9HODE stimulates cell proliferation and extracellular matrix synthesis in human mesangial cells via PPARgamma

Plasma oxidized low-density lipoprotein (OX-LDL) levels are elevated in patients with renal diseases, including diabetic nephropathy. We examined effects of OX-LDL on cell proliferation and extracellular matrix (ECM) production by using normal human mesangial cells. Furthermore, we examined possible involvement of peroxisome proliferator-activated receptor gamma (PPARgamma). Mesangial cell proliferation with OX-LDL, 9-hydroxy-10,12-octadecadienoic acid (9HODE), and 13-hydroxy-9,11-octadecadienoic acid (13HODE), the major components of OX-LDL, were determined by 5-bromo-2'-deoxyuridine (BrdU) or 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) incorporation. The effect of OX-LDL on mesangial cell proliferation with PD98059 pretreatment was determined by BrdU incorporation. Type IV collagen, fibronectin, and PPARgamma expression with OX-LDL or 9HODE or 13HODE was determined by Western blotting. Type IV collagen expression with antisense oligonucleotide against PPARgamma pretreatment was also determined by Western blotting. The effect of PD98059 pretreatment on PPARgamma expression was determined by Western blotting. In mesangial cells exposed to isolated OX-LDL from human plasma, BrdU incorporation was increased, and this increase was deleted by PD98059. Type IV collagen expression was significantly increased by OX-LDL. 9HODE and 13HODE increased BrdU and MTT incorporation into mesangial cells and also increased expressions of Type IV collagen and fibronection, the major components of ECM. PPARgamma expression in mesangial cells was stimulated by 9HODE. The reduction of PPARgamma synthesis by pretreatment of antisense oligonucleotide against PPARgamma remarkably attenuated Type IV collagen synthesis induced by 9HODE. PPARgamma expression induced by 9HODE was also reduced by PD98059 pretreatment. These findings demonstrate that 9HODE, the major component of OX-LDL, stimulates cell proliferation and ECM production of human mesangial cells. In addition, the stimulatory effects are, at least in part, mediated by PPARgamma, which may exist in downstream of ERK1/2 pathway.     

Regulation of rat mammary gene expression by extracellular matrix components

In the mammary gland the induction and maintenance of differentiation are dependent on both lactogenic hormones and the extracellular matrix (ECM). Since mammary epithelial cells differentiate on a basement membrane in vivo we have examined the effects of basement membrane components on the expression of milk protein genes in primary rat mammary cultures. We examined the effects of a basement membrane gel derived from the Englebreth-Holm-Swarm tumor as well as its major component, laminin, on the expression of a group of milk protein genes. We demonstrate that the basement membrane gel induces alpha-casein and alpha-lactalbumin (alpha-LA) accumulation up to 160- and 70-fold, respectively, of that on tissue culture plastic. Laminin, a major component of the basement membrane, also caused significant induction of these same proteins. In order to determine whether these ECM effects occurred at a translational or post-translational level, pulse-chase experiments were performed. These experiments demonstrated that a laminin substratum selectively effects milk protein turnover and secretion. In order to demonstrate whether ECM effects occurred at the level of steady state accumulation of mRNA we performed dot blot and Northern analyses using cloned cDNA probes for alpha-, beta-, and gamma-caseins and alpha-LA. These studies demonstrated that ECM components induced alpha- and beta-caseins up to 10-fold, and alpha-LA up to 3-fold, with no significant effect on gamma-casein. These results demonstrate that milk protein genes are not coordinately regulated by ECM components. Furthermore, since the amount of induction of milk proteins exceeds the amount of induction of mRNAs for these proteins, we conclude that in our system a major effect of ECM components is at the translational and/or post-translational levels. Based on these findings we propose a model in which basement membrane components effect mammary gene expression at multiple levels.     

Collagen I upregulates extracellular matrix gene expression and secretion of TGF-beta 1 by cultured human mesangial cells

Progressive renal diseases are characterized by an increased synthesis of extracellular matrix (ECM) components. The mechanisms involved in the development of these alterations are not completely known, but a crucial role for TGF-1 has been suggested. Moreover, the ability of the ECM to modulate the phenotypic expression of different cell types has been widely described. In experiments presented here, human mesangial cells (HMC) were grown on collagen type I (COL I) or IV (COL IV). ECM protein and TGF-1 mRNA expression were evaluated by Northern blot analysis, and TGF-1 secretion was evaluated by ELISA. The involvement of tyrosine kinase and serine-threonine kinase pathways was studied by Western blot analysis, immunofluorescence, and in vitro kinase assays. HMC cultured on COL I showed an increased mRNA expression of COL I and COL IV, fibronectin, and TGF-1. Both tyrosine phosphorylation and integrin-linked kinase (ILK) activity increased when HMC were cultured on COL I, and blockade of these pathways inhibited the increased secretion of TGF-1. In conclusion, the present results support a role for extracellular COL I in the regulation of TGF-1 synthesis during progressive renal sclerosis and fibrosis and the subsequent increase in newly synthesized ECM proteins. In addition, ILK, along with the tyrosine kinases, participates in the genesis of this effect. fibrosis; tyrosine phosphorylation; integrin-linked kinase     

Differential expression of extracellular matrix components in rat Sertoli cells.

We studied expression of laminin, fibronectin, and Type IV collagen in the testis by means of immunofluorescence and immunoblot analysis and also examined gene expression of fibronectin using the ribonuclease protection assay. By immunofluorescence on sections from 20-day-old rats, laminin, fibronectin, and Type IV collagen were found in the basement membrane of the seminiferous tubules and in the interstitial regions of the testis. No localization of any extracellular matrix components was found inside the sectioned cells. However, when Sertoli cells were cultured on glass coverslips, laminin and Type IV collagen were both found inside the cells, suggesting new synthesis. In cultured peritubular cells, Type IV collagen, laminin, and fibronectin were found within the cells. When examined by immunoblot analysis, freshly isolated Sertoli and peritubular cells from 20-day-old rats did not demonstrate production of laminin or fibronectin. After 5 days in culture, peritubular cells produced both laminin and fibronectin, whereas cultured Sertoli cells produced only laminin. In contrast, freshly isolated and cultured Sertoli and peritubular cells all produced Type IV collagen. Moreover, the ribonuclease protection assay indicated that the bulk of fibronectin gene expression occurs within the first 10 days of postnatal development, with lower maintenance levels occurring thereafter. These results indicate that in the testis the highest levels of expression of laminin and fibronectin occur during development and in primary cell culture, whereas expression of Type IV collagen is higher at later stages.     

Developmental expression of extracellular matrix components in intramuscular connective tissue of bovine semitendinosus muscle

 We have investigated the expression patterns of extracellular matrix components in intramuscular connective tissue during the development of bovine semitendinosus muscle by means of indirect immunofluorescence techniques. Types I, III, V, and VI collagen and fibronectin were located in the endomysium and the perimysium. Type IV collagen, laminin, and heparan sulfate proteoglycans (PGs) were exclusively located in the endomysium, and dermatan sulfate PGs existed only in the perimysium. The localization of these components in the intramuscular connective tissue of semitendinosus muscle remained unchanged throughout prenatal and postnatal growth of cattle, suggesting that they are essential for forming and maintaining structures of the endomysium and perimysium in bovine semitendinosus muscle. On the other hand, decorin was undetectable in the endomysium of neonates, although other matrix components were already expressed. It was expressed slightly in the endomysium of 2-month-old calves, and clearly detectable in the endomysium of cattle more than 6 months old. Chondroitin sulfate PGs were barely detectable in the perimysium of fetuses and neonatal calves, and progressively appeared during postnatal development of the muscle. It seems likely that these PGs are closely related to the postnatal development of the endomysium and perimysium. Accepted: 30 October 1996     

Use of extracellular matrix components for cell culture

Extracellular matrix components when used as a substratum in vitro can greatly influence cell behavior. The response observed is dependent on the type of cell and matrix used. Cells in vitro usually respond best to the matrix components with which they are normally in contact in vivo. More differentiated phenotypes are observed and cells generally survive longer on such matrices. In some cases, the presence of such matrices allows cells to be cultured in the absence of serum and growth factors. As more investigators try the matrices and matrix components described, as well as new components and combinations of them, it is anticipated that improvement in the culture of many cells can be expected.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Interleukin-6 stimulates c-myc expression and proliferation of cultured vascular smooth muscle cells

The effects of interleukin-6 (IL-6), a cytokine recently found to be secreted by monocytes and macrophages, on c-myc expression and proliferation of cultured vascular smooth muscle cells (VSMC) were investigated. Treatment with IL-6 caused rapid increase in the c-myc mRNA level of VSMC. It also stimulated DNA synthesis and proliferation of the cells significantly and dose-dependently at concentrations of more than 10 U/ml. These results suggest that IL-6 may be important in the proliferation of VSMC, which is a key event in the development of arteriosclerosis, as a factor mediating immune cell-VSMC interaction.     

Basal lamina and other extracellular matrix produced by bovine granulosa cells in anchorage-independent culture

Bovine granulosa cells from 3–7 mm follicles were cultured without anchorage in soft agar/methylcellulose solution for 14 days, with or without 50 ng/ml basic fibroblast growth factor. The granulosa cells divided to form colonies of cells. These were analysed by light and electron microscopy, immunohistochemistry and Western immunoblotting. In approximately 20% of the colonies extracellular matrix was clearly visible at the light-microscope level. Ultrastructurally the matrix resembled a basal lamina 30–100 nm thick and was composed of tangled fibres or cords. Unidentified spherical structures of less than 50 nm diameter were sometimes present and attached to this basal lamina. The basal lamina of follicles had similar features, except that the basal lamina produced in vitro was a large aggregate of many convoluted layers. The cells produced collagen type IV and the cellular form of fibronectin. Intercellular areas not associated with basal lamina were identified. Ruthenium red staining revealed these areas to be rich in proteoglycan granules. Free granules were clustered near the cell surface, and the lumina of these areas were rich in fibres decorated with ruthenium red. This material did not resemble follicular fluid of antral follicles. Thus, granulosa cells in anchorage-independent cultures have a follicular cell morphology and secrete two distinct extracellular matrices, one similar to the follicular basal lamina.This study was funded by the Flinders Medical Centre Research Foundation, Flinders University, and the National Health and Medical Research Council of Australia     

Tensin is potentially involved in extracellular matrix production in mesangial cells

Tensin, a focal adhesion protein, is expressed in renal tubular epithelial cells (TECs). Tensin-null mice develop multiple large cysts in the renal proximal tubules. However, the role of tensin in human glomeruli remains unclear. In this study, we assessed tensin localization in human kidney and interaction between tensin and other adhesion components. In human mesangial cells (MCs) and TECs, we confirmed mRNA and protein expressions of tensin by RT-PCR and immunoprecipitation. In normal kidney, immunohistochemistry revealed that tensin was localized in MCs and parietal epithelial cells as well as TECs. In biopsy specimens, the expression of tensin was significantly increased in areas of mesangial expansion in patients with IgA nephropathy and diabetic nephropathy. These results suggest that the expression of tensin is associated with extracellular matrix (ECM) production. In vitro, immunocytochemistry revealed that MCs express tensin mainly at the ends of actin stress fibers and apparently in the focal adhesion areas. Integrin 5, but not 1 and 3, colocalized with tensin. Vinculin and focal adhesion kinase (FAK) were coprecipitated by tensin, suggesting that tensin can mediate signal transduction between cell and ECM through these molecules. Tensin may play important roles in mesangial ECM production through an adhesion complex with integrin 5, FAK, and vinculin.     

Roles of extracellular matrix components in differentiating teratocarcinoma cells

F9 embryonal carcinoma cells treated with 5 X 10(-8) M retinoic acid and cultured in suspension for 8 days form aggregates consisting of an outer epithelial layer of alpha-fetoprotein-producing visceral endoderm cells. We have previously shown (Grover, A., Oshima, R. G., and Adamson, E. D. (1983) J. Cell Biol. 96, 1690-1696) that the differentiation of F9 cells to visceral endoderm is accompanied by the activation of several genes, and increased laminin synthesis is one of the earliest events. Here we analyze in detail the syntheses and secretion of fibronectin, type IV collagen, and laminin during the 8-day process. Employing immunoprecipitation and enzyme-linked immunosorbent assay, we show that the levels of all three components change with different patterns. Unstimulated F9 cells synthesize and secrete relatively high levels of fibronectin and low levels of type IV collagen. Fibronectin synthesis and secretion decreases to 10% of its original level whereas type IV collagen synthesis rises approximately 3-fold during the differentiation process. Laminin synthesis also rises at least 2-fold, and the proportions of its subunits change as the syntheses of B1 and A accelerate starting on day 2. However, unlike fibronectin and type IV collagen, laminin is largely accumulated in the aggregates. The data suggest that fibronectin has a role in aggregation whereas laminin is important in the differentiation process.     

Turnour necrosis factor stimulates endothelin-1 gene expression in cultured bovine endothelial cells

We have studied the effect of human recombinant tumour necrosis factor-alpha (TNF-alpha) on gene expression and production of endothelin-1 in cultured bovine aortic endothelial cells. TNF-alpha (10 and 100 ng ml(-1)) increased in a time dependent manner the preproendothelin-1 mRNA levels in respect to unstimulated endothelial cells. TNF-alpha induced endothelin-1 gene expression was associated with a parallel increase in the release of the corresponding peptide in the culture medium. These findings suggest that the enhanced synthesis and release of endothelin-1 occurring in conditions of increased generation of TNF, may act as a modulatory factor that counteracts the hypotensive effect and the excessive platelet aggregation and adhesion induced by TNF.     

The influence of extracellular matrix and prolactin on global gene expression profiles of primary bovine mammary epithelial cells in vitro

An in vitro bovine mammosphere model was characterized for use in lactational biology studies using a functional genomics approach. Primary bovine mammary epithelial cells cultured on a basement membrane, Matrigel, formed three-dimensional alveoli-like structures or mammospheres. Gene expression profiling during mammosphere formation by high-density microarray analysis indicated that mammospheres underwent similar molecular and cellular processes to developing alveoli in the mammary gland. Gene expression profiles indicated that genes involved in milk protein and fat biosynthesis were expressed, however, lactose biosynthesis may have been compromised. Investigation of factors influencing mammosphere formation revealed that extracellular matrix (ECM) was responsible for the initiation of this process and that prolactin (Prl) was necessary for high levels of milk protein expression. CSN3 (encoding κ-casein) was the most highly expressed casein gene, followed by CSN1S1 (encoding αS1-casein) and CSN2 (encoding β-casein). Eighteen Prl-responsive genes were identified, including CSN1S1 , SOCS2 and CSN2, however, expression of CSN3 was not significantly increased by Prl and CSN1S2 was not expressed at detectable levels in mammospheres. A number of novel Prl responsive genes were identified, including ECM components and genes involved in differentiation and apoptosis. This mammosphere model is a useful model system for functional genomics studies of certain aspects of dairy cattle lactation.     

Organization of extracellular matrix components during differentiation of adipocytes in long-term culture

Summary Scanning electron microscopy (SEM) observation showed that fully differentiated spherical adipocytes were embraced by a network of collagens and fibroblastic preadipocytes. The properties of both the collagen networks and the preadipocytes allow the adipocytes to be interconnected, forming a fat-cell cluster, which can anchor to the bottom of a culture dish. In this network structure, collagen fibrils and fibrillar bundles were closely arranged and stratified. We found that immunostained collagens appeared to form extracellular network structures, which can be observed by SEM. The extracellular network of fibronectin was the first to develop among the extracellular matrix (ECM) components, though it became degraded with the progress of adipocyte differentiation. The type I collagen network was the last to develop and remained well organized through the late stage of adipocyte differentiation. The extracellular networks of type III, V, and VI collagen developed by the mid-stage and remained in the late stage of adipocyte differentiation. The network structures of type IV collagen and laminin became degraded during the differentiation process and localized at the surface of spherical cells. In addition to these basement membrane components, types III, V, and VI collagens also showed pericellular spherical staining patterns. These results demonstrated that the constitution and distribution of the ECM are altered during adipocyte differentiation, suggesting that the organization of each ECM component into a suitable structure is a requirement for the differentiation and maintenance of unilocular adipocytes.