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Molecular mechanisms underlying lipolysis, as defined by mobilization of fatty acids from adipose tissue, are not fully understood. A database search for enzymes with α/β hydrolase folds, the GXSXG motif for serine esterase and the His-Gly dipeptide motif, has provided a previously unannotated gene that is induced during 3T3-L1 adipocytic differentiation. Because of its remarkable structural resemblance to triacylglycerol hydrolase (TGH) with 70.4% identity, we have tentatively designated this enzyme as TGH-2 and the original TGH as TGH-1. TGH-2 is also similar to TGH-1 in terms of tissue distribution, subcellular localization, substrate specificity, and regulation. Both enzymes are predominantly expressed in liver, adipose tissue, and kidney. In adipocytes, they are localized in microsome and fatcake. Both enzymes hydrolyzed p-nitophenyl butyrate, triolein, and monoolein but not diolein, cholesteryl oleate, or phospholipids; hydrolysis of short-chain fatty acid ester was 30,000-fold more efficient than that of long-chain fatty acid triacylglycerol. Fasting increased the expression of both genes in white adipose tissue, whereas refeeding suppressed their expression. RNA silencing of TGH-2 reduced isoproterenol-stimulated glycerol release by 10% in 3T3-L1 adipocytes, while its overexpression increased the glycerol release by 20%. Thus, TGH-2 may make a contribution to adipocyte lipolysis during period of increased energy demand.

Background: Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1) is a scaffolding protein at tight junctions (TJs). We have recently identified junctional adhesion molecule 4 (JAM4) as a MAGI-1-interacting protein. JAM4 belongs to the immunoglobulin superfamily and mediates Ca2+-independent adhesion. In this study, we examined the subcellular localization of JAM4 in various tissues and the involvement of JAM4 in the localization of MAGI-1. Moreover, we investigated into roles of immunoglobulin-like loops (Ig-loops) of JAM4. Results: JAM4 was localized at TJs but also on apical membranes of epithelial cells in jejunum, ileum, and renal proximal tubules. In Madine Darby canine kidney (MDCK) cells, the localization of JAM4 at TJs depended on the first Ig-loop and did not require the MAGI-1-interacting region. JAM4 determined the subcellular localization of MAGI-1 in MDCK cells. In ileum, however, MAGI-1 was localized at TJs where JAM4 was not detected. Both of Ig-loops were necessary for homophilic interactions, but cis interactions depended on the first Ig-loop. Conclusion: JAM4 may be primarily targeted to apical membranes, and subsequently recruited to TJs through the first Ig-loop–mediated molecular interaction. JAM4 determines the localization of MAGI-1 in MDCK cells, but the in vivo localization of MAGI-1 does not necessarily depend on JAM4.

L-arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts L-arginine to urea and L-ornithine. L-Ornithine is the principal precursor for the production of polyamines and L-proline, which are required for cell proliferation and collagen synthesis. Endothelial NOS is expressed in the human endometrial glandular epithelium, but the expression and physiological roles of arginase in the human endometrium are not clear. The objective of this study was to investigate the expression and distribution patterns of arginases Ⅰ (A-Ⅰ) and Ⅱ (A-Ⅱ) in the human endometrium by using immunohistochemistry, reverse transcription-polymerase chain reaction (RTPCR), and western blotting. A-Ⅰ and A-Ⅱ were detected by immunohistochemistry in human endometrial epithelial cells during the proliferative and secretory phases of the menstrual cycle. RT-PCR showed that A-Ⅰ and A-Ⅱ mRNA were expressed in human endometrial tissue. Western blotting analysis results showed the expression of A-Ⅱ protein. Immunohistochemistry and western blotting results showed that expression levels of A-Ⅱ were significantly higher in the secretory phase than in the proliferative phase. Increased A-Ⅱ levels in the secretory phase may be responsible for endometrial growth by increasing polyamines and proline products.

Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.

MAGI-1 and CASK are membrane-associated guanylate kinases of epithelial junctions. MAGI-1 is localized at tight junctions in polarized epithelial cells, whereas CASK is localized along the lateral membranes. We obtained the KIAA0769 gene product through the yeast two-hybrid screening using MAGI-1 as a bait and named it Carom. Carom has a coiled-coil domain in the middle region, and two src homology 3 domains and a PSD-95/Dlg-A/ZO-1 (PDZ)-binding motif in the C-terminal region. Carom binds to the fifth PDZ domain of MAGI-1 and the calmodulin kinase domain of CASK in vitro. MAGI-1 and CASK bind to the distinct sequences in the C-terminal region of Carom, but still compete with each other for Carom binding. The study using a stable transformant of Madine Darby canine kidney (MDCK) cells expressing GFP-Carom revealed that Carom was partially overlapped by MAGI-1 in MDCK cells, which have not yet established mature cell junctions, but became separated from MAGI-1 and colocalized with CASK in polarized cells. Carom was highly resistant to Triton X-100 extractions and recruited CASK to the Triton X-100-insoluble structures. Carom is a binding partner of CASK, which interacts with CASK in polarized epithelial cells and may link it to the cytoskeleton.

Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1) is a scaffolding protein at tight junctions (TJs). We have recently identified junctional adhesion molecule 4 (JAM4) as a MAGI-1-interacting protein. JAM4 belongs to the immunoglobulin superfamily and mediates Ca2+-independent adhesion. In this study, we examined the subcellular localization of JAM4 in various tissues and the involvement of JAM4 in the localization of MAGI-1. Moreover, we investigated into roles of immunoglobulin-like loops (Ig-loops) of JAM4.JAM4 was localized at TJs but also on apical membranes of epithelial cells in jejunum, ileum, and renal proximal tubules. In Madine Darby canine kidney (MDCK) cells, the localization of JAM4 at TJs depended on the first Ig-loop and did not require the MAGI-1-interacting region. JAM4 determined the subcellular localization of MAGI-1 in MDCK cells. In ileum, however, MAGI-1 was localized at TJs where JAM4 was not detected. Both of Ig-loops were necessary for homophilic interactions, but cis interactions depended on the first Ig-loop.JAM4 may be primarily targeted to apical membranes, and subsequently recruited to TJs through the first Ig-loop-mediated molecular interaction. JAM4 determines the localization of MAGI-1 in MDCK cells, but the in vivo localization of MAGI-1 does not necessarily depend on JAM4.

MAGI-1 is a membrane-associated guanylate kinase protein at tight junctions in epithelial cells. It interacts with various molecules and functions as a scaffold protein at cell junctions. We report here a novel MAGI-1-binding protein that we named junctional adhesion molecule 4 (JAM4). JAM4 belongs to an immunoglobulin protein family. JAM4 was colocalized with ZO-1 in kidney glomeruli and in intestinal epithelial cells. Biochemical in vitro studies revealed that JAM4 bound to MAGI-1 but not to ZO-1, whereas JAM1 did not bind to MAGI-1. JAM4 and MAGI-1 interacted with each other and formed clusters in COS-7 cells when coexpressed. JAM4 mediated calcium-independent homophilic adhesion and was accumulated at cell-cell contacts when expressed in L cells. MAGI-1, ZO-1, and occludin were recruited to JAM4-based cell contacts. JAM4 also reduced the permeability of CHO cell monolayers. MAGI-1 strengthened JAM4-mediated cell adhesion in L cells and sealing effects in CHO cells. These findings suggest that JAM4 together with MAGI-1 provides an adhesion machinery at tight junctions, which may regulate the permeability of kidney glomerulus and small intestinal epithelial cells.