Prokineticins, multifunctional secreted proteins, activate two endogenous G protein-coupled receptors PKR1 and PKR2. From in situ analysis of the mouse brain, we discovered that PKR2 is predominantly expressed in the olfactory bulb (OB). To examine the role of PKR2 in the OB, we created PKR1-and PKR2-gene-disrupted mice (Pkr1 ؊/؊ and Pkr2 ؊/؊ , respectively). Phenotypic analysis indicated that not Pkr1 ؊/؊ but Pkr2 ؊/؊ mice exhibited hypoplasia of the OB. This abnormality was observed in the early developmental stages of fetal OB in the Pkr2 ؊/؊ mice. In addition, the Pkr2 ؊/؊ mice showed severe atrophy of the reproductive system, including the testis, ovary, uterus, vagina, and mammary gland. In the Pkr2 ؊/؊ mice, the plasma levels of testosterone and follicle-stimulating hormone were decreased, and the mRNA transcription levels of gonadotropin-releasing hormone in the hypothalamus and luteinizing hormone and follicle-stimulating hormone in the pituitary were also significantly reduced. Immunohistochemical analysis revealed that gonadotropin-releasing hormone neurons were absent in the hypothalamus in the Pkr2 ؊/؊ mice. The phenotype of the Pkr2 ؊/؊ mice showed similarity to the clinical features of Kallmann syndrome, a human disease characterized by association of hypogonadotropic hypogonadism and anosmia. Our current findings demonstrated that physiological activation of PKR2 is essential for normal development of the OB and sexual maturation.
Degradation of extracellular matrix (ECM) proteins in the aorta is a critical step for the development of atherosclerosis. Expression of matrix metalloproteinase (MMP)-12 (macrophage elastase), an elastin-degrading proteinase in the MMP family, was investigated in the thoracic aorta of rabbits fed a 1% cholesterol-containing diet for 16 weeks. In the atherosclerotic lesions, MMP-12 was produced abundantly at both the mRNA and protein levels, whereas no expression was observed in the normal rabbit aortas. The principal source of MMP-12 was macrophage foam cells (MFCs) that had infiltrated the atherosclerotic intima; this was demonstrated in both in vitro culture studies of MFCs purified from atherosclerotic lesions and immunohistochemical studies of aortic lesions. Additional biochemical studies using recombinant rabbit MMP-12 revealed that MMP-12 digested elastin, type IV collagen, and fibronectin and also activated MMP-2 and MMP-3. Expression of MMP-12 by human macrophage cell lines was increased by stimulation with acetylated low-density lipoprotein, implying augmentation of MMP-12 production during foam cell formation. Increased expression of MMP-12 in atherosclerotic lesions, concomitant with foam cell generation, which triggers the acceleration of ECM breakdown, is likely to be a critical step in the initiation and progression of the atherosclerotic cascade.
The extra domain-A (EDA), present in fibronectin (FN) molecules arising from alternatively spliced transcripts, appears only during specific biological and pathogenic processes. However, its function is poorly understood. To define the physiologic role of this domain in joint connective tissue, the biological effects on rabbit cartilage explants, chondrocytes, and synovial cells were studied. A recombinant EDA protein (rEDA) increased proteoglycan release (3.6-fold) in cartilage explant cultures and markedly induced production of matrix metalloproteinase (MMP)-1 in chondrocytes. In addition, rEDA induced MMP-1, MMP-3, and MMP-9 in synovial cells. These effects were elicited only by rEDA, while its neighboring type III repeats, III 11 or III 12 , scarcely had any such effects. Interestingly, reorganization of F-actin stress fibers accompanied MMP-1 expression in synovial cells treated with rEDA, suggesting alteration of cellular phenotype. Subsequent Northern blotting revealed expression of pro-inflammatory cytokines, including interleukin (IL)-1␣ and IL-1, was induced by rEDA prior to MMP-1 expression. Delayed MMP-1 expression suggests that rEDA-induced IL-1s promote MMP-1 expression in an autocrine manner. This hypothesis is supported by the reduction of EDA-induced MMP-1 production by IL-1 receptor antagonist. The effect of EDA on MMP-1 production was reduced by connection with an adjacent type III repeat on either the NH 2 or COOH side of EDA and was abolished by connection on both sides of EDA, suggesting that exposure of either the NH 2 or COOH terminus of EDA domain by proteolytic cleavage releases the inducing activity. In agreement with these results, full-length cellular FN did not induce MMP-1 production. Furthermore, a 160-kDa EDA-positive FN fragment, which was purified from human placental tissue and corresponds to the region from NH 2 terminus through the EDA, induced MMP-1 production. Taken together, these results suggest that the EDA in FN fragments triggers alterations of cell physiology and plays a role in matrix degradation in joint connective tissue. Fibronectin (FN)1 is a multifunctional glycoprotein abundant in plasma and widely distributed in the extracellular matrix (1). It is a dimer of subunits cross-linked by disulfide bonds. Each FN monomer is comprised of three types of repeating units designated type I, II, and III (2). Some of these repeats bind to cell surface and extracellular matrix components such as integrins, collagens, heparin, and fibrin. Several of these binding activities have been assigned to the motif sequences in FN, including the Arg-Gly-Asp (RGD) motif in the type III 10 domain (3), the Pro-His-Ser-Arg-Asn (PHSRN) motif in the type III 9 domain (4), and the CS-1 sequence in the III-CS region (5). Consequently, this multifunctional glycoprotein mediates a variety of cellular functions including cell adhesion, cell migration, and cell differentiation. FN molecules have multiple isoforms generated from a single gene by alternative splicing of combinations of 3 exons: e...
The G protein-coupled receptor (GPCR) family is highly diversified and involved in many forms of information processing. SREB2 (GPR85) is the most conserved GPCR throughout vertebrate evolution and is expressed abundantly in brain structures exhibiting high levels of plasticity, e.g., the hippocampal dentate gyrus. Here, we show that SREB2 is involved in determining brain size, modulating diverse behaviors, and potentially in vulnerability to schizophrenia. Mild overexpression of SREB2 caused significant brain weight reduction and ventricular enlargement in transgenic (Tg) mice as well as behavioral abnormalities mirroring psychiatric disorders, e.g., decreased social interaction, abnormal sensorimotor gating, and impaired memory. SREB2 KO mice showed a reciprocal phenotype, a significant increase in brain weight accompanying a trend toward enhanced memory without apparent other behavioral abnormalities. In both Tg and KO mice, no gross malformation of brain structures was observed. Because of phenotypic overlap between SREB2 Tg mice and schizophrenia, we sought a possible link between the two. Minor alleles of two SREB2 SNPs, located in intron 2 and in the 3 UTR, were overtransmitted to schizophrenia patients in a family-based sample and showed an allele load association with reduced hippocampal gray matter volume in patients. Our data implicate SREB2 as a potential risk factor for psychiatric disorders and its pathway as a target for psychiatric therapy.gene manipulation ͉ memory ͉ SNPs T he SREB (superconserved receptor expressed in brain) family of SREB1 (GPR27), SREB2 (GPR85), and SREB3 (GPR173) is a unique subfamily of G protein-coupled receptor (GPCR) selectively expressed in neurons (1-5). Intriguing features of the SREB family include its high degree of sequence conservation throughout vertebrate evolution and its abundant expression in brain structures showing high levels of plasticity, for example the hippocampal dentate gyrus. Among these three members, SREB2 is the most conserved-the primary amino acid sequence is 100% identical among humans, rats, and mice. SREB1 and SREB3 are also highly conserved in mammals. Despite the extraordinary conservation rate in vertebrates, SREB orthologues are not encoded in the genome sequence of Caenorhabditis elegans or Drosophila melanogaster (3).The history of drug discovery has proven that GPCRs are excellent therapeutic targets (6, 7). Although efforts have been made to identify endogenous ligand(s) for SREB, they have been unsuccessful (3). Recent progress in understanding of GPCR physiology has, however, enabled screening of drug candidates for promising GPCRs without knowledge of their endogenous ligands, e.g., screening compounds by using constitutively active mutants (8) or ligand-induced conformational change (9). Thus, if their physiological function is clarified, and their link to the pathophysiology of diseases is demonstrated, then newly discovered GPCRs, even orphan GPCRs like SREB2, become promising drug targets. The distinct features of SREB2, namel...
Abstract. Histamine H 4 receptor is considered as a novel therapeutic target for allergic diseases. To enhance the knowledge about species difference, which is essential for drug discovery research, monkey H 4 receptor was identified. Monkey H 4 receptor was characterized to have comparable similarity with its human counterpart. Discovery of monkey H 4 receptor will contribute to a better interpretation of effective drug discovery. Keywords: histamine, H 4 receptor, G-protein coupled receptorHistamine H 4 receptor is the most recently identified histamine receptor through human genomic DNA analysis (1, 2). H 4 receptor is a member of the G-protein coupled receptor (GPCR) family and can activate Gαi-type heterotrimeric G-proteins to inhibit the intracellular accumulation of cyclic AMP. H 4 receptor is expressed predominantly on eosinophils, neutrophils, and mast cells at the cell level; and it induces inflammatory symptoms including chemotaxis, cell-shape change, and adhesion molecule expression for these cells (3 -7). The analysis using H 4 -receptor gene disrupted mouse indicated that the H 4 receptor plays pathological as well as physiological roles in the immune tissues, leading to the speculation that the H 4 receptor functions in chronic allergy symptoms (8). In order to develop an antiinflammatory drug acting on the H 4 receptor, it is indispensable to evaluate candidate chemical drugs with appropriate animal models. However, the fact that there is low conservation of the H 4 receptor in the rodents (9) and swine (10) impeded effective drug discovery research. From this perspective, it is significant to identify the animal H 4 receptor, which has pharmacological and molecular characteristics similar to those of the human counterpart.In order to analyze the homology conservation of the H 4 receptor through mammalian species, we performed a multiple-species Southern blot analysis, so-called "zoo blot analysis", for genomic DNA obtained from multiple mammalian species (human, monkey, porcine, bovine, rabbit, rat, mouse) (all purchased from Clontech, Palo Alto, CA, USA). Using the 32 P-labeled full-length coding region cDNA of human H 4 or H 3 receptor, the genome-blotted membranes were hybridized at 42°C for 16 h in a solution containing 50% formamide, 5 × SSPE, 5 × Denhardt's solution, 0.5% SDS, and 200 µg / ml salmon sperm DNA and then washed four times at 57°C for 15 min with 0.5% SSPE and 0.1% SDS. As shown in Fig. 1a, the Southern zoo blot analysis probed with human H 4 receptor only detected the counterpart genes in the monkey and porcine genomic DNA. In contrast, probing the analysis with human H 3 receptor, which has 37% homology with the H 4 receptor and is the closest subtype molecule of the H 4 receptor in the histamine receptor family, detected the corresponding gene for all the species tested (Fig. 1b). There is more than 94% homology between the human and mouse H 3 receptor (11), indicating that the H 3 receptor is well conserved over multiple mammalian species. On the other hand, human H 4 rece...
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