Interstitial collagens I, III, and VI sequester and modulate the multifunctional cytokine oncostatin M.
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The binding of certain growth factors and cytokines to components of the extracellular matrix can regulate their local availability and modulate their biological activities. We show that oncostatin M (OSM), a profibrogenic cytokine and modulator of cancer cell proliferation, specifically binds to collagen types I, III, IV, and VI, immobilized on polystyrene or nitrocellulose. Single collagen chains inhibit these interactions in a dose-dependent manner. Cross-inhibition experiments of collagen-derived peptides point to a limited set of OSM-binding collagenous consensus sequences. Furthermore, this interaction is found for OSM but not for other interleukin-6 type cytokines. OSM binding to collagens is saturable, with dissociation constants around 10(-8) m and estimated molar ratios of 1-3 molecules of OSM bound to one molecule of triple helical collagen. Furthermore, collagen-bound OSM is biologically active and able to inhibit proliferation of A375 melanoma cells. We conclude that abundant interstitial collagens dictate the spatial pattern of bioavailable OSM. This interaction could be exploited for devising collagenous peptide-antagonists that modulate OSM bioactivity in tumor growth and fibrotic disorders like rheumatoid arthritis and hepatic fibrosis. (+info)
Flexibility and packing in proteins.
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Structural flexibility is an essential attribute, without which few proteins could carry out their biological functions. Much information about protein flexibility has come from x-ray crystallography, in the form of atomic mean-square displacements (AMSDs) or B factors. Profiles showing the AMSD variation along the polypeptide chain are usually interpreted in dynamical terms but are ultimately governed by the local features of a highly complex energy landscape. Here, we bypass this complexity by showing that the AMSD profile is essentially determined by spatial variations in local packing density. On the basis of elementary statistical mechanics and generic features of atomic distributions in proteins, we predict a direct inverse proportionality between the AMSD and the contact density, i.e., the number of noncovalent neighbor atoms within a local region of approximately 1.5 nm(3) volume. Testing this local density model against a set of high-quality crystal structures of 38 nonhomologous proteins, we find that it accurately and consistently reproduces the prominent peaks in the AMSD profile and even captures minor features, such as the periodic AMSD variation within alpha helices. The predicted rigidifying effect of crystal contacts also agrees with experimental data. With regard to accuracy and computational efficiency, the model is clearly superior to its predecessors. The quantitative link between flexibility and packing density found here implies that AMSDs provide little independent information beyond that contained in the mean atomic coordinates. (+info)
Differential gene expression in human abdominal aorta: aneurysmal versus occlusive disease.
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OBJECTIVE: Inflammation and atherosclerosis are present in both abdominal aortic aneurysm (AAA) and arterial occlusive disease (AOD). Changes in gene expression that underlie the development of AAA versus AOD are poorly defined. This study evaluated differences in gene expression in AAA, AOD, and control aortic tissue with human gene array technology. METHODS: RNA was isolated from human aortic specimens (seven AAA, five AOD, and five control), and complementary DNA (cDNA) probes were generated. The cDNA probes were hybridized to a human cell interaction array of 265 genes and quantitated with phosphorimaging. The data were corrected for background and were standardized to housekeeping genes. Statistical differences in individual gene expression were determined with the Kruskal-Wallis test. RESULTS: Of 265 genes studied, 11 showed statistically different expression in diseased aorta as compared with control. The following three genes were downregulated in AAA: collagen VI alpha1 (P <.037), glycoprotein IIIA (P <.006), and alpha2-macroglobulin (P <.020). The following two genes were upregulated in AOD: laminin alpha4 (P <.034) and insulin-like growth factor 2 receptor (P <.049). The following three genes were upregulated in both AAA and AOD: matrix metalloproteinase-9 (MMP-9; P <.005), intercellular adhesion molecule-1 (P <.012), and tumor necrosis factor--beta receptor (P <.022). The following three genes were downregulated in both AAA and AOD: integrin alpha5 (P <.012), ephrin A5 (P <.037), and rho/rac guanine nucleotide exchange factor (P <.028). Of 16 MMPs evaluated, only MMP-9 was significantly (P <.005) upregulated in both AAA and AOD. Evaluation results of four tissue inhibitors of metalloproteinases showed no significant difference in expression for all tissue types, although tissue inhibitor of metalloproteinase-1 trended toward upregulation in AAA (P =.053). Eight of the fifteen most highly expressed genes in all the groups were extracellular matrix or secreted proteins. Of these, only collagen VI alpha1 (P <.037) showed a significant change, although biglycan trended toward downregulation in AAA (P =.076). CONCLUSION: This study used cDNA array technology in the comparison of human control and pathologic aortic tissue. Six genes had similar differential expression in both AAA and AOD as compared with control. Even more interesting were differences between AAA and AOD in the expression of five genes. These data suggest a similarity in genetic expression for both AAA and AOD, with altered expression of several genes playing a role in disease differentiation. (+info)
Antibody response against perlecan and collagen types IV and VI in chronic renal allograft rejection in the rat.
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Chronic rejection is the leading cause of late renal transplant failure. Various structural lesions are observed in grafts undergoing chronic rejection including glomerular basement membrane (GBM) duplications. The well-established Fisher (F344) to Lewis (LEW) rat renal transplant model for chronic rejection was used to assess the presence and role of the humoral immune response against graft antigens during chronic rejection. LEW recipients of F344 allografts develop transplant glomerulopathy and produce IgG1 antibodies directed against F344 GBM preparations that are detectable 3 weeks after transplantation. Glomerular IgG1 deposition was observed that in vitro co-localized with a rabbit anti-rat GBM antiserum in rejecting F344 grafts; elution experiments of isolated glomeruli yielded IgG1 antibodies reactive in vitro with F344 GBM, but not LEW GBM. Prevention of acute rejection by transient treatment of the recipients with cyclosporin A completely abrogated the production of anti-GBM antibodies. Using proteomic techniques we identified the antigens recognized by the LEW posttransplant sera as being the heparan sulfate proteoglycan perlecan and the alpha1 chain of collagen type VI in association with the alpha5 chain of collagen type IV. In conclusion, LEW recipients of F344 kidney grafts produce IgG1 antibodies against donor type perlecan and alpha1(VI)/alpha5(IV) collagen and develop transplant glomerulopathy. These data implicate an important role for the humoral immune response in the development of glomerulopathy during chronic rejection. (+info)
The influence of the pericellular microenvironment on the chondrocyte response to osmotic challenge.
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OBJECTIVE: To examine whether differences in the pericellular microenvironment of different chondron preparations influence the chondrocyte volume regulatory response to experimental osmotic challenge. DESIGN: Mechanically extracted chondrons (MC), enzymatically extracted chondrons (EC) and isolated chondrocytes (IC) were seeded into agarose and sampled at 1, 3 and 7 days. Samples mounted in a perfusion chamber were subjected to osmotic challenge. The cross-sectional areas of the chondrocyte and pericellular microenvironment were measured under isotonic, hypertonic and hypotonic conditions, and percentage change calculated. Separate samples were immunolabeled for type VI collagen and keratan sulfate. RESULTS: Initially, the microenvironment of MC represented 60% of the chondron area and was occupied by type VI collagen and keratan sulfate. In EC, the microenvironment comprised 18% of the chondron area with narrow bands of type VI collagen and keratan sulfate. IC had no visible microenvironment, with small amounts of type VI collagen and keratan sulfate present. All preparations sequestered additional pericellular macromolecules during culture. Under isotonic conditions, the EC and IC chondrocytes were larger than those of MC. All chondrocytes shrank under hypertonic conditions and swelled under hypotonic conditions. MC were the least responsive, displaying the most efficient volume regulation. IC showed the largest response initially but this decreased with time. EC exhibited intermediate responses that decreased as the microenvironment increased in size. CONCLUSIONS: The composition and structural integrity of the pericellular microenvironment do influence the cellular response to experimental osmotic challenge. This suggests that the microenvironment functions in situ to mediate the chondrocyte response to physicochemical changes associated with joint loading. (+info)
Mutations in COL6A3 cause severe and mild phenotypes of Ullrich congenital muscular dystrophy.
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Ullrich congenital muscular dystrophy (UCMD) is an autosomal recessive disorder characterized by generalized muscular weakness, contractures of multiple joints, and distal hyperextensibility. Homozygous and compound heterozygous mutations of COL6A2 on chromosome 21q22 have recently been shown to cause UCMD. We performed a genomewide screening with microsatellite markers in a consanguineous family with three sibs affected with UCMD. Linkage of the disease to chromosome 2q37 was found in this family and in two others. We analyzed COL6A3, which encodes the alpha3 chain of collagen VI, and identified one homozygous mutation per family. In family I, the three sibs carried an A-->G transition in the splice-donor site of intron 29 (6930+5A-->G), leading to the skipping of exon 29, a partial reduction of collagen VI in muscle biopsy, and an intermediate phenotype. In family II, the patient had an unusual mild phenotype, despite a nonsense mutation, R465X, in exon 5. Analysis of the patient's COL6A3 transcripts showed the presence of various mRNA species-one of which lacked several exons, including the exon containing the nonsense mutation. The deleted splice variant encodes collagen molecules that have a shorter N-terminal domain but that may assemble with other chains and retain a functional role. This could explain the mild phenotype of the patient who was still ambulant at age 18 years and who showed an unusual combination of hyperlaxity and finger contractures. In family III, the patient had a nonsense mutation, R2342X, causing absence of collagen VI in muscle and fibroblasts, and a severe phenotype, as has been described in patients with UCMD. Mutations in COL6A3 are described in UCMD for the first time and illustrate the wide spectrum of phenotypes which can be caused by collagen VI deficiency. (+info)
Isolation of up- or down-regulated genes in PPARgamma-expressing NIH-3T3 cells during differentiation into adipocytes.
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Adipocyte differentiation is a complex process in which the expression of many transcription factors and adipocyte-specific genes is regulated under a strict program. The peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the steroid/thyroid nuclear hormone receptor superfamily of ligand-activated transcription factors, is an important regulator of adipocyte gene expression and differentiation. In this study, we tried to identify downstream target genes of PPARgamma, by using PPARgamma-expressing cells and a subtractive cloning strategy, and isolated cDNA clones which were up-regulated or down-regulated by PPARgamma. Northern blot analyses revealed that the expression levels of the aldehyde dehydrogenase-2-like, type VI collagen alpha 3 subunit, cellular retinoic acid binding protein 1 and thrombospondin 1 are changed during the differentiation of mouse 3T3-L1 preadipocyte cells, indicating that these genes might be downstream targets of PPARgamma in adipocyte differentiation. (+info)
Identification of known and novel genes whose expression is regulated by endogenous retinoic acid during early embryonic development of the mouse.
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Retinoic acid (RA) derived from vitamin A is necessary for, among other things, mammalian embryonic development. Although the impact of RA-dependent gene-regulation on embryonic development has been examined through genetic disruption of the retinoid receptors, the understanding of the underlying molecular mechanism remain unclear, in part, due to the difficulty in identifying RA-regulated genes in an intact embryo. We report here that RA-regulated genes can be identified from total RA-deficient embryos created by retinol-binding protein antisense (RBP-AS) oligodeoxynucleotide treatment in conjunction with differential display. Of the 28 genes isolated, 15 genes matched known genes in the GenBank database and the others either represented EST sequences or encoded novel genes. Semi-quantitative reverse transcriptase-polymerase chain reaction verified that the mRNA levels of mouse DN 38, COL VI 3 alpha, cul-1, alpha-tropomyosin, and PP2A-C alpha were substantially increased, whereas mouse Msh 2, Ndufa2, Ribosomal protein S19, sFRP-1, GDAP-10 and mSmcD were significantly decreased in vitamin A deficient (VAD) embryos compared to the control embryos. The utility of the method is exemplified by our finding that several genes in the Wnt signaling pathway are vitamin A regulated in day 9.0 post coitum (p.c.) embryos. (+info)