Two types of cDNAs for human macrophage scavenger receptors were cloned from a cDNA library derived from the phorbol ester-treated human monocytic cell line THP-1. The type I and type II human scavenger receptors encoded by these cDNAs are homologous (73% and 71% amino acid identity) to their previously characterized bovine counterparts and consist of six domains: cytoplasmic (I), membrane-spanning (II), spacer (Ill), ae-helical coiled-coil (IV), collagen-like (V), and a type-specific C-terminal (VI). The receptor gene is located on human chromosome 8. The human receptors expressed in CHO-K1 cells mediated endocytosis of modified low density lipoproteins. Two mRNAs, 4.0 and 3.2 kiobases, have been detected in human liver, placenta, and brain. Immunohistochemical studies using an anti-peptide antibody which recognizes human scavenger receptors indicated the presence of the scavenger receptors in the macrophages of lipid-rich atherosclerotic lesions, suggesting the involvement of scavenger receptors in atherogenesis.The macrophage scavenger receptors are trimeric membrane glycoproteins implicated in the pathologic deposition of cholesterol in arterial walls during atherogenesis (1,2). The molecular cloning of bovine scavenger receptor cDNAs has revealed several unexpected features (3, 4). Two types of receptor subunits exist, and both of these receptor proteins contain two extracellular domains that are predicted to form a long triple-stranded a-helical coiled coil and a collagen-like triple helix (3, 4). These receptors mediate the endocytosis of a diverse group of macromolecules, including modified low density lipoproteins (LDLs) (1-5). When cultured macrophages are exposed to appropriately modified LDLs, they are converted to cholesteryl ester-rich foam cells which are strikingly similar to the foam cells found in atherosclerotic plaques (1). Recent in vivo studies support the suggestion that modified LDLs, possibly internalized via the scavenger receptors, may play a critical role in the development of atherosclerosis (6)(7)(8).To study the role of human scavenger receptors in atherogenesis, we have cloned human scavenger receptor cDNAs.tt From the deduced amino acid sequence, we generated rabbit anti-peptide antiserum that recognizes human macrophage receptors. Immunohistochemical studies using this antibody have revealed the presence of immunoreactive scavenger receptor protein in the macrophages ofatherosclerotic lesions, suggesting the involvement of scavenger receptors in atherogenesis. MATERIALS AND METHODSMolecular Cloning of Human Scavenger Receptor cDNAs. THP-1 cells were cultivated in the presence of 200 nM phorbol 12-myristate 13-acetate (PMA) for 4 days (2). Poly(A)+ RNA was isolated from these cells and was used to construct a size-fractionated oligo(dT)-primed cDNA library in A ZAP 11 (3). The Xba I-Sph I fragment of pBSR7 corresponding to the collagen-like domain of the bovine receptor (3) labeled by random priming was used as a hybridization probe to screen 6 x 105 plaques. Two positive...

Our study indicates that the Akt/mTOR pathway is a key signaling cascade in Gas6-mediated mesangial and glomerular hypertrophy and revealed a crucial role of Gas6/Axl and the Akt/mTOR pathway in the development of diabetic nephropathy.

Monocyte chemoattractant protein (MCP-1) is an important mediator for macrophage recruitment in atherosclerosis and various glomerulonephritis. However, the role of MCP-1 and its receptor CCR2 in the progression of diabetic nephropathy remains unknown. Using a type 1 diabetic nephropathy model that shows noticeable glomerulosclerosis, we examined the role of MCP-1/CCR2 by propagermanium (Pro; CCR2 antagonist) treatment, and confirmed it by transfection of plasmids carrying the 7ND (a mutant of MCP-1) gene. We measured the mesangial matrix expansion, type IV collagen (Col4), transforming growth factor (TGF)-beta1 positive area, and macrophage infiltration in glomeruli after 12 weeks. Mesangial matrix expansion and macrophage infiltration were increased in diabetic mice and inhibited by Pro or 7ND-treatment. Increased glomerular expression of Col4 and TGF-beta1 in diabetic mice was also ameliorated. Thus blocking the MCP-1/CCR2 pathway ameliorated glomerulosclerosis, indicating that the MCP-1/CCR2 pathway plays a crucial role in the progression of diabetic nephropathy.

Objective-Recently, adipose tissue inflammation induced by macrophage infiltration through MCP-1/C-C chemokine receptor-2 (CCR2) pathway is considered to play a role in the development of visceral obesity and insulin resistance.In the present study, to further examine the role of CCR2 in the development of obesity and type 2 diabetes, we studied the effect of pharmacological inhibition of CCR2 from the early stage of obesity in db/db mice. Methods and Results-Db/ϩm (lean control) and db/db mice were fed with a standard diet with or without 0.005% propagermanium, as a CCR2 inhibitor for 12 weeks from 6 weeks of age. Propagermanium treatment decreased body weight gain, visceral fat accumulation, and the size of adipocytes only in db/db mice. Further, propagermanium suppressed macrophage accumulation and inflammation in adipose tissue. Propagermanium treatment also ameliorated glucose tolerance and insulin sensitivity, and decreased hepatic triglyceride contents in db/db mice. Key Words: cytokines Ⅲ diabetes mellitus Ⅲ insulin resistance Ⅲ macrophages Ⅲ receptors T he metabolic syndrome, characterized by a clustering of visceral obesity, impaired glucose tolerance, hypertension, and dyslipidemia, is a major cause of type 2 diabetes mellitus and cardiovascular disease. 1 Visceral obesity and insulin resistance are thought to represent common underlying factors of the syndrome. 2 Therefore, it is very important to clarify the mechanism of the development of obesity and insulin resistance and to establish the therapeutic method based on its mechanism. Conclusions-PropagermaniumMany reports have shown that obesity is associated with a state of chronic, low-grade inflammation, suggesting that inflammation may be a potential mechanism whereby obesity leads to insulin resistance. 3 Indeed, obesity and insulin resistance are strongly associated with systemic markers of inflammation, and clinically, inflammation has been recognized as a major predictor of atherosclerotic disease. 3,4 The adipose tissue is an important endocrine organ that secretes many biologically active molecules, such as leptin, adiponectin, tumor necrosis factor (TNF-␣), and monocyte chemoattractant protein 1 (MCP-1), which are collectively termed adipocytokines. 5-8 Dysregulated production of proinflammatory and antiinflammatory adipocytokines seen in visceral fat obesity is associated with the metabolic syndrome, 5,9 suggesting that inflammatory changes in the adipose tissue may contribute to the development of many aspects of the metabolic syndrome and result in type 2 diabetes and atherosclerosis.Recent studies have demonstrated that obese adipose tissue is characterized by increased infiltration of macrophages, suggesting that they are important sources of inflammation in adipose tissue. 10,11 C-C chemokine receptor-2 (CCR2), known as a receptor for MCP-1, play a role in monocyte/ macrophage recruitment and macrophage-dependent inflammatory response and the development of atherosclerosis. 12 Mouse models have demonstrated that adipose tissue macrop...

Nephropathy is one of the most common complications of diabetes mellitus. Glomerular hypertrophy is a hallmark in the early phase of the nephropathy. The mechanism of glomerular hypertrophy, however, remains incompletely understood. We have reported that Gas6 (growth arrest-specific gene 6) and its receptor, Axl, play a key role in the development of glomerulonephritis. Here we show the important role of Gas6/Axl in the pathogenesis of diabetic glomerular hypertrophy. In streptozotocin (STZ)-induced diabetic rats, mesangial and glomerular hypertrophy and an increase in the glomerular filtration rate (GFR) and albuminuria were observed after 12 weeks of STZ injection. The glomerular expression of Gas6 and Axl was increased in those rats. Administration of warfarin inhibited mesangial and glomerular hypertrophy and the increase in GFR and albuminuria in STZ rats. Moreover, we found less mesangial hypertrophy in STZ-treated Gas6 knockout mice than control mice. In vitro we found that stimulation of mesangial cells with Gas6 resulted in mesangial cell hypertrophy. Thus we have found a novel mechanism of glomerular hypertrophy through the Gas6/Axl-mediated pathway in the development of diabetic nephropathy.

The function(s) and RNA binding properties of vigilin, a ubiquitous protein with 14 KH domains, remain largely obscure. We recently showed that vigilin is the estrogen-inducible protein in polysome extracts which binds specifically to a segment of the 3 untranslated region (UTR) of estrogen-stabilized vitellogenin mRNA. In order to identify consensus mRNA sequences and structures important in binding of vigilin to RNA, before vigilin was purified, we developed a modified in vitro genetic selection protocol. We subsequently validated our selection procedure, which employed crude polysome extracts, by testing natural and in vitro-selected RNAs with purified recombinant vigilin. Most of the selected up-binding mutants exhibited hypermutation of G residues leading to a largely unstructured, single-stranded region containing multiple conserved (A) n CU and UC(A) n motifs. All eight of the selected down-binding mutants contained a mutation in the sequence (A) n CU. Deletion analysis indicated that approximately 75 nucleotides are required for maximal binding. Using this information, we predicted and subsequently identified a strong vigilin binding site near the 3 end of human dystrophin mRNA. RNA sequences from the 3 UTRs of transferrin receptor and estrogen receptor, which lack strong homology to the selected sequences, did not bind vigilin. These studies describe an aproach to identifying long RNA binding sites and describe sequence and structural requirements for interaction of vigilin with RNAs.The steps between gene transcription and mRNA translation, which include nuclear RNA processing, mRNA trafficking, and cytoplasmic mRNA degradation, are increasingly seen as important regulatory sites in diverse cellular processes (5, 6, 52). Many of these steps in mRNA metabolism appear to be regulated by RNA binding proteins containing K-homology (KH) domains (14). While a detailed picture of KH-domain-RNA interaction is not yet available, in several cases proteins containing KH domains have been shown to bind RNA (13,14,21,57). Some KH-domain proteins are clinically significant, including FMR1 protein (57), which is involved in fragile X syndrome, the major cause of heritable human mental retardation, and Nova-1, which is important in the motor control disorder paraneoplastic opsoclonus-ataxia (12, 13). KH-domain proteins which affect nuclear RNA splicing include Mer1, SF1, and PSI (1, 46, 55), while the ␣-poly(C) binding protein plays a role in the cytoplasmic stability of globin mRNA (68). Prokaryotic KH-domain proteins are highly diverse and include NusA, polyribonucleotide:orthophosphate nucleotidyltransferase, CsrA, and ribosomal protein S3 (27,40,58). Since the RNA binding sites and mechanisms of action of many KH-domain proteins remain obscure, the question of whether KH-domain proteins can preferentially recognize and bind to specific RNA binding sites remained unresolved. In an important recent paper, Buckanovich and Darnell used in vitro genetic selection with purified recombinant Nova-1 to identify RNAs which ...

Although liver is an estrogen target tissue, the number of hepatic genes known to be directly induced by estrogen is very small. We identified proteinase inhibitor 9, or PI-9, as being rapidly and strongly induced by estrogen in an estrogen receptor-positive human liver cell line (HepG2-ER7). Since PI-9 mRNA was also induced by estrogen in a human liver biopsy sample, PI-9 is a genuine estrogen-regulated human gene. PI-9 is a potent inhibitor of granzyme B and of granzyme B-mediated apoptosis. Estrogens induced PI-9 mRNA within 2 h, PI-9 mRNA levels reached a plateau of 30 -40-fold induction in 4 h, and induction was not blocked by cycloheximide, indicating that induction of PI-9 mRNA is a primary response. The antiestrogen trans-hydroxytamoxifen was a partial agonist for PI-9 mRNA induction, whereas the antiestrogen ICI 182,780 was a pure antagonist. Western blot analysis showed that estrogen strongly increases PI-9 protein levels. Inhibition of transcription with actinomycin D resulted in identical rates of PI-9 mRNA decay in the presence and absence of estrogen. We isolated genomic clones containing the PI-9 promoter region, identified a putative transcription start site, and carried out transient transfections of PI-9-luciferase reporter gene constructs. The estrogen, moxestrol, elicited a robust induction from the PI-9-luciferase reporter. Mutational inactivation of three potential imperfect estrogen response elements in the PI-9 5-flanking region had no effect on moxestrol estrogen receptor induction.

Diabetic nephropathy is the leading cause of end-stage renal disease, and glomerular mesangial matrix expansion is the hallmark in diabetic nephropathy. However, the precise mechanism for the development of mesangial matrix expansion has remained unknown. The key component involved in mesangial matrix expansion is type IV collagen (Col4). Recently, we have reported that Smad1 transcriptionally regulates expression of Col4 under diabetic conditions in vitro. Here we show that this direct regulator of Col4 also plays a crucial role for mesangial matrix expansion in vivo. Streptozotocin-induced diabetic rats are the model of incipient diabetic nephropathy, and showed various levels of mesangial matrix expansion at 24 weeks. The glomerular expression of Smad1 was significantly increased in diabetic rats with more mesangial matrix expansion by Western blot and immunohistochemical analysis. Furthermore, the glomerular expression of Smad1 was closely correlated with the glomerular expression of Col4 and smooth muscle alpha actin (a-SMA), while albuminuria or glomerular filtration rate was not correlated with mesangial matrix expansion. We also found that urinary excretion of Smad1 was closely associated with the severity of mesangial matrix expansion. In cultured mesangial cells expression of Smad1 upregulated the transcriptional activity of key molecules in mesangial matrix expansion, such as Col4 and a-SMA. These data indicate the critical involvement of Smad1 in mesangial matrix expansion in the early phase of diabetic nephropathy. Our data imply that urinary Smad1 might be a representative diagnostic marker for mesangial matrix expansion in diabetic nephropathy.