Effect Of Basement Membrane Components Research Articles

Quantitative Analysis of the Effects of Extracellular Matrix Proteins on Membrane Dynamics Associated with Corneal Epithelial Cell Motility

Interaction of corneal epithelial cells with components of the basement membrane regulates cell morphology, adhesion, and migration. The authors examined the effects of basement membrane components on the behavior of human corneal epithelial (HCE) cells, including movement of the cell membrane in relation to the direction of cell migration. HCE cells were plated on fibronectin, laminin, collagen types I and IV, and bovine serum albumin (BSA). Cell adhesion was evaluated by staining with crystal violet. Cell movement was monitored by time-lapse microscopy, and an image processing program was developed for quantitative analysis of movement of the cell membrane in relation to the direction of cell migration. Actin cytoskeleton, focal adhesions, and the K+ channel Kv2.1 were detected by fluorescence microscopy. Laminin, fibronectin, and collagen types I and IV each promoted cell adhesion compared with that apparent with BSA. Fibronectin and collagen types I and IV, but not laminin, increased cell motility. Laminin had no effect on membrane movement compared with that observed in the presence of BSA. Collagen types I and IV each increased membrane movement but in a manner independent of the direction of cell migration. Fibronectin induced membrane movement in the direction of cell migration. Fibronectin induced the formation of thick bundles of F-actin and focal adhesions at the cell periphery and the localization of Kv2.1 at the leading edge of the cell, whereas laminin and collagen types I and IV elicited the formation of thinner bundles of F-actin and smaller focal adhesions. Fibronectin may modulate the directional migration of corneal epithelial cells.

Effect of basement membrane components on assembly of 3‐pyroglutamylamyloid β‐protein

Abstract3‐pyroglutamyl amyloid β‐protein is major component of senile plaques in Alzheimer’s disease. Basement membrane (BM) components co‐localize with amyloid in the plaque. We investigated the potential of BM components, such as type IV collagen (collagen IV), laminin and entactin, to prevent assembly of 3‐pyroglutamyl amyloid β‐protein 3‐42 (Aβ3pE‐42) and to induce a disassembly of Aβ3pE‐42 fibril. For this we used thioflavin T fluorometric assays. All BM components significantly inhibited Aβ3pE‐42 fibril formation (Molar ratio; Aβ:collagen IV = 136:1, Aβ:laminin = 180:1, Aβ:enactin = 20:1). The inhibitory effect of BM components was in a dose‐dependent manner. Collagen IV and laminin significantly disrupted pre‐formed AβpE‐42 fibril, whereas enactin without effect. We propose that BM components may be involved in senile plaque formation by regulating both AβpE‐42 fibril formation and disassembly of the fibril.

Reconstituted basement membrane induces glandular-like morphogenesis but no difference in ACTH synthesis of anterior pituitary cells.

Basement membrane, a thin extracellular matrix, promotes tissue integrity and differentiated phenotype. This study was performed in order to investigate the effect of basement membrane components on adrenocorticotropin (ACTH) synthesis and to observe its relationship with cell morphology. Rat anterior pituitary cells were cultured on plastic culture plate coated with either Matrigel or poly-D-lysine. Phase-contrast microscopy and scanning electron microscopy showed that cells cultured on Matrigel appeared as a three-dimensional glandular-like cell aggregate, while those cultured on plastic showed a flat, confluent monolayer. Reverse-transcription polymerase chain reaction (RT-PCR) and Northern blot analysis revealed that ACTH synthesis in the Matrigel culture was not significantly different from that in the plastic culture. Our results suggest that the relationship between the morphological changes caused by cell-substrate interaction and pituitary hormone synthesis does not exist in all pituitary cell types and that other factors associated with cell-substrate interaction influence the hormone synthesis of some pituitary cells.

Pexicrine effects of basement membrane components on paracrine signaling by renal tubular cells

Paracrine interactions between tubular epithelium and interstitial cells have been assumed to be mediated largely by soluble cytokines. While the role of extracellular matrix (ECM) and matrix metalloproteinases (MMPs) in modifying cell function is widely appreciated, the role of the renal tubular basement membrane in modulation of tubulointerstitial function has not been studied. To establish whether those components of the ECM which support tubular epithelial cells also influence cell function (that is, a pexicrine effect), we studied their effects on paracrine signaling between epithelium and fibroblasts. Primary cultures of rat renal proximal tubular epithelial cells (PTE) were cultured on laminin (LN), collagen types-IV and -I (COL-IV, COL-I) and fibronectin (FN). PTE attained confluence more rapidly when grown on LN = COL-IV > COL-I = FN = plastic. On all substrates PTE produced the MMPS, gelatinase-A and -B and collagenase with an apparent increase in gelatinase-A and -B production when cultured on LN. MMPs were found to be secreted both apically and basally with basal secretion predominating, except on LN where secretion was primarily from the apical surface. Cultures of rat renal cortical interstitial fibroblasts were established and characterized. Cortical fibroblasts (CF) were found to secrete gelatinase-A and collagenase. Conditioned medium (CM) from PTE cultured on COL-IV stimulated proliferation of CF but proliferation was unaltered by CM from PTE grown on other substrates. By contrast, co-culture of PTE on LN with CF suppressed collagenase and gelatinase activity in both cell types, indicating a bi-directional, paracrine modulation of MMP production. Thus in the tubulointerstitium, the BM components LN and COL-IV not only fulfill a structural role but act as signaling molecules with differential effects which modify the function of the tubular epithelium and its paracrine interaction with adjacent fibroblasts. The initiation of interstitial fibrosis induced by injury to the tubular basement membrane may reside in the perturbation of this interaction.

Basement membrane components are potent promoters of rat intestinal epithelial cell differentiation in vitro

Basement membranes have been implicated in morphogenesis and cell differentiation. In this study, the effect of basement membrane components on intestinal epithelial cell maturation in a mesenchyme-free environment was investigated. Fetal rat small intestinal epithelial cells (from the 14th–17th day of gestation) were exposed to basement membrane-derived proteins (laminin, collagen type IV, and a complex basement membrane-enriched extract from the Engelbreth-Holm-Swarm sarcoma) and other extracellular matrix proteins (collagen type I and fibronectin) coated onto Petri dishes. The cells attached readily only to fibronectin and basement membrane proteins. For 5 days the developing epithelial colonies were monitored in vitro, assessing morphological and functional parameters of cell maturation. Colonies grown on laminin and the basement membrane extract were larger and of greater cell density. An increase in alkaline phosphatase and lactase activity was observed after 3–4 days in these colonies which could be enhanced to yield 90%–100% positive cells by the addition of dexamethasone to the medium while no sucrase-isomaltase activity was elicited. Electron microscopy confirmed a high degree of cellular polarization illustrated by tight junctions and apical microvilli in epithelial cells grown on a basement membrane-like support. In contrast, none of the other proteins stimulated the cells to mature in vitro. The authors conclude that certain basement membrane components actively promote fetal intestinal epithelial cell differentiation.

Regulation of mammary differentiation by the extracellular matrix.

In multicellular organisms cell growth and differentiation are influenced by soluble factors, cell-cell interactions and cell-extracellular matrix interactions. We have used the rat mammary gland as a model system to study the role of extracellular matrix components in the regulation of milk protein gene expression. Since mammary epithelial cells differentiate on a basement membrane in vivo, we investigated the effects of basement membrane components on the expression of the milk protein genes, alpha-casein, alpha-lactalbumin, and transferrin. We have demonstrated that a basement membrane gel, as well as its major basement membrane component, laminin, induced alpha-casein and alpha-lactalbumin expression as much as 160-fold compared to tissue culture plastic. We demonstrate that laminin affects mRNA stability as well as having an effect on protein stability and secretion. Laminin interacts with mammary epithelial cells via an 68 kD cell surface receptor which is capable of interacting with the cellular cytoskeleton. In order to provide evidence that laminin affects on mammary differentiation are mediated through this receptor via the cytoskeleton, we examined the effects of cytoskeletal disrupting agents on milk protein gene expression. We demonstrate that cytochalasin D or colchicine selectively block laminin-mediated milk protein gene expression by affecting mRNA stability. Based on these experiments, we propose a model in which laminin affects mammary gene expression through interaction with cell surface receptors which interact with the cytoskeleton resulting in stabilization of mRNAs for milk protein genes.

Modulation of type IV collagenase and plasminogen activator in a hamster fibrosarcoma by basement membrane components and lung fibroblasts.

The effect of basement membrane components (laminin, fibronectin and type IV collagen) and lung fibroblasts on type IV collagenase and plasminogen activator activity was investigated in a primary HSV-2-induced hamster fibrosarcoma, and its in vivo derived sublines and in vitro derived clones of varying metastatic potential. Fibronectin and type IV collagen were ineffective at influencing the expression of either type IV collagenase or plasminogen activator activity. Laminin, however, at concentrations of 1-10 micrograms ml-1 added to the serum-free culture supernatants, increased the release of type IV collagenase by up to 100% for the parental cell line. Three highly metastatic sublines (two from in vivo origin and one from in vitro cloning) showed increases of up to 300%. Non-metastatic sublines (two from in vivo origin and one from in vitro cloning), however, showed no increase in type IV collagenase activity. Plasminogen activator release from either the parental line cell or its metastatic sublines and clones, was unaffected by the addition of laminin. Addition of tumour cells to lung fibroblast monolayers resulted in an increased expression of PA activity in the supernatant, whilst type IV collagenase activity was reduced.

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 α-casein and α-lactalbumin (α-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 α-, β-, and γ-caseins and α-LA. These studies demonstrated that ECM components induced α- and β-caseins up to 10-fold and α-LA up to 3-fold, with no significant effect on γ-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.

Synthesis and effects of basement membrane components in cultured rat Schwann cells.

Schwann cells, the myelin-forming cells of the peripheral nervous system, are surrounded by a basement membrane. Whether cultured rat Schwann cells synthesize the basement membrane-specific components, laminin and collagen type IV, and whether these components influence the adhesion, morphology, and growth of these cells have been investigated. Both laminin and collagen type IV were detected in the cytoplasm of Schwann cells by immunofluorescence. After ascorbate treatment, laminin and collagen type IV were both found in an extracellular fibrillar matrix bound to the Schwann cell surface. Laminin was further localized on the Schwann cell surface by electron microscopy using gold immunolabeling. Anti-laminin IgG-labeled gold particles were scattered over the cell surface, and linear rows of particles and small aggregates were found along the cell edges and at points of contact with other cells. When added to the culture medium, laminin acted as a potent adhesion factor, stimulating Schwann cell adhesion as much as eightfold above control levels on type IV collagen. In the presence of laminin, the cells became stellate and by 24 hr had extended long, thin processes. Laminin also stimulated cell growth in a dose-dependent manner and anti-laminin IgG completely inhibited cell attachment and growth in the absence of exogenous laminin. Thus, cultured Schwann cells synthesize laminin and collagen type IV, two major components of basement membrane, and laminin may trigger Schwann cell differentiation in vivo during early stages of axon-Schwann cell interaction before myelination.