Laminin-like glycoproteins in extracellular matrix of endodermal cells

Extracellular matrix from a mouse endodermal cell line consisted mainly of two polypeptides with molecular weights of about 200,000 (200K) and 400,000 (400K). Both poly-peptides incorporated radioactivity from [³H]proline and [³H]glucosamine and were solubilized from the matrix by treatment with bacterial collagenase or 0.5 m sodium chloride. These polypeptides appeared similar to those of laminin (R. Timpl, H. Rohde, P. G. Robey, S. I. Rennard, J.-M. Foidart, and G. R. Martin, 1979, J. Biol. Chem., 254, 9933–9937) in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate but the laminin polypeptides seemed slightly larger than the 200K and 400K polypeptides, respectively. The amino acid compositions of the isolated 200K and 400K polypeptides resembled one another and the previously published amino acid composition of laminin. Antibodies prepared against the solubilized extracellular matrix protein (mixture of 200K and 400K components) as well as those against the isolated 400K component precipitated both the 400K component and the 200K component from culture media. These antisera and antisera to laminin showed identical reactivities in immunodiffusion and in immunofluorescence of tissue sections where they stained basement membranes. The immunofluorescent staining pattern was similar to that obtained with antifibronectin except in the liver where antifibronectin stained the biliary ducts and the liver sinusoids, while laminin-like immunoreactivity was not present in the sinusoidal areas. Such differences in distribution of matrix components could be involved in generation of signals for differentiation and growth of the adjacent cells.     

Regulation of autophagy by extracellular matrix glycoproteins in HeLa cells

Macroautophagy is a major lysosomal degradation pathway for cellular components in eukaryotic cells. Baseline macroautophagy is important for quality control of the cytoplasm in order to avoid the accumulation of cytotoxic products. Its stimulation by various stressful situations, including nutrient starvation, is important in maintaining cell survival. Here we demonstrate that macroautophagy is regulated differently depending on whether HeLa cells adhere to collagen I or collagen IV, proteins typical of connective tissue and basal membrane, respectively. We observed that the basal levels of macroautophagy were higher in cells plated on collagen IV than in cells plated on collagen I or on uncoated substrate. However, the stimulation of macroautophagy by nutrient starvation, as reflected by the buildup of autophagosomes and the increase in the autophagic flux, was higher in cells plated on collagen I than in cells plated on collagen IV. These contrasting results were not due to differences in the starvation-dependent inhibition of mTOR complex 1 signaling. Interestingly, cells plated on collagen IV formed numerous focal adhesions (FAs), whereas fewer FAs were observed in cells plated on the other substrates. This implies that focal adhesion kinase (FAK) was more robustly activated by collagen IV. Silencing the expression of FAK by siRNA in cells plated on collagen IV shifted the autophagic phenotype of these cells to an "uncoated substrate autophagic phenotype" under both basal and starvation-induced conditions. Moreover, cells plated on collagen IV were less dependent on autophagy to survive in the absence of nutrients. We conclude that extracellular matrix components can modulate macroautophagy and mitigate its role in cell survival.     

Acetylcholine decreases formation of myofibroblasts and excessive extracellular matrix production in an in vitro human corneal fibrosis model

Acetylcholine (ACh) has been reported to play various physiological roles, including wound healing in the cornea. Here, we study the role of ACh in the transition of corneal fibroblasts into myofibroblasts, and in consequence its role in the onset of fibrosis, in an in vitro human corneal fibrosis model. Primary human keratocytes were obtained from healthy corneas. Vitamin C (VitC) and transforming growth factor‐β1 (TGF‐β1) were used to induce fibrosis in corneal fibroblasts. qRT‐PCR and ELISA analyses showed that gene expression and production of collagen I, collagen III, collagen V, lumican, fibronectin (FN) and alpha‐smooth muscle actin (α‐SMA) were reduced by ACh in quiescent keratocytes. ACh treatment furthermore decreased gene expression and production of collagen I, collagen III, collagen V, lumican, FN and α‐SMA during the transition of corneal fibroblasts into myofibroblasts, after induction of fibrotic process. ACh inhibited corneal fibroblasts from developing contractile activity during the process of fibrosis, as assessed with collagen gel contraction assay. Moreover, the effect of ACh was dependent on activation of muscarinic ACh receptors. These results show that ACh has an anti‐fibrotic effect in an in vitro human corneal fibrosis model, as it negatively affects the transition of corneal fibroblasts into myofibroblasts. Therefore, ACh might play a role in the onset of fibrosis in the corneal stroma.     

Ontogenesis of the murine hepatic extracellular matrix: an immunohistochemical study

To define the role of the extracellular matrix (ECM) in hepatogenesis, we examined the temporal and spatial deposition of fibronectin, laminin and collagen types I and IV in 12.5-21.5 day fetal and 1, 7 and 14 day postnatal rat livers. In early fetal liver, discontinuous deposits of the four ECM components studied were present in the perisinusoidal space, with laminin being the most prevalent. All basement membrane zones contained collagen type IV and laminin, including those of the capsule (mesothelial), portal vein radicles and bile ductules. Fibronectin had a distribution similar to that of collagen type IV early in gestation. However, at later gestational dates, fibronectin distribution in the portal triads approached that of collagen type I, being present in the interstitial connective tissues; whereas, collagen type IV and laminin were restricted to vascular and biliary basement membrane zones in those regions. The cytoplasm of some sinusoidal lining cells and hepatocytes reacted with antibodies to extracellular matrix components. By electron microscopy the immunoreactive material was localized in the endoplasmic reticulum, indicating the ability of these cells to synthesize these ECM proteins. Biliary ductular cells had prominent intracytoplasmic staining for laminin and collagen type IV from day 19.5 gestation until 7 days of postnatal life, but lacked demonstrable fibronectin or collagen type I. These results demonstrate that by 12.5 days of gestation the rat liver anlage has deposited a complex extracellular matrix in the perisinusoidal space. The prevalence of laminin in the developing hepatic lobules suggests a possible role for this glycoprotein in hepatic morphogenesis. In view of the intimate association of the hepatic lobular extracellular matrix with the developing vasculature, we hypothesize that laminin provides a scaffold of the developing liver, but once the ontogenesis is complete, intrahepatic perisinusoidal laminin expression is suppressed.     

Sulfated glycoproteins and extracellular matrix of cultured human pulmonary endothelial cells

Endothelial cells derived from human pulmonary arteries incorporate (³H)-glucosamine and ³⁵SO₄ into glycosaminoglycans and into the carbohydrate side chains of glycoproteins. These ³H/³⁵S-carbohydrate chains were isolated from cells and culture medium after Pronase digestion. The ³H/³⁵S-glycosaminoglycans were separated from the ³H/³⁵S glycopeptides by chromatography on Sephadex G-50. The distribution of cellular glycosaminoglycans and glycopeptides indicated that 30–60% of the cellular ³⁵S-glycopeptides may be associated with the matrix components that are synthesized by the cell and attached to a plastic substratum. Human pulmonary arterial endothelial cells were grown on collagen or on a matrix derived from vascular smooth muscle cells in order to investigate how smooth muscle cell extracellular matrix components may regulate the synthesis of endothelial cell glycoconjugates. Endothelial cells grown on plastic release various proportions of the glycoconjugates they synthesize into the culture medium. However, these same cells, when grown on substratum composed of extracellular matrix materials, synthesized altered proportions of cell-associated glycosaminoglycans and reduced the levels of total glycosaminoglycans they released into the culture medium. Thus the growth of endothelial cells on a matrix of smooth muscle cell components indicates that the glycosaminoglycan materials released into the culture medium by cells grown on a plastic substratum may not be an accurate reflection of the levels or composition of extracellular matrix materials made by endothelial cells in vivo.     

Functional peptide sequences derived from extracellular matrix glycoproteins and their receptors

Peptides derived from extracellular matrix proteins have the potential to function as potent therapeutic reagents to increase neuronal regeneration following central nervous system (CNS) injury, yet their efficacy as pharmaceutical reagents is dependent upon the expression of cognate receptors in the target tissue. This type of codependency is clearly observed in successful models of axonal regeneration in the peripheral nervous system, but not in the normally nonregenerating adult CNS. Successful regeneration is most closely correlated with the induction of integrins on the surface of peripheral neurons. This suggests that in order to achieve optimal neurite regrowth in the injured adult CNS, therapeutic strategies must include approaches that increase the number of integrins and other key receptors in damaged central neurons, as well as provide the appropriate growth-promoting peptides in a “regeneration cocktail.” In this review, we describe the ability of peptides derived from tenascin-C, fibronectin, and laminin-1 to influence neuronal growth. In addition, we also discuss the implications of peptide/receptor interactions for strategies to improve neuronal regeneration.     

Evidence for autocatalytic cross-linking of hydroxyproline-rich glycoproteins during extracellular matrix assembly in Volvox

The alga Volvox carteri is one of the simplest multicellular organisms, yet it has a surprisingly complex extracellular matrix (ECM), making Volvox suitable as a model system in which to study ECM self-assembly. Here, we analyze the primary structures and post-translational modifications of two main ECM components synthesized in response to sexual induction as well as wounding. These proteins are members of the pherophorin family with as yet unknown properties. They contain polyhydroxyproline spacers as long as 500 and 2750 residues. Even the highly purified proteins retain the capacity to self-assemble and cross-link, producing an insoluble fibrous network in an apparently autocatalytic reaction. This pherophorin-based network is located within the deep zone of the ECM. A molecular genetic search for additional members of the pherophorin family indicates that at least nine different pherophorin species can be expected to serve as precursors for ECM substructures. Therefore, the highly diversified members of the pherophorin family represent region-specific morphological building blocks for ECM assembly and cross-linking.     

Regulation of autophagy by extracellular matrix glycoproteins in HeLa cells

Macroautophagy is a major lysosomal degradation pathway for cellular components in eukaryotic cells. Baseline macroautophagy is important for quality control of the cytoplasm in order to avoid the accumulation of cytotoxic products. Its stimulation by various stressful situations, including nutrient starvation, is important in maintaining cell survival. Here we demonstrate that macroautophagy is regulated differently depending on whether HeLa cells adhere to collagen I or collagen IV, proteins typical of connective tissue and basal membrane, respectively. We observed that the basal levels of macroautophagy were higher in cells plated on collagen IV than in cells plated on collagen I or on uncoated substrate. However, the stimulation of macroautophagy by nutrient starvation, as reflected by the buildup of autophagosomes and the increase in the autophagic flux, was higher in cells plated on collagen I than in cells plated on collagen IV. These contrasting results were not due to differences in the starvation-dependent inhibition of mTOR complex 1 signaling. Interestingly, cells plated on collagen IV formed numerous focal adhesions (FAs), whereas fewer FAs were observed in cells plated on the other substrates. This implies that focal adhesion kinase (FAK) was more robustly activated by collagen IV. Silencing the expression of FAK by siRNA in cells plated on collagen IV shifted the autophagic phenotype of these cells to an “uncoated substrate autophagic phenotype” under both basal and starvation-induced conditions. Moreover, cells plated on collagen IV were less dependent on autophagy to survive in the absence of nutrients. We conclude that extracellular matrix components can modulate macroautophagy and mitigate its role in cell survival.     

Synthesis of extracellular matrix glycoproteins by a differentiated thyroid epithelial cell line

We examined the synthesis of extracellular matrix macromolecules by the differentiated rat thyroid epithelial cell line FRTL-5. As shown by electron microscopy, the extracellular material produced by these cells is deposited at the basolateral surface and focally organized in the form of a basement membrane. Biochemical and biosynthetic studies demonstrated that laminin, type IV collagen, and fibronectin are synthesized and deposited in the culture monolayer. Secretion of fibronectin into the culture medium also occurred. By immunofluorescence we observed some peculiarities in the distribution patterns of the basement membrane glycoproteins; while fibronectin and laminin had an almost superimposable distribution, type IV collagen displayed a rather different pattern. Type IV collagen and laminin localization at sites where extracellular material was detected was confirmed by immuno electronmicroscopy using the protein A-colloidal gold technique. The results indicate that under appropriate culture conditions the differentiated thyroid epithelial cell line FRTL-5 synthesizes, secretes and organizes an extracellular matrix where some basement membrane glycoproteins are present.     

Synthesis of lysyl oxidase in experimental hepatic fibrosis.

The synthesis of lysyl oxidase, which initiates the cross-linking of collagen and elastin, was investigated in carbon tetrachloride (CCl4) induced fibrotic liver of rat. Lysyl oxidase activity of the fibrotic liver was 4 times greater than that of normal liver. mRNAs from the livers of normal and CCl4-treated rats were prepared for in vitro protein synthesis and the products were analyzed by immunoprecipitation with a monoclonal antibody against lysyl oxidase. The mRNAs from the fibrotic liver gave more than 3 times higher level of messenger copies for lysyl oxidase than did mRNAs from normal liver. The molecular weight of the nascent lysyl oxidase was 48,000.     

Relationship between connective tissue cells and fibronectin in a sequential model of experimental hepatic fibrosis

The cellular and non-cellular components of fibrous septa formed at early and late stages in a sequential model of experimental hepatic fibrosis have been investigated using ultrastructural and immunocytochemical techniques. In the early septa, cells with intermediate features between lobular Ito cells and active fibroblasts were formed. These cells frequently displayed subplasmalemmal microfilaments (myofibroblast-like cells). Macrophages were also present. Scanty typical fibroblasts were present in the late septa. This cellular recruitment might be related to an extracellular glycoprotein-fibronectin-which is at present under investigation as a chemotactic factor for fibroblasts. Strong positivity for fibronectin in early septa and its sharp decrease in late septa seems to support this view. Fibroblasts and/or macrophages are the likely source of fibronectin synthesis.     

The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum

In this report we examine the accumulation of glycoconjugates in the extracellular medium and insoluble matrices surrounding developing cells of the cellular slime mold Dictyostelium discoideum. Conditions were employed which permitted advanced development (slug stage and beyond) in suspension culture. Under these conditions, up to one-third of the total culture protein appeared as non-sedimentable, extracellular material over the course of 48 h of incubation. Most of the secreted molecules expressed carbohydrate antigens (glycoantigens) as detected by Western blotting, using a panel of six monoclonal antibodies. Since the glycoantigens are secreted, immunoelectron microscopy was used to localize the glycoantigens in the extracellular matrices surrounding normally developing cells, including the slime sheath, stalk tube, inner spore coat, outer spore coat, and intercellular fluid between spores. Each glycoantigen had a characteristic distribution, and each extracellular matrix space contained a unique combination of glycoantigens. Thus, although each of these matrices (except inter-spore fluid) contains cellulose as a primary component, they could be distinguished on the basis of their glycoantigen and, by inference, glycoprotein compositions. Furthermore, there were differences between anterior and posterior regions of both slime sheaths and stalk tubes. These observations show that secretion as detected in suspension culture occurs under normal conditions as a part of the process of depositing extracellular matrices around the cells. The distributions show that the cell aggregate positionally regulates the expression and deposition of secretory glycoproteins; the resultant patterns of expression of unique protein-linked carbohydrate structures imply a functional role in matrix organization and possibly cell activity which can now be explored.     

Regulation of hepatic fibrosis and extracellular matrix genes by the th response: new insight into the role of tissue inhibitors of matrix metalloproteinases.

Hepatic fibrosis is the hallmark of Schistosoma mansoni infection and often results in portal hypertension and bleeding from esophageal varices. The fibrotic process is highly dependent on type 2 cytokines, yet their role in the regulation of extracellular matrix remodeling genes remains largely unknown. Here, we examined the expression of matrix metalloproteases (MMP) -2, -3, -9, -12, and -13 and their inhibitors, tissue inhibitor of metalloproteases (TIMP) -1, -2, and -3, in the livers of infected mice and correlated their expression profiles with fibrosis and type 2 cytokine production. Expression of MMP-2, -3, -9, -12, and -13 and of TIMP-1 and -2 mRNA rapidly increased at the onset of egg laying in infected mice, while TIMP-3 was unchanged. Because TIMP are presumed to be important regulators of the extracellular matrix, and their expression correlated with the development of fibrosis, we studied their role in fibrogenesis by infecting TIMP-1- and TIMP-2-deficient mice. Strikingly, our data revealed no role for TIMP-1 or -2 in the fibrotic pathology induced by S. mansoni eggs. Because of these findings, we infected IL-10/IFN-gamma-deficient mice that develop an exaggerated fibrotic response to determine whether changes in type 2 cytokine dominance influence the pattern of MMP and TIMP expression. Fibrosis and type 2 cytokine production correlated with increased MMP-2/MMP-9 vs TIMP-1/TIMP-2 expression. These data, in addition to our knockout studies, demonstrate that TIMP-1/TIMP-2 play no essential role in fibrogenesis in schistosomiasis. Indeed, our findings suggest that inhibiting profibrotic cytokines or specific MMP may be a more effective strategy to ameliorate fibrotic pathology.     

Involvement of extracellular matrix proteins in the course of experimental paracoccidioidomycosis

We aimed at determining involvement of extracellular matrix proteins (ECMp) and an ECM-binding adhesin (32-kDa protein) from Paracoccidioides brasiliensis, in the course of experimental paracoccidioidomycosis. BALB/c mice were infected with P. brasiliensis conidia previously incubated with soluble laminin, fibronectin and fibrinogen or a mAb against the fungal adhesin. Inflammatory response, chitin levels and cytokine production at different postinfection periods were determined. Chitin was significantly decreased in lungs of mice infected with ECMp-treated conidia when compared with controls at week 8, especially with laminin and fibrinogen. Contrariwise, when animals were infected with mAb-treated conidia no differences in chitin content were found. The observed inflammatory reaction in lungs was equivalent in all cases. IFN-gamma increased significantly in lungs from mice infected with soluble ECMp - (at day 4 and week 12) or mAb-treated conidia (at week 12) when compared with animals infected with untreated conidia. Significant increased levels of tumour necrosis factor-alpha were observed at 8 weeks in animals infected with ECMp-treated conidia while no differences were observed during the remaining periods. These findings point toward an inhibitory effect of ECMp on P. brasiliensis conidia infectivity and suggest that these proteins may interfere with conidia initial adhesion to host tissues probably modulating the immune response in paracoccidioidomycosis.