RA175/TSLC1/SynCAM/IGSF4A (RA175), a member of the immunoglobulin superfamily with Ca 2؉ -independent homophilic trans-cell adhesion activity, participates in synaptic and epithelial cell junctions. To clarify the biological function of RA175, we disrupted the mouse Igsf4a (Ra175/Tslc1/SynCam/Igsf4a Ra175) gene. Male mice lacking both alleles of Ra175 (Ra175 ؊/؊ ) were infertile and showed oligo-astheno-teratozoospermia; almost no mature motile spermatozoa were found in the epididymis. Heterozygous males and females and homozygous null females were fertile and had no overt developmental defects. RA175 was mainly expressed on the cell junction of spermatocytes, elongating and elongated spermatids (steps 9 to 15) in wild-type testes; the RA175 expression was restricted to the distal site (tail side) but not to the proximal site (head side) in elongated spermatids. In Ra175 ؊/؊ testes, elongated and mature spermatids (steps 13 to 16) were almost undetectable; round spermatids were morphologically normal, but elongating spermatids (steps 9 to 12) failed to mature further and to translocate to the adluminal surface. The remaining elongating spermatids at improper positions were finally phagocytosed by Sertoli cells. Furthermore, undifferentiated and abnormal spermatids exfoliated into the tubular lumen from adluminal surfaces. Thus, RA175-based cell junction is necessary for retaining elongating spermatids in the invagination of Sertoli cells for their maturation and translocation to the adluminal surface for timely release.RA175/TSLC1/SynCAM/IGSF4A (RA175) is a new member of the immunoglobulin superfamily that has three immunoglobulin domains in its extracellular region and EYFI, a type II PDZ (postsynaptic density 95/disk large/zonula occluders-1)-binding domain, in its C-terminal intracellular region and has Ca 2ϩ -independent homophilic trans-cell adhesion activity (10). RA175 was at first isolated as one of the genes preferentially expressed during the neuronal differentiation of P19 mouse embryonal carcinoma (P19 EC) cells induced by retinoic acid (9, 21) and is localized in the mouse developing nervous system and epithelium of various organs (2,10,11,29). In the nervous system, RA175 is designated the synaptic cell adhesion molecule (SynCAM) and has been suggested to be involved in the formation of functional synapses (2). The localization of RA175 also suggests its biological role in the migration of neurons and the fasciculation of axons (11). In the developing lung epithelium, RA175 is localized in the celladherent region of the basolateral membrane in the polarized cells lining the lumen (10). Defects in IGSF4A/tumor suppressor gene in lung cancer 1 (TSLC1), an orthologue of mouse Igsf4a (Ra175), promote the metastasis of lung carcinoma cells (18). However, little is known about its real biological function during organ development. To clarify the biological function of RA175, we disrupted the murine Ra175 gene. Male mice lacking both alleles of Ra175 (Ra175 Ϫ/Ϫ ) were infertile, whereas heterozygous males...

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Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.

Hepatic stellate cells (HSCs) were changed in their morphology, proliferative activity, and functions by culturing on type I collagen gel, as compared to the culture on polystyrene surface. HSCs have been found to produce extracellular matrix components and matrix metalloproteinases (MMPs). In this study, we have assessed the effects of several types of substrata on the expression of MMPs in HSC culture. MMP-1 expression was detectable in HSC culture on polystyrene surface and on type I collagen gel by immunofluorescence staining and reverse transcriptase-polymerase chain reaction (RT-PCR). The results from in situ zymography revealed the presence of interstitial collagenase activity around HSCs and along their cellular processes. Although proMMP-2 and proMMP-9 were detectable by gelatin zymography in the conditioned medium from both cultures using type I collagen gel and Matrigel as substratum, an active form of MMP-2 but not of MMP-9 was detected only in the culture using type I collagen as a substratum. Tissue inhibitor of metalloproteinase-2 expression was observed by RT-PCR in HSCs cultured on or in type I collagen gel, suggesting the suppression of MMP-2 activity detected in HSC culture using type I collagen. These results indicate a differential expression of MMP activity, hence the remodeling of extracellular matrix components is dependent on the substratum used for HSC culture. The HSC culture using several types of substrata appears to be a useful in vitro model to study the mechanism of extracellular matrix remodeling.

Intestinal ischemia/reperfusion (IIR) is a critical and triggering event in the development of distal organ dysfunction, frequently involving the lungs. Respiratory failure is a common cause of death and complications after intestinal I/R. Stress protein heme oxygenase-1 (HO-1) confers the protection against a variety of oxidant-induced cell and tissue injuries. The aim of this study was to investigate the hypothesis that the induced HO-1 expression by pharmacological preconditioning with anticancer drug doxorubicin (Dox) could protect the lung injury induced by intestinal I/R. Intravenous administration of Dox induced HO-1 expression in the lungs and high levels of the expression were sustained at least to 48 h after the injection. Therefore, as pharmacological preconditioning, a low dose of Dox was injected intravenously into rats at 48 h before the start of intestinal ischemia. Rats underwent intestinal I/R by superior mesenteric artery occlusion for 120 min followed by 120 min of reperfusion. Preconditioning with Dox significantly ameliorated the lung injury induced by the intestinal I/R. Administration of a specific inhibitor of HO activity reduced the efficacy of the preconditioning. Our results suggest that this improvement may be mediated at least in part by the HO-1 induction. These findings may offer interesting perspectives for patient management In Intestinal surgical operation and intestine transplantation.

methylation reactions in vivo and also serves the propylMammalian S-adenosylmethionine (AdoMet) synthetase examino donor in polyamine biosynthesis. AdoMet synthetase ists as two isozymes, liver-type and nonhepatic-type enzymes, exists as two isozymes, liver-type and nonhepatic-type enwhich are the products of two different genes. It is known that zymes, respectively. The liver-type isozyme is confined to the liver-type isozyme is only expressed in adult liver. Whereas, the liver, whereas the nonhepatic-type isozyme is widely disthe nonhepatic-type isozyme is widely distributed in various tributed in various tissues examined and faintly detected in tissues. In addition to the liver-type isozyme, a minor amount adult liver. [2][3][4][5][6][7][8] We have recently reported that the nonhepaticof the nonhepatic-type isozyme is also detected in adult liver. To investigate the distribution of these two isozymes in the type isozyme messenger RNA (mRNA) predominantly exists liver in detail, the localization of these two isozymes was exam-in fetal human liver and is replaced by the liver-type isozyme ined in each cell type of liver using a combination of cell mRNA during development. 9 In addition, we have also refractionation technique and Western blot analysis. In the paren-ported that the nonhepatic-type isozyme mRNA is obviously chymal cells, the liver-type isozyme protein was predominantly induced by 3-methyl-4-dimethylaminoazobenzene adminisexpressed, and a small amount of the nonhepatic-type isozyme tration as a carcinogen in adult rat livers.10 From these results protein was also detected. On the other hand, in the stellate we proposed that the nonhepatic-type isozyme seemed to be cells the nonhepatic-type isozyme protein was exclusively or an oncodevelopmental marker in the hepatocarcinogenesis. only expressed. Interestingly, a large amount of both isozymes However, these results were obtained from whole liver tiswere present in endothelial and Kupffer cell fraction. Using sues. Particularly, in the case of tumor tissues we could not both antibodies to anti-rat nonhepatic-type and liver-type iso-exclude the possibility that specimens of hepatomas used zymes, respectively, immunohistochemical analysis clearly con-contained normal parts near the tumor region. In addition, firmed these results. In addition, in cultured hepatocellular liver tissue is mainly composed of several cell types, parencarcinoma cells (FAA-HTC1), the nonhepatic-type isozyme pro-chymal cells (hepatocytes), endothelial cells, Kupffer cells, tein only was detected, and the liver-type isozyme protein com-pit cells, and stellate cells (fat-storing cells, Ito cells, or lipopletely disappeared. This result indicates that the changes in cytes). Therefore, it seems of particular interest to clarify the the isozyme expression is regulated within the parenchymal localization of these two isozymes in normal rat liver using cells. Administration of hepatotoxic drug carbon tetrachloride more appropriate methods. (CCl 4 ) to rats resulted in about 40% to 50%...

Cultured hepatic stellate cells were induced to elongate long, multipolar cellular processes by interstitial collagen gel used as a substratum, as compared to flattened or round cell shapes on polystyrene surface or on Matrigel containing the basement membrane components, respectively. The process induction was inhibited by several reagents as follows: (1) anti-integrin alpha2 antibody; (2) an oligopeptide, DGEA, an integrin-binding sequence in type I collagen molecule; (3) wortmannin, a phosphatidylinositol 3-kinase inhibitor. Protein tyrosine phosphorylation was enhanced throughout cells including cellular processes by culturing on type I collagen gel. Dual fluorescence staining showed that the core of the processes contained microtubules, whereas the periphery of the processes comprised fibrillar actin. Thus, the process extension was found to depend on integrin-binding to type I collagen fibres, followed by signal transduction and cytoskeleton assembly. The cellular processes included interstitial collagenase and vitamin A-containing lipid droplets. The lipid droplets and vitamin A-autofluorescence were increased by retinyl acetate addition to the culture medium, suggesting an important role of processes in hepatic stellate cell function.

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