Masao Katoh

The Fibronectin Extra Domain A Activates Matrix Metalloproteinase Gene Expression by an Interleukin-1-dependent Mechanism

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, III11 or III12, 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)-1α and IL-1β, 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 NH2 or COOH side of EDA and was abolished by connection on both sides of EDA, suggesting that exposure of either the NH2 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 NH2terminus 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.

Identification of soluble type of membrane-type matrix metalloproteinase-3 formed by alternatively spliced mRNA.

Homology screening for human membrane-type MMP (MT-MMP) was carried out, and cDNA encoding a soluble type of MT3-MMP (SM3), which is considered to be an alternatively spliced variant of MT3-MMP, was obtained. SM3 had a novel sequence consisting of 50 amino acids after Lys407 instead of amino acids containing the transmembrane domain of MT3-MMP. When SM3 tagged with a FLAG epitope (SM3-flag) was expressed in COS-7 cells, SM3-flag was present in the conditioned medium in its activated form. The enzymatic activity of SM3 was studied using a recombinant enzyme expressed in E. coli (SM3-e). The fluorogenic peptide substrate hydrolyzing activity of SM3-e was inhibited by EDTA and by the tissue inhibitor of metalloproteinase-2 (TIMP-2), whereas TIMP-1 had only relatively weak inhibitory ability. SM3-e was able to activate proMMP-2, and this activity was also inhibited by TIMP-2 but not by TIMP-1. SM3-e was able to cleave type III collagen, and also digested fibronectin. In view of the homology of the primary structures, MT3-MMP was considered to have the same catalytic activity as SM3. The results of studies of SM3s activity on extracellular matrix (ECM) protein suggests that MT3-MMP plays a role in ECM turnover not only by activating proMMP-2 but also by acting directly on ECM macromolecules.

The evolutionarily conserved G protein-coupled receptor SREB2/GPR85 influences brain size, behavior, and vulnerability to schizophrenia

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.

INVOLVEMENT OF MMP-1 AND MMP-3 IN COLLAGEN DEGRADATION INDUCED BY IL-1 IN RABBIT CARTILAGE EXPLANT CULTURE

To determine whether matrix metalloproteinase-1 (MMP-1) or MMP-3 is involved in cartilage collagen degradation, polyclonal antibodies were separately raised against MMP-1 and MMP-3 and their effects on collagen degradation were assessed in rabbit cartilage explant culture. We found that anti-MMP-1 antibodies completely inhibited collagen degradation induced by the combination of interleukin-1 (IL-1) and plasminogen. Anti-MMP-3 antibodies showed 40% inhibition at maximum concentration. These results indicate that MMP-1, and possibly MMP-3, are involved in collagen degradation in cartilage explant culture.

Absence of Increased α1-Microglobulin in IgA Nephropathy Proteinuria

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only α1-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of α1-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of α1-microglobulin forms a complex with IgA in serum. These results suggest that α1-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that α1-microglobulin might have a relationship with IgA nephropathy pathology.

Effect of YM-126414 on glucose uptake and redistribution of glucose transporter isotype 4 in muscle cells

We discovered a novel compound, YM-126414 [1,3, 3-trimethyl-2-(2-phenylaminovinyl)-3H-indolium perchlorate], which stimulates glucose uptake in skeletal muscle cells in vitro. This compound increased the rate of consumption of glucose by C2C12 mouse myoblast cells in a dose-dependent manner (EC(50)=10 nM). To investigate the mechanism of this stimulation, we determined the redistribution of insulin-regulatable glucose transporter isotype 4 (Glut4). When fully differentiated C2C12 cells stably expressing myc-tagged Glut4 protein were treated with YM-126414, redistribution was dramatically increased in a dose-dependent manner (EC(50)=21 nM). These results indicate that YM-126414 is a novel glucose uptake stimulator for muscle cells by causing up-regulation of Glut4 redistribution in differentiated muscle cells. Our findings for the in vitro effects of YM-126414 suggest a direction for the development of new drugs for the treatment of type 2 diabetes.

Combination of MS Protein Identification and Bioassay of Chromatographic Fractions to Identify Biologically Active Substances from Complex Protein Sources

Purification of biologically active proteins from complex biological sources is a difficult task, usually requiring large amounts of sample and many separation steps. We found an active substance in a serum response element-dependent luciferase reporter gene bioassay in interstitial cystitis urine that we attempted to purify with column chromatography and the bioassay. With anion-exchange Mono Q and C4 reversed-phase columns, apparently sharp active peaks were obtained. However, more than 20 kinds of proteins were identified from the active fractions with MS, indicating that the purification was not complete. As further purification was difficult, we chose a candidate molecule by means of studying the correlation between MS protein identification scores and bioassay responses of chromatographic fractions near the active peaks. As a result, epidermal growth factor (EGF) was nominated as a candidate molecule among the identified proteins because the elution profile of EGF was consistent with that of the bioassay, and the correlation coefficient of EGF between MS protein identification scores and bioassay responses was the highest among all the identified proteins. With recombinant EGF and anti-EGF and anti-EGF receptor antibodies, EGF was confirmed to be the desired substance in interstitial cystitis urine. This approach required only 20 ml of urine sample and two column chromatographic steps. The combination of MS protein identification and bioassay of chromatographic fractions may be useful for identifying biologically active substances from complex protein sources.

Glucose uptake stimulator YM‐138552 activates gene expression and translocation of glucose transporter isotype 4

In this study, we examined the effect of YM‐138552 on the glucose uptake, gene expression, and transport activities of the insulin‐regulatable glucose transporter isotype 4 (Glut4) in skeletal muscle cells. YM‐138552 stimulated medium glucose consumption in a dose‐dependent manner (EC50 = 0.07 μM) in myoblast muscle C2C12 cells under differentiation conditions with 2% horse serum supplement. The stimulatory effect of glucose consumption was verified by radiolabeled 2‐DG uptake assay. The compound showed dose‐dependent stimulation of 2‐DG uptake in G8 myoblast muscle cells up to a 1 μM concentration (EC50 = 0.19 μM). To investigate the mechanism of glucose uptake stimulation by YM‐138552, the mRNA level of Glut4 was determined using real‐time quantitative RT‐PCR. The Glut4 mRNA level expressed in C2C12 cells treated with YM‐138552 increased up to at least 24 h (227% vs. control), after which it gradually decreased to the initial level at 36 h. In addition, we established that C2C12 cells stably expressed the myc‐tagged Glut4 protein (C2C12‐Glut4myc) and measured the Glut4 translocation activity. The Glut4 translocation activity was stimulated by treatment of YM‐138552 without insulin in a dose‐dependent manner (EC50 = 0.62 μM), and no insulin effect (100 nM) was observed. This suggests that YM‐138552 has an insulin‐like effect. These results suggest that the stimulation of glucose uptake by YM‐138552 in muscle cells was partly due to upregulation of the Glut4 gene expression and its translocation activation. Our findings on the in vitro effects of YM‐138552 glucose uptake stimulation through the Glut4 transporter may suggest a direction for the development of new drugs for the treatment of NIDDM. Drug Dev. Res. 51:43–48, 2000.