta-induced EMT-associated phenotype changes in ARPE-19 cells and the invasiveness of ARPE-19 cells into a collagen gel matrix are mediated, at least in part, by uPA. (C) 2013 Elsevier Ltd. All rights reserved.

Fibrosis is a state, in which excess amounts of extracellular matrix are deposited in the tissue. Fibrosis can occur in various organs, including the liver, lung, kidney and heart. The progression of fibrosis involves interstitial hypercellularity, accumulation of extracellular matrix, and atrophy of epithelial structures, resulting in a loss of normal function. Myofibroblasts play a crucial role in the development and progress of fibrosis. When stimulated, myofibroblasts actively synthesize connective tissue components and cause organ fibrosis. As a result, the process and the mechanism of myofibroblast activation represent a target for antifibrotic treatment. As yet, however, an effective treatment has not been developed, and new treatment modalities are expected. Because activation of myofibroblasts is a key event during fibrosis development, there is great interest in identifying and characterizing proteins whose expression is changed after this activation. In this review, fibrosis is outlined and the role of myofibroblasts in this disorder is described. Furthermore, the search for candidate proteins to target for treatment and the prospects of antifibrotic therapy are discussed.

Background: Corneal ulceration leading to perforation is associated with infectious and non-infectious destructive conditions in the cornea. The fibrinolytic (plasminogen/plasmin) system is considered to contribute to tissue remodeling in the wound healing process and it is believed to play an important role in proteolysis and fibrosis. To determine the localization of urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and alpha 2-antiplasmin (alpha 2AP) in the tissue of a corneal perforation, we investigated immunohistochemical expressions of u-PA, u-PAR, alpha 2AP, CD68, and alpha-smooth muscle actin (alpha-SMA) in a patient with corneal perforation that developed from an ulcer of no clear cause. Case presentation: The patient was a 77-year-old woman who presented with a perforated corneal ulcer in her right eye. The cause of her corneal ulcer was unknown. Double immunohistochemistry was performed for the combinations of u-PA with u-PAR, CD68 or alpha-SMA and alpha 2AP with CD68 or alpha-SMA to detect the localization of u-PA and alpha 2AP. u-PA and u-PAR co-localization was seen in the corneal ulceration area. u-PA was mainly observed in CD68-positive cells and in some alpha-SMA positive cells. On the other hand, alpha 2AP was not expressed in CD68-positive cells, but was expressed in alpha-SMA positive cells. Conclusion: We identified expression of the u-PA/u-PAR complex and alpha 2AP in a patient with a corneal ulcer. These two molecules are believed to play a crucial role in inflammatory cell recruitment, ECM synthesis and degradation during corneal wound healing.

Pulmonary fibrosis is a devastating condition resulting from excess extracellular matrix deposition that leads to progressive lung destruction and scarring. In the pathogenesis of fibrotic diseases, activation of myofibroblasts by transforming growth factor-beta (TGF-beta) plays a crucial role. Since no effective therapy for pulmonary fibrosis is currently recognized, finding an effective antifibrotic agent is an important objective. One approach might be through identification of agents that inactivate myofibroblasts. In the current study we examined the potential of conditioned medium obtained from several types of cells to exhibit myofibroblast inactivating activity. Conditioned media from lung cancer cell lines A549 and PC9 were found to have this action, as shown by its ability to decrease alpha-smooth muscle actin expression in MRC-5 cells. Subsequently the inhibitory factor was purified from the medium and identified as 5'-deoxy-5'-methylthioadenosine (MTA), and its mechanism of action elucidated. Activation of protein kinase A and cAMP responsive element binding protein (CREB) were detected. MTA inhibited TGF-beta-induced mitogen-activated protein kinase activation. Furthermore, the gain-of-function mutant CREB caused inactivation of myofibroblasts. These results show that A549 and PC9 conditioned media have the ability to inactivate myofibroblasts, and that CREB-phosphorylation plays a central role in this process. (C) 2011 Elsevier Inc. All rights reserved.

Corneal wound healing is a complex process involving the integrated actions of various growth factors, cytokines and extracellular matrix produced by corneal cells and inflammatory cells. Connective tissue growth factor (CTGF) has been linked to wound healing, and fibronectin (FN) is a major component of the extracellular matrix. However, the functions of CTGF and FN in corneal epithelial cells are not well understood. We therefore investigated the coordinated function of CTGF and FN in the attachment and migration of corneal epithelial cells. Treatment of human corneal epithelial cells (HCECs) with transforming growth factor (TGF) beta 1 up-regulated the expression of CTGF, but did not noticeably affect FN expression, as judged by immunoblot analysis of cell lysates. In contrast, the amount of FN accumulated in the cultured media was increased in a time-dependent manner, but CTGF was undetectable in the cultured media. The expression level of FN was decreased by the knockdown of CTGF expression with a specific short hairpin RNA, indicating that CTGF acts as an upstream mediator of FN expression. CTGF augmented the FN-mediated increase in the attachment of HCEC by about twofold, although CTGF alone did not influence the attachment. Moreover, the migration assay with rabbit corneal blocks revealed that CTGF (390 nM) alone or in combination of FN (10 mu g/mL) promoted corneal epithelial migration; the mean migration distances of control, CTGF, and CTGF + FN were 272, 325, and 626, pm, respectively. In conclusion, CTGF cooperates with FN in enhancing the attachment and migration of corneal epithelial cells.

Myofibroblasts are defined as fibroblasts that express certain features of smooth muscle differentiation. Activation of myofibroblasts plays a central role in fibrosis. Transforming growth factor-beta (TGF-beta) is a potent activator of myofibroblasts; namely, TGF-beta causes changes in myofibroblast phenotypes including morphological alterations and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. Because it has been well known that humoral factors, especially, TGF-beta, and extracellular matrix components cause myofibroblast activation, we examined the expression of integrin and related proteins during activation of MRC-5 human myofibroblasts with TGF-beta. Western blot analysis revealed that TGF-beta treatment for 3 days increased the expression of alpha-SMA, which was also immunocytochemically observed as actin stress fibers. In the early phase of TGF-beta treatment, fibronectin expression was greatly increased, followed by the increased expression of integrin alpha v and alpha 11 and integrin beta 1 and beta 3. Co-immunoprecipitation assays revealed that the integrin av subunit was co-precipitated with integrin beta 1 and beta 3, and that integrin beta 1 was co-precipitated with all, alpha v, alpha 2, and alpha 5. The expression of focal adhesion kinase and integrin-linked kinase proteins was also upregulated by treatment with TGF-beta. In addition, the expression of type I collagen mRNA was increased by TGF-beta, but not type III collagen mRNA, as judged by real-time PCR analysis. These results suggest the possibility that TGF-beta induces fibronectin expression in MRC-5 cells, which subsequently induces the expression of integrin receptors, alpha v beta 3, alpha v beta 1, and alpha 11 beta 1. This report also shows that expression of integrin alpha 11 is upregulated during the TGF-beta-mediated activation of myofibroblasts.

Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role. (c) 2006 Elsevier B.V. All rights reserved.

It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in ostcoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1. (c) 2006 Elsevier Inc. All rights reserved.

Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 mu g/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 mu g/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 mu g/mL ox-LDL significantly suppressed chondrocytes viability (84.6% 3.4% and 80.9% 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% 7.1% and 85.7% 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 mu g/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% 4.9% and 48.7% 6.7% of control at 24 h, respectively (n 3; p < 0.01 when compared with individual application of cyclic loading or 10 mu g/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis. (c) 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1.(ADAMTS-4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified alpha 1-antitrypsin, a member of the family of plasma serine protemase inhibitors, as a prey. Recombinant aggrecanase-1 and alpha 1-antitrypsin were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length aggrecanase-1, and alpha 1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of otiantitrypsin against elastase, and a 1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and alpha 1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and alpha 1-antitrypsin remains unclear. (c) 2005 Elsevier B.V. All rights reserved.

5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (AIAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.

Objective:Because articular cartilage has no vascular supply, chondrocytes are hypoxic under normal physiological conditions. Nitric oxide (NO) plays an important role in chondrocyte damage, such as apoptosis. Although oxygen stress with hydrogen peroxide was found to cause chondrocyte damage, these data were obtained under normoxic (21% O-2) conditions. We investigated the effects of hypoxia on hydrogen peroxide-induced chondrocyte damage Methods:Bovine articular chondrocytes were used in this study. Proteoglycan (PG) synthesis and the induction of apoptosis were analyzed with [S-35]-sulfate incorporation and annexin V staining, respectively. The induction of NO was examined using a fluorescent probe and RT-PCR. Results:Cells maintained at 5% O-2 had the maximum PG synthesis. Under normoxic conditions, hydrogen peroxide inhibited PG synthesis and induced annexin V positive cells in a dose-dependent fashion. However, in those cells cultured under hypoxic (5%) conditions, the hydrogen peroxide-induced annexin V expression was attenuated. Chondrocytes exposed to hypoxia showed induction of NO. When the hypoxia-induced NO was inhibited, the hypoxia-enhanced PG synthesis was abolished and hydrogen peroxide clearly induced cell damage. Conclusions:Endogenous NO induced by hypoxia protects chondrocytes from apoptosis induced by an oxidative stress.

Objective: To examine the effect of oxidized low-density lipoprotein (ox-LDL) on the intracellular production of reactive oxygen species (ROS) in bovine articular chondrocytes (BACs) and to investigate whether this increase occurs through binding to the receptor lectin-like ox-LDL receptor-1 (LOX-1). Furthermore, to ascertain whether the binding of ox-LDL to LOX-1 results in NF-kappaB activation. Design: BACs were preincubated with 2',7'-dichlorofluorescin diacetate (DCFH-DA), a dye that allows the monitoring of intracellular ROS production for DCF by spectrofluorometry. BACs were incubated with native LDL and ox-LDL (10, 50, and 100 mug/ml) for 5 min at 37degreesC and DCF formation was observed. BACs were also preincubated with anti-LOX-1 mAb (40 mug/ml) or ascorbic acid (10 t!V!). Nuclear extracts from BACs treated for the indicated periods with 50 mug/ml ox-LDL, and preincubated with anti-LOX-1 mAb or ascorbic acid, were prepared and analyzed by electrophoretic mobility shift assay (EMSA). Results: ox-LDL induced a significant dose-dependent increase in ROS production after 5-min incubation with BACs (P<0.001). ROS formation was markedly reduced in BACs preincubated with anti-LOX-1 mAb and ascorbic acid (P<0.001). Activation in BACs of the transcription factor NF-κB was evident after 5-min incubation with ox-LDL and was attenuated by anti-LOX-1 mAb and ascorbic acid. Conclusion: ox-LDL binding to LOX-1 in BACs increased the production of intracellular ROS and activated NF-κB. Reduction of NF-κB activation by ascorbic acid indicates that the activation, at least in part, is ROS-dependent. These observations support the hypothesis that hypercholesterolemia is one of several risk factors for arthritis, and that lipid peroxidation products such as ox-LDL are involved in cartilage matrix degradation. © 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Objective:Mechanical stress is an essential factor in the pathogenesis of osteoarthrosis. We sought to determine whether the strain-mediated alteration in proteoglycan (PG) synthesis was modulated by nitric oxide (NO) synthesis. Methods:Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [S-35] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used. Results:Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced. Conclusions:Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis.

Objective. We have previously demonstrated that reactive oxygen species (ROS) are involved in cartilage degradation. Decreased size of hyaluronan (HA), the major macromolecule in synovial fluid, to which it imparts viscosity, is reported in patients with arthritis. The purpose of this study was to determine the alteration in the molecular weight range of HA as a result of mechanical deformation loaded on the chondrocytes, as well as the involvement of ROS in this action. Methods. ROS were generated via the oxidation of hypoxanthine by xanthine oxidase. Cyclic tensile stretch was loaded using a vacuum-operated instrument. Levels of HA were measured using a sandwich enzyme-binding assay. Superoxide dismutase (SOD) activity and ROS were measured using water-soluble tetrazolium and a chemiluminescent probe, respectively. Results. ROS depolymerized HA molecules. Cyclic tensile stretch depolymerized HA and induced ROS. SOD inhibited not only ROS induction but also HA depolymerization caused by the mechanical stress. Conclusion. ROS play an important role in mechanical stress-induced HA depolymerization.

Decorin is a member of the family of small leucine-rich proteoglycans found in the extracellular matrix and has an important role in promoting fiber formation and in controlling cell proliferation. Here, we have investigated whether the leucine-rich repeat (LRR) region of decorin interacts with proteins from human lung fibroblasts by using a yeast two-hybrid assay. We report that the LRR region of decorin interacts with the cytoskeletal protein, filamin-A (ABP-280), a peripheral cytoplasmic protein. This interaction is dependent on the 288 carboxyl-terminal amino acids of filamin-A, which correspond to repeats 22-24 of its conserved beta-sheet structure. We also show that the recombinant LRR region of decorin binds to filamin-A in vitro, and that the deglycosylated core protein of decorin coprecipitates with filamin-A, whereas intact decorin does not. Together, these results suggest that proteins containing the LRR motif that interact with filamin-A may be present in the cytoplasm or at the plasma membrane. (C) 2002 Societe francaise de biochimie et biologie moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.

A tutorial education system for medical students was introduced at Kinki University in 1998. To evaluate the efficacy and to identify problems of the system, questionnaires were given to both students and tutors. Many students (approximately 80%) enjoyed the system and felt that tutorial lectures were effective. Many students believed that their selflearning time had increased and that they had developed the ability to think scientifically. However, they also thought that the material for tutorials was insufficient and that some tutors lacked teaching ability. Tutors thought that students had developed motivation (52%), problem-solving ability (58%), and debating skills (77%). Tutors also pointed out several problems, e.g., that some students had not developed self-leaning ability. Also, some tutors were poorly motivated. These findings suggest that we need to improve tutorial materials and the quality of tutors as well as fostering the self-learning ability of students.

Contrapsin is a member of the serpin superfamily and inhibits trypsin much more strongly than alpha(1)-antiproteinase. Mouse and rat contrapsins, however, have similarity in sequence to human alpha(1)-antichymotrypsin. In order to test the hypothesis that reactive site regions of contrapsin family evolved under strong selective pressure, cDNA sequence of C57BL/6 mouse contrapsin was determined and compared with that of ICR mouse. The cDNA sequence of C57BL/6 mouse contrapsin was found to contain an open reading frame encoding polypeptide consisting of 418 amino acid residues. The work reported in this paper shows that the reactive site is not hypervariable as compared with the rest of molecule.

Sequences of fetuin cDNA and its deduced amino acid residues from the African green monkey cell line Vero were found to differ by 7.3% and 12.9%, respectively, from the corresponding human sequences. Most amino acid substitutions were clustered within a small segment of the third domain (D3). Calculations of nonsynonymous and synonymous nucleotide substitution rates suggest that this small segment was mutated under positive selection. cDNAs encoding alpha(1)-antitrypsin, beta-actin and the sequences of intron 4 of alpha(1)-antitrypsin gene in human liver and Vero cells were also investigated. The results substantiated the positive selection imposed on the D3 segment.

A 70-kDa protein, P70, found mostly in the pyramidal cells of the cerebral cortex of cobalt-induced epileptogenic rats, has been-implicated in epileptogenesis. The presence of a P70-like substance was searched for immunohistochemically in the cerebral cortex of MGS/ldr, a seizure-sensitive strain of the Mongolian gerbil (Meriones unguiculatus) that we previously established. Immunoreactive aggregates were observed in the pyramidal neurons of the motor cortex and the primary somatosensory cortex. Analysis using confocal laser:scanning microscopy revealed that the aggregates were often colocalized with a second type of aggregate with red autofluorescence at the marginal zone of the cell somata. Both aggregates appeared and increased before the appearance of generalized tonic-clonic convulsion. These may be involved in some change of physiological function of the cerebral cortex but their presence itself is not enough to determine the occurrence of epileptic seizure because the gerbils that showed no such seizure had both aggregates. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

cDNA encoding alpha(1)-microglobulin/bikunin (AMBP) was amplified from guinea pig (Cavin porcellus) liver mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods, cloned and sequenced. The deduced amino acid sequence was found to be homologous to the sequence of AMBP of other mammals (69-76% amino acid identity). It has two Kunitz-type trypsin inhibitor domains in the bikunin part as reactive sites, one in the N-terminal region and another in the C-terminal region. The N-terminal inhibitor domain sequence is well-conserved, but the P1 residue of the C-terminal inhibitor domain sequence was found to be Gin rather than Arg, a residue highly conserved in the AMBP of seven other mammals examined to date. By RT-PCR and nested PCR, AMBP mRNA was detected not only in liver tissue, previously known to be a site of its synthesis, but also in pancreas, stomach, small intestine, colon, lung, spleen, kidney, testis, skeletal muscle, and leukocytes, but not in brain or heart. We examined the AMBP mRNA levels in guinea pig liver by RT-PCR, comparing normal levels and those in a state of inflammation. The mRNA levels, however, did not significantly change. (C) 1999 Elsevier Science Inc. All rights reserved.

cDNA encoding alpha(2)-HS glycoprotein was amplified from guinea pig liver mRNA by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends, cloned and sequenced. By RT-PCR and nested PCR, alpha(2)-HS glycoprotein mRNA was detected not only in liver tissue but also in pancreas, stomach, small intestine, colon, spleen, kidney, testis, skeletal muscle, brain, heart and leukocytes, but not in the lung. The alpha(2)-HS glycoprotein mRNA levels in the liver were reduced to half at 48 h after subcutaneous injection of turpentine oil.

Of 18 monoclonal Bence Jones proteins purified from urine of patients with multiple myeloma, 4 were found to have a DNA-nicking activity. In contrast, all Bence Jones proteins tested showed detectable amidase activity against carbobenzoxy-L-valyl-glycyl-L-arginine p-nitroanilide. No correlation between the two activities was found. Four patients excreting Bence Jones protein with the DNA-nicking activity showed somewhat severe symptoms, suggesting that this activity may be related to the progressive deterioration of clinical status. (C) 1998 Academic Press.

Complementary DNA clones coding for countertrypin were isolated from a liver cDNA library of the Mongolian gerbil, and sequenced, They contained one open reading frame encoding 348 amino acid residues, which were assigned to consist of an 18-residue signal peptide and a 330-residue mature protein, The amino acid sequence was about 74% identical with mouse countertrypin and rat fetuin, 60% with bovine fetuin, and 55% with human alpha(2)HS glycoprotein, indicating that this protein belongs to the mammalian fetuin family, The members of this family are known to consist of three domains, i.e., two tandemly arranged cystatin domains (D1 and D2) and an unrelated domain (D3) located at the C-terminal region, When compared with the other members of this family, D3, especially its N-terminal half, varies greatly with deletion or insertion as well as nucleotide substitutions even among three rodent species, i.e., gerbil, rat, and mouse, The sequence comparison also suggests that the conformation of human alpha(2)HS glycoprotein differs greatly from that of other members of this family, A molecular phylogenetic tree of 7 members, constructed on the basis of the synonymous substitution rate of D1 and D2, shows that the gerbil gene diverged prior to the separation of mouse and rat.

We have sequenced serum albumin cDNA from liver of the Mongolian gerbil, Memories unguiculatus. The deduced amino acid sequence showed 82.6% and 73.6% identity with the corresponding proteins from rats and humans, respectively. Identical cDNA was detected in pancreas by reverse transcription followed by polymerase chain reaction (RT-PCR). Further amplification of cDNA by nested PCR revealed the presence of the same cDNA in the brain and kidney. These results indicate that serum albumin is expressed in some extrahepatic tissues. In rats, an albumin-related 70-kDa protein (P70) has been proposed to be associated with cobalt-induced epilepsy (Onozuka et al. (1995) Neurochem. Res., 20, 901-905). We intensively searched for a P70-like protein in the brain of an epilepsy-prone gerbil strain, MGS/Idr, by the RT-PCR and nested PCR using several pairs of primers based on the albumin cDNA sequence. However, we found only mRNA for albumin itself.

Members of mammaliar, fetuin family, such as human alpha(2)HS glycoprotein and bovine fetuin, consist of three domains, two tandemly arranged cystatin-like domains and an unrelated domain, but they have no inhibitory activity against cysteine proteinases. We found that countertrypin, a novel trypsin inhibitor, is mouse counterpart of human alpha(2)HS glycoprotein, and that human alpha(2)HS glycoprotein and bovine fetuin which were prepared without use of ethanol are capable of inhibiting trypsin (Yamamoto, K. and Sinohara, H. (1993) J. Biol. Chem, 268, 17750-17753). In the present study, cDNA encoding countertrypin was isolated and sequenced, and evidence is presented, based on the site-directed mutagenesis, that lysine-231 in the second cystatin domain is the P1 site for trypsin inhibition.

Complementary DNA clones encoding plasma alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) were isolated from Syrian hamster liver cDNA library and sequenced. The deduced amino acid sequence of putative reactive site (P3-P'3) was Ile-Pro-Met-Ser-Val-Pro, characteristic of alpha-1-antiproteinase of orthodox type (Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932). A molecular phylogenetic tree of all known orthologous proteins was constructed based on the synonymous substitution rate. The result shows that the hamster has branched off first before the divergence among mice, rats, and gerbils, and that the rabbit is the closest relative of the guinea pig which is separated from the rodents. Although this tree differs largely from the classical phylogeny based on the morphology (hamsters and gerbils belong to the same family, Cricetidae, and the guinea pig belongs to the order Rodentia), it lends support to recent concepts that the hamster and guinea pig differ, in a number of biochemical features, not only from each other but also from mice and rats, and that the guinea pig may belong to an order distinct from Rodentia.

alpha-1-Antiproteinase (also called alpha-1-proteinase inhibitor or alpha-1-antitrypsin) with a molecular mass of 56 kDa was purified from plasma of the Mongolian gerbil, Meriones unguiculatus, to apparent homogeneity. It inhibited trypsin, chymotrypsin, elastase, and plasmin, but not kallikrein or thrombin. Eight cDNA clones coding for this protein were isolated from a liver cDNA library and sequenced. They contained the same coding regions consisting of a 24-residue signal peptide and a 382-residue mature protein. The reactive site sequence (P3-P'3) was Val-Pro-Met-Ser-Ile-Pro, characteristic of alpha-1-antiproteinase of orthodox type [Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932]. A molecular phylogenetic tree of 11 orthologous inhibitors, constructed on the basis of the synonymous substitution rate, shows (i) that the reactive site region is highly conserved as compared to the other part of the molecule, which contrasts with the generally accepted view that the reactive site region of serpins is strongly hypervariable, and (ii) that the myomorphs (gerbil, rat, and two species of mouse, i.e. Mus domesticus and Mus caroli) and the caviomorph (guinea pig) fail to consist of a monophyletic order, which also contradicts the traditional taxonomy based on the morphology. In the present tree, the guinea pig joins the lagomorph (rabbit), and is rather widely separated from the myomorph branch. The result, however, supports the recent hypothesis based on the molecular evolution of several other proteins that the guinea pig does not belong to the same order as the myomorph, and the caviomorphs should be elevated in taxonomic rank and conferred an ordinal status distinct from the rodents.

The cDNAs encoding two isoforms, S (slow) and F (fast), of α1-antiproteinase (also referred to as α1-antitrypsin or α1-proteinase inhibitor) as well as contrapsin were obtained by screening λgt11 cDNA library prepared from inflamed guinea pig liver. The sequence analyses of these cDNAs and NH2-terminal peptides of the purified proteins revealed that both isoforms of α1-antiproteinase consist of 405 amino acid residues including a signal peptide of 24 residues and that contrapsin consists of 410 amino acid residues with the same length of the signal peptide. Guinea pig contrapsin had 89, 88, 62, 42, and 41% homology to its own α1-antiproteinases F and S, rat α1-antiproteinase, mouse and rat contrapsins, respectively. This suggests that guinea pig contrapsin is not orthologous to mouse and rat contrapsins and that it developed from a much later duplication of α1-antiproteinase gene after the guinea pig had diverged from the murine lineage. The available data suggest that the reactive site region of α1-antiproteinase can be categorized into orthodox and unorthodox types: the former has P3-P′3 consensus sequence of Xaa-Pro-Met-Ser-Xaa-Pro, where Xaa is Leu, Ile, Val, or Met, while the latter, which occurs in species having multiple α1-antiproteinase isoforms, has the sequence whose P1 Met has changed to other amino acids. Thus, the reactive site region of the orthodox type, which occurs in all seven mammals examined to date, is highly conserved. This is in marked contrast to the fact that the same region is hypervariable among the paralogous proteins belonging to the serpin superfamily.

We reported a rare case of plasma cell dyscrasia with a variety of μ-heavy chains in the serum. Monoclonal 19S (pentamer) and 7S (monomer) IgM-κ, Bence-Jones protein-κ and μ- heavy chains polymers were detected in the serum. Molecular weights of these μ-heavy chains were revealed as proteins with 86K, 69K 55K and 46K by immunoblotting method. 74K μ-heavy chain with normal molecular weight was not detected in the serum.
The bone marrow was occupied (42%) by plasma cell, which had a few large vacuoles in the cytoplasm. The cell contained μ-heavy chains, whose molecular weights were revealed 74K of normal μ-heavy chain and 56K of free μ-heavy chain. In the urine, Bence-Jones protein-κ and 33K μ-heavy chains were detected. Having obtained these data described above, it was suggested that some modification of processing for μ-heavy chain synthesis and secretion occured in the cytoplasm. Although plasma cells in bone marrow increased (52%), he had no symptoms without therapy. It was considered that this case was very rare in plasma cell dyscrasia, and was interested which mechanism of IgM segmentation occured.

[背景/目的]癌間質組織における細胞成分のなかで最も多く存在する癌関連線維芽細胞(CAF: Cancer-associated fibroblast)は、癌細胞と連携し癌を進展させると考えられている。癌間質へCAFを動員させる制御因子の解明が、効率的な癌治療に繋がることが期待される。[対象/方法]非小細胞肺癌組織よりCAFの分離培養を行い、正常肺線維芽細胞を対照とした遊走能を解明した。[結果]CAGE(Cap Analysis of Gene Expression)によるトランスクリプトームの網羅的解析では、ITGA11(integrin alpha 11)の発現が蛋白レベルとともに上昇した。免疫組織染色では、肺癌組織間質中におけるITGA11の発現が術後再発や病期分類と関連した。また、CAFはfibronectinに対する遊走能が亢進しており、その遊走能はITGA11の発現との関連を認めた。さらに肺癌細胞株の培養上清液は肺線維芽細胞のITGA11の発現をより上昇させ、CAFの培養上清液は肺癌細胞の遊走をより亢進させた。[結語]CAFは、癌細胞との相互作用においてITGA11-fibronectinカスケードを介して癌間質へ動員され、このカスケードは癌進展の制御機序となりうることがわかった。

臓器線維症の原因の1つとして上皮系の細胞が間葉系の性質を獲得する上皮間葉転換(EMT)がある。 EMTは TGF-βによって誘導される。また epigallocatechin gallate (EGCG)はカテキンの1種で抗癌作用、抗アレ ルギー作用など様々な生理活性を持つ物質である。細胞性線溶は細胞表面に存在する受容体を介し細胞機能に影 響を与えることが明らかになりつつある。細胞性線溶に関与する urokinase-type plasminogen activator receptor (uPAR)の遺伝子を導入し、 uPARを高発現させたヒト角膜上皮細胞(HCEC： Human Corneal Epithelial Cell)を用いて、 TGF-βの EMT誘導作用および EGCGの TGF-β活性に及ぼす効果について、細胞形態の観察やウ エスタンブロット法などにより検討した。(方法)プラスミドベクター pEBに uPAR 遺伝子を挿入したものを HCECに導入し、 uPARを安定発現するようになった細胞（ HCEC＋ uPAR）を用いた。 pEBのみを導入した細胞 （ HCEC+Empty）を対照として使用した。 HCEC+Emptyと HCEC+uPARをそれぞれ培養し、培地中に TGF-β を1ng/mlと EGCGを様々な濃度で同時に添加したとき、 EMTにどのような影響を与えるかを検討した。試薬を添 加して48時間経過後にセルライセート調製を行った。また、 TGF-βの経時的な作用の検討を行うため、添加 24時間、48時間、72時間でそれぞれセルライセート調製を行った。ウエスタンブロット法にてマーカータンパク 質の発現量を解析した。間葉系細胞のマーカーとして Fibronectin(FN)、α-smooth muscle actin(SMA)、上皮系 細胞のマーカーとして E-cadherinを用い、 Glyceraldehyde 3-phosphate dehydrogenase(GAPDH)を内部標準 とした。(結果) タンパク質発現量の定量化を行うと、 TGF-βと EGCGを同時添加した際、+Emptyと比較して +uPARにおいて FNと E-cadherinついては大きな差異は見られず、α-SMAについては+uPARにおいて発現が抑制 される傾向にあった。また、 TGF-βの経時的な影響については E-cadherinは、+Emptyで減少し、+uPARで増加 した。(考察) TGF-βと EGCGを同時添加すると FNおよび E-cadherinの発現については HCEC+Emptyと HCEC+uPARに差異は認められなかったことから EGCGは TGF-β活性に影響を与えなかったものと考える。一 方、α-SMAの発現については HCEC+uPARにおいて抑制される傾向にあることから EGCGが TGF-βによる EMTに影響を与えたと考えられる。+Emptyにおいて TGF-βが時間依存的に E-cadherinの発現を減少させたこと から uPARは E-cadherinの発現を促進させると考えられる。また、細胞形態においてはいずれも変化は見られな かった。

尿細管上皮細胞の上皮間葉転換(Epithelial Mesenchymal Transition : EMT)は、腎線維化の一因であると考えられている。EMTを誘導する因子の1つとしてとして、transforming growth factor (TGF)-βが知られている。カテキンの一種であるエピガロカテキンガレート(EGCG : epigallocatechin gallate)は、様々な生理作用活性をもつが、EGCGの抗線維化作用についてはよくわかっていない。NRK-52E(ラット由来尿細管上皮細胞)を用いて、TGF-βによるEMTの誘導、また、EGCGによるEMTの抑制効果について検討し、EGCGの腎線維化治療への応用を模索する。培地にTGF-βのみを添加した場合、FN、α-SMAの発現が増加した。また、EMT誘導の程度はTGF-β濃度に依存的であった。E-cadの発現は、変化しなかった。これらの結果より、TGF-βは、NRK-52Eに対しEMTを誘導したと考えられる。TGF-βとEGCGを同時に添加した場合、FN、E-cadの発現解析結果よりEGCGはTGF-βの作用を抑制したと考えられる。また、EGCGはα-SMAの発現にはほとんど影響しなかった。このことはEGCGがTGF-βのシグナル伝達経路のすべてを抑制するわけではないことを示唆する。以上の結果から、EGCGは腎線維化の治療薬候補になりうると考える。

臓器線維症はコラーゲンをはじめとする細胞外マトリックスが過剰に増加し、臓器が機能不全に陥る疾患で、その有効な治療法は未だ確立されていない。臓器線維化を惹起するサイトカインとしてTGF-βがあり、緑茶に含まれるエピガロカテキンガレート(epigallocatechin gallate: EGCG)はTGF-βの作用を抑制することが知られている。しかし、上皮間葉転換(epithelial mesenchymal transition: EMT)におけるTGF-βとEGCGの関係には不明な点が多い。そこで本研究では培養ヒト角膜上皮細胞(human corneal epithelial cells: HCECs)を用いてTGF-βのEMT誘導作用、EGCGのEMT抑制効果、EMTの可逆性について検討し、EGCGによる臓器線維症治療の可能性を探る。TGF-βはHCECsに対してEMTを誘導したと考えられるが、典型的なEMT様変化は呈さなかった。EGCGはEMTを抑制するが、高濃度では細胞毒性をもつ可能性がある。

TGF-βはHCECに対しEMTを誘導したと考えられるが、典型的なEMT様変化は呈さなかった。EGCGはそのEMTを抑制する作用をもつと推察される。

Transforming growth factor-beta (TGF-β)は、epithelial-mesenchymal transition (EMT)の主要関連分子のひとつであり、TGFβはEMTを促進し、細胞の移動、浸潤に関与している。今回我々は uPAのヒト培養網膜色素上皮細胞(ARPE19)のTGFβを介したEMTおよびtype1コラーゲンゲル内遊走に与える影響について検討した。ゲル内にARPE19を包埋し、uPAがTGFbeta2による細胞形態変化に与える影響および、ゲル内細胞遊走評価モデルにより、TGFb処理ARPE19のゲル内遊走抑制効果について検討した。TGFb刺激によるuPA, uPA receptor (uPAR)の発現をfibrin enzymography, western blot法およびreal time PCR法を用いて検討した。ARPE19とuPAの結合反応を生体分子間相互作用解析装置でそれぞれ検討した。ゲル内の細胞はTGFb刺激により線維芽細胞様の形態を示し,その形態変化はuPA阻害剤により抑制された。TGFb(10ng/ml)刺激によりuPAの産生は増加した。ゲル内に遊走する細胞数はuPA阻害剤により有意に抑制された（p<0.01）。uPA, uPARは、TGFbの刺激により依存的に発現上

原因不明の角膜潰瘍による角膜穿孔症例に対するurokinase type plasminogen activator(uPA),uPA receptor(uPAR),alpha-2-antiplasmin(A2AP)の角膜内局在について検討した。 角膜移植時に得られた角膜片のパラフィン切片を作成し、HE染色、蛍光染色を行った。HE染色では角膜潰瘍部に多数の炎症細胞と線維芽細胞を認めた。蛍光二重染色では同部位にuPA/uPAR陽性の細胞浸潤を認めた。A2APは角膜潰瘍周囲にみられた角膜瘢痕部に存在するαSMA細胞に多く共発現を認めた。

臓器の線維化は、組織が障害された後の修復過程においてコラーゲンをはじめとする細胞外マトリックス（ECM）が過剰に沈着し、臓器の機能障害をきたす病態である。plasminはfibrinを分解して血栓溶解作用を示すが、それ以外にECMを構成するフィブロネクチンやプロテオグリカンを分解する機能をもつ。その活性はセリンプロテアーゼインヒビター（セルピン）のひとつであるα2-antiplasmin(AP)によって抑制される。APが皮膚組織における線維化を促進することが報告されているが、その機序については不明な点も多い。APが組織線維化にどのように関与するのかを明らかにするために、APと相互作用するタンパク質を探索した。 APのsignal peptideを除いた部分をbaitタンパク質として、ヒト肺線維芽細胞 (WI-38)由来cDNA libraryに対して酵母two-hybrid法によるスクリーニングを行った。 その結果、マトリックスメタロプロテイナーゼ2 (MMP-2) がpreyタンパク質のひとつとして同定された。APとMMP-2の結合は

インテグリンα11β1はコラーゲン結合性のインテグリンで、歯根膜線維芽細胞において歯の萌出時に必要とされること、また、αサブユニットであるインテグリンα11 (ITGA11) は非小細胞肺癌において高発現していることなどが報告されているが、その他の生理的・病理的役割についてはよくわかっていない。線維化を促進する主要な分子であるTGF-βでヒト肺線維芽細胞を刺激したとき、ITGA11の発現が著明に亢進することを我々はプロテオーム解析で見出した。今回我々はITGA11の細胞内ドメインと相互作用するタンパク質を探索し、線維化との関連を検討した。 ITGA11の細胞内ドメインをbaitとして、ヒト肺線維芽細胞cDNA libraryに対して酵母two-hybrid法によるスクリーニングを行った。その結果、calcium and integrin binding protein 1 (CIB1) がpreyタンパク質のひとつとして同定された。ITGA11との結合にはCIB1のC末側で2か所のEF-hand motifを含む領域が必要であった。ITGA11とCIB1の結合は免疫沈降法でも確認された

connective tissue growth factor(CTGF)がフィブロネクチンによる角膜上皮細胞の接着と角膜上皮の伸長にどのような作用を与えるかを培養ヒト角膜上皮細胞（ＨＣＥＣ）とウサギ角膜ブロックを用いた角膜器官培養法により検討した。

目的： connective tissue growth factor（CTGF）はフィブロネクチン（FN）と結合し細胞活性に影響を与えることが指摘されている。今回、角膜上皮の創傷治癒に対するCTGFとFNの相互作用を検討した。対象と方法：不死化ヒト培養角膜上皮細胞（HCEC）を用いた。CTGFを特異的に抑制するshRNAプラスミドを用いて、HCECのCTGF、FN産生量を検討した。またHCECの接着能に対するFN、CTGFの相互作用を検討するため、FNとBSAをコーティングしたプレートに、CTGF を含む培養液で24時間培養したHCEC1x103を播種し、45分後に接着細胞数をcountした。角膜上皮の伸長に対するFNとCTGFの相互作用を検討するため、角膜器官培養法を用い、CTGFとFNを単独または同時に添加し、24時間後に角膜片の伸長を測定した。結果：CTGF特異的shRNAプラスミド導入後48時間でCTGF、FNの産生量は低下した。細胞接着能はFNコート群、BSAコート群のいずれにおいてもCTGFの刺激により、接着能は増強した。角膜器官培養では、CTGF単独群、CTGF＋FN群ともに

MRC-5細胞において、細胞骨格タンパク質の1種であるtransgelinをノックダウンするとtransgelinのみならずα-smooth muscle actin(SMA)の発現も低下し細胞形態が変化した。MRC-5細胞をTGF-β処理した場合のSMAやfibronectinの誘導発現も抑制された。

近畿大学薬学部・医学部合同学習会におけるPBL教育に関する報告。

結合組織成長因子(CTGF)がフィブロネクチン(FN)と結合することを酵母two-hybrid法を用いて見出した。両者の結合に関わる領域はCTGFのC-terminal domainとFNのtype I repeat領域であった。この結合は表面プラズモン法とELISA法でも確認された。また、CTGFはFNのfibrinへの結合を促進し親和性を増強した(英文）。

To identify an endogenous inhibitor of aggrecanase-1 (ADAMTS-4), we performed a yeast two-hybrid screen using the catalytic domain of human ADAMTS-4 as a bait, and transformed an EGY48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified α1-antitrypsin (AT), a member of the family of plasma serine proteinase inhibitors, as a prey. Recombinant ADAMTS-4 and AT were expressed in mammalian cells and the cell lysates were used in coimmunoprecipitation experiments, which showed that full-length ADAMTS-4 and AT are also associated in vivo. However, ADAMTS-4 did not interfere with the inhibitory activity of AT against elastase, and AT had no effect on the proteolytic activity of ADAMTS-4. Taken together, these data suggest that ADAMTS-4 and AT bind in vivo, although the physiological significance of the interaction between ADAMTS-4 and AT remains unclear.(英文)

結合組織成長因子(CTGF)がフィブロネクチンと結合することを酵母ツーハイブリド法で発見した。その結合にはCTGFのドメイン4とフィブロネクチンのtypeI repeat領域が関与する。（英文）

アグリカナーゼ-1(ag-1)と相互作用するタンパク質を酵母two-hybrid法を用いて探索したところα1-antitrypsin(AT)がpreyタンパク質として同定された。この結合は免疫沈降法でも確認された。ag-1はATのエラスターゼ阻害活性に影響を与えず、ATはag-1のaggrecan分解活性を阻害しなかった（英文）。

20週齢のWKY，SHRSPの胸部大動脈を用いて，NADPH oxidaseサブユニット（Nox1, Nox2, p22phox, p47phox）mRNA発現を比較したところ，すべてのサブユニットにおいてSHRSPで発現の亢進が認められた．また，胸部大動脈のsuperoxide産生もSHRSPで亢進していたことから，高血圧の進展に活性酸素種が関与していることが明らかになった．

６および２０週齢のWKYおよびSHRSPを用いて，胸部大動脈のNO合成酵素mRNAの発現について比較したところ，差異は認められなかった．

アグリカンの分解に関与する主要な酵素であるaggrecanase-1と相互作用するタンパク質を酵母two-hybrid法を用いて探索したところ、ヒト肝臓cDNAライブラリーからα1-antitrypsin、α1-antichymotrypsinなどのセリンプロテアーゼインヒビターがpreyタンパク質として同定された（英文）。

プロテオグリカンであるデコリンのleucine-rich repeat配列と相互作用するタンパク質の探索を酵母two-hybrid法を用いて行った。preyタンパク質としてアクチン架橋タンパク質のひとつであるfilamin-Aが同定された。

アグリカンの分解に関与する主要な酵素であるaggrecanase-1と相互作用するタンパク質を酵母two-hybrid法を用いて探索したところ、ヒト肝臓cDNAライブラリーからα1-antitrypsin、α1-antichymotrypsinなどがpreyタンパク質として同定された。

デコリンのロイシンリッチリピート配列と相互作用するタンパク質を酵母two-hybrid法を用いて探索したところ、ヒト線維芽細胞cDNAライブラリーからfilamin(AB-280)、Mss4などが同定された。

In EMT (epithelial-messenchymal transition) study, retinal pigment epithelial cells have been the main research subject and findings of EMT on retinal pigment epithelial cells have been accumulated. Contrarily, there are few reports, so far, on EMT induced by uPAR through the fibrinolytic system. Therefore, in this study, uPAR was stably introduced into human retinal pigment epithelial cells (ARPE-19), and EMT induced by uPAR was investigated. In consequence, EMT-like changes could not be recognized in ARPE-10 by stably introduced uPAR alone, and collagen gel contraction could not be recognized, either. EMT could not be induced in the retina with a single stimulation of increased expression of uPAR. Involvement of growth factors like TGF-β and cytokines like interleukin should also be considered in the future study.

When corneal fibroblast (keratocyte) was stimulated by TGF-beta, the keratocytes showed myofibroblast-like morphology. In addition, the expression of integrin alpha11 (ITGA11), alpha-smooth muscle actin (alpha-SMA), fibronectin in keratocytes was increased by TGF-beta treatment. Sensitivity to TGF-beta was up-regulated in the cells constitutively expressing ITGA11 or calcium-and integrin-binding protein 1. It was suggested that ITGA11 and CIB1 could be the target protein of the organ fibrosis treatment.Furthermore, we found that the green tea component epigallocatechin-3-gallate (EGCG) binds to TGF-beta type II receptor and inhibits the action of TGF-beta.

Activation of myofibroblasts was inhibited when the expression of transgelin was knocked down. When human lung fibroblasts were stimulated by TGF-β, the integrin α11 (ITGA11) expression was markedly increased. Calcium and integrin binding protein 1 (CIB1) bound to the intracellular domain of ITGA11. The expression of CIB1 increased in human lung fibroblasts treated with TGF-β and in bleomycin-treated mice pulmonary tissues. These results suggest that CIB1 may be involved in the fibrosis.

Previously we showed that the expression of integrin aω, β1, β3 in MRC-5 cells increased by TGF-β. Increased expression of fibronectin and focal adhesion kinase was also shown. Here, we show the results of proteomics analysis of activation and inactivation of myofibroblast cell line, MRC-5. Transforming growth factor-b stimulated MRC-5 cell extract was compared with that from non-stimulated cells. Several proteins differentially expressed between stimulated and non-stimulated cells including actin-bindin proteins and molecular chaperones.As previously reported the culture supernatant of A-549 inactivates myofibroblast MRC-5. RNA was obtained from MRC-5 cells after inactivation by the conditioned medium of A-549 and the expressions were analyzed using low density arrays. Expression of some proteins concerned in cell protrusions was increased. The cells treated with forskolin were subjected to proteome analysis. Inceased expression of actin capping protein was observed.These results, together with previous our findings, suggests that signal transduction mediated by interantions between extracellular matrix and integrins are important in activation and inactivation of myofibroblasts.

Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role."

Aggrecan is greatly responsible for bad-bearing and shock-absorbing junction of the joint cartilage. In degradative articular diseases such as rheumatoid arthritis(RA) and osteoarthritis(OA), loss of extracellular matrices occurs resulting in the destruction of joint cartilage. Aggrecan proteolysis is one of the early events leading to the breakdown of the extracellular matrix. Matrix metalloproteinases have been reported to be the key enzymes in cartilage degeneration. Recent studies show that aggrecanase-1 and aggrecanase-2 are members of the ADAMTS(a disintegrin and metalloproteinase with thrombospondin motifs) family, and these enzymes are considered to play a pivotal role in the abrasion of cartilage aggrecan in RA or OA. Tissue inhibitor of matrix metalloproteinase-3(TIMP-3) is a potent inhibitor of aggrecanase-1 and -2, but whether TIMP-3 is the only inhibitor of aggrecanases remains unknown. The identification of a specific endogenous aggrecanase inhibitor therefore would be of critical importance in considering new medication for degradative joint diseases. The aims of the current study were to identify the endogenous inhibitor of aggrecanase-1 and to characterize the mechanism of interaction between aggrecanase-1 and its endogenous inhibitor We performed the yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait, and an EGY48 yeast strain containing the bait plasmid was transformed with a human liver cDNA library plasmid. We identified alpha-1-antitrypsin and alpha-1-antichymotrypsin, members of plasma serine proteinase inhibitors, as preys.

5-aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS-N) is regulated by heme. Recently, a short amino acid sequence, the heme regulatory motif (HRM), has been shown to be involved in the hemin inhibition of protein transport in vitro. To elucidate the role of HRM in the heme regulation of ALAS transport in vivo, we constructed a series of mutants of rat ALAS-N in which the specific cysteine residues within the HRMs were converted to serines by site-directed mutagenesis. Wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, followed by analyses of the mitochondrial import of the enzymes. The heme inhibition, which was observed in the wild-type ALAS-N, abolished completely when all the three HRMs in the enzyme were mutated, indicating that the HRM is actually required for the heme inhibition of ALAS-N transport within the cells. In contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS (ALAS-E) under the comparable experimental conditions. Mitochondrial import of the deletion mutant of ALAS-N in which 40 amino acids between HRM2 and HRM3 are removed was not affected by heme. Then, fusion protein of presequnce of ALAS-E and mature enzyme of ALAS-N was made. Heme inhibited the mitochondrial import of the fusion enzyme. These results may reflect the difference in the physiological function between two ALAS isoforms.