Basement Membrane Glycoprotein Laminin Research Articles

Haemophilus influenzae Protein E Binds to the Extracellular Matrix by Concurrently Interacting With Laminin and Vitronectin

Nontypeable Haemophilus influenzae (NTHi) causes otitis media and is commonly found in patients with chronic obstructive pulmonary disease (COPD). Adhesins are important for bacterial attachment and colonization. Protein E (PE) is a recently characterized ubiquitous 16 kDa adhesin with vitronectin-binding capacity that results in increased survival in serum. In addition to PE, NTHi utilizes Haemophilus adhesion protein (Hap) that binds to the basement-membrane glycoprotein laminin. We show that most clinical isolates bind laminin and that both Hap and PE are crucial for the NTHi-dependent interaction with laminin as revealed with different mutants. The laminin-binding region is located at the N-terminus of PE, and PE binds to the heparin-binding C-terminal globular domain of laminin. PE simultaneously attracts vitronectin and laminin at separate binding sites, proving the multifunctional nature of the adhesin. This previously unknown PE-dependent interaction with laminin may contribute to NTHi colonization, particularly in smokers with COPD.

The Respiratory PathogenMoraxella catarrhalisBinds to Laminin via Ubiquitous Surface Proteins A1 and A2

Moraxella catarrhalis is one of the leading causes of exacerbations in chronic obstructive pulmonary disease (COPD). In the present article, we show that moraxella (n=15) binds to the major basement-membrane glycoprotein laminin, which is thickened in the airways of smokers. Using clinical strains of M. catarrhalis and their corresponding ubiquitous surface protein (Usp) A1/A2 mutants, we demonstrate that UspA1 and UspA2 are important for the laminin interaction. Binding assays with recombinant proteins demonstrated that the binding regions are localized within the N-terminal fragments, where both proteins form a globular head. Thus, UspA1/A2-dependent interactions with laminin might promote bacterial adhesion, particularly in smokers with COPD.

Identification of Apoptotic and Antiangiogenic Activities of Terazosin in Human Prostate Cancer and Endothelial Cells

Identification of Apoptotic and Antiangiogenic Activities of Terazosin in Human Prostate Cancer and Endothelial Cells

Increased expression of galectin-1 in carcinoma-associated stroma predicts poor outcome in prostate carcinoma patients.

Galectin-1, a member of the beta-galactoside-binding galectin family, is a pleiotropic dimeric protein participating in a variety of normal and pathological processes, including cancer progression. Modulation of the interactions with the basement membrane glycoprotein laminin and induction of apoptosis in activated T lymphocytes are well-known functions of this galectin. In this study, the expression of galectin-1 was examined in 148 human primary prostate carcinoma samples. Immunohistochemical staining of paraffin sections of prostate tissues revealed that galectin-1 was not detected in normal, PIN (prostatic intraepithelial neoplasia) or carcinoma cells, but accumulated in the stroma and associated fibroblasts. Galectin-1 expression was significantly increased in the tumour-associated stroma compared with the non-neoplastic gland-associated stroma in 21.3% of the cases (Mantel-Haenszel test, p=0.001; Wilcoxon signed rank test, p<0.0001). Increased galectin-1 expression in the cancer-associated stroma compared to the normal gland-associated stroma (p=0.03) was identified by multivariate analysis as a strong independent predictor of prostate-specific antigen (PSA) recurrence, just after the pathological stage (p<0.0001). The association between accumulation of galectin-1 in the stroma of the malignant tissue and aggressiveness of the tumour adds weight to the body of evidence that identifies a role for galectin-1 in the acquisition of the invasive phenotype. In addition to modulating cancer cell interactions with laminin, galectin-1 accumulated around the cancer cells may act as an immunological shield by inducing activated T-cell apoptosis. This exciting hypothesis warrants further investigation.

Changes in the distribution pattern of galectin-1 and galectin-3 in human placenta correlates with the differentiation pathways of trophoblasts

Human placentation is a complex biological phenomenon that results from precisely regulated interactions between cells and the extracellular matrix. Galectin- 1 and galectin-3 belong to a newly defined family of galactose-binding lectins that can bind several glycoconjugates such as the basement membrane glycoprotein laminin, and are involved in many biological events including cell adhesion. In this study, the expression of these two galectins in first and third trimester normal human placenta was examined using single and double immunohistochemical staining and specific antibodies for galectins and cytokeratins. Galectin-3 was detected in all trophoblastic lineages including villous cytotrophoblasts and extravillous trophoblasts (trophoblastic cell columns, infiltrating trophoblasts, endovascular trophoblasts and placental bed giant cells). On the contrary, galectin-1 distribution was restricted to endometrium. A reduction of galectin-3 expression was observed from the villous trophoblasts to the trophoblastic cell columns. This pattern correlated with the switch from a proliferative to a migratory phenotype. Galectin-1 and galectin-3 were both detected in maternal decidual cells. Our data demonstrate a specific pattern of galectin-1 and galectin-3 expression in trophoblastic tissue, and suggest these lectins could contribute to cell-cell and cell matrix interactions of trophoblast during placentation.

Peptide G, Containing the Binding Site of the 67-kDa Laminin Receptor, Increases and Stabilizes Laminin Binding to Cancer Cells

We investigated the effect of peptide G, a synthetic peptide derived from the sequence of the 37-kDa laminin receptor precursor, on the interaction of laminin in two tumor cell lines one of which produces laminin and one of which does not. Addition of peptide G to the culture medium induced a significant increase in the amount of endogenous laminin detectable on the cell membrane of both cell lines. Moreover, pretreatment of exogenous laminin with peptide G dramatically increased laminin binding on both cell lines. Kinetics analysis of membrane-bound labeled laminin revealed a 3-fold decrease in the kd of peptide G-treated laminin compared with untreated or unrelated or scrambled peptide-treated laminin. Moreover, the affinity constant of peptide G-treated laminin increased 2-fold, with a doubling of the number of laminin binding sites, as determined by Scatchard analysis. Expression of the VLA6 integrin receptor on the cell membrane increased after incubation with peptide G-treated laminin. However, the lower binding inhibition of peptide G-treated laminin after anti-VLA6 antibody or cation chelation treatment indicates that membrane molecules in addition to integrin receptors are involved in the recognition of peptide G-modified laminin. These "new" laminin-binding proteins also mediated cell adhesion to laminin, the first step in tumor invasion. Together, the data suggest that peptide G increases and stabilizes laminin binding on tumor cells, involving surface receptors that normally do not take part in this interaction. This might explain the abundant clinical and experimental data suggesting a key role for the 67-kDa laminin receptor in the interaction between cancer cells and the basement membrane glycoprotein laminin during tumor invasion and metastasis.

Evidence of a Laminin Binding Protein on the Surface ofLeishmania donovani

Both the promastigote and amastigote forms of the intracellular parasite,Leishmania donovanibind the basement membrane glycoprotein laminin with high affinity (Kd= 3.56 × 10−9M and 3.98 × 10−9M respectively) with ∼9000 and ∼800 sites per cell. Bound laminin was identified by direct autoradiography and the binding protein through analysis of the parasite extract by SDS-PAGE and immunoblotting. A major component of 67 kDa was detected. The same protein was obtained when parasite outer membrane proteins were adsorbed to laminin-sepharose affinity matrix and subsequently eluted with SDS. The binding affinity of the isolated receptor was similar to that of the whole cells. Such a receptor isolated inLeishmaniafor the first time, may function as one of the bridging molecules for extracellular matrix recognition.

Decreased expression of galectin-3 is associated with progression of human breast cancer.

Galectin-3, a member of the beta-galactoside-binding lectin family, is involved in several biological events including binding to the basement membrane glycoprotein laminin. Although the exact role of galectin-3 during the interactions between cells and laminin is not yet known, it has recently been observed that its expression is down-regulated at both the protein and the mRNA level in colon cancer tissues in correlation with progression of the disease. This study investigated the possibility that breast cancer cells might also exhibit decreased galectin-3 expression in association with their aggressiveness. The expression of galectin-3 was examined by immunoperoxidase staining, using a polyclonal antibody raised against recombinant galectin-3, in a collection of 98 human breast lesions including 12 fibroadenomas, 15 fibrocystic disease lesions, 22 in situ carcinomas, and 49 infiltrating ductal carcinomas, 19 of which had positive axillary lymph nodes. Normal breast tissue adjacent to the lesions was present in 59 biopsies. Normal breast tissue expressed high levels (3+) of galectin-3. High expression (2+ to 3+) was also found in most benign lesions examined. The expression of galectin-3 was significantly decreased in in situ carcinoma and this down-regulation was more pronounced in invasive ductal carcinoma, particularly when associated with infiltration of axillary lymph nodes. These data constitute the first observation that galectin-3 is down-regulated in breast cancer and suggest the decreased expression of this galactoside-binding lectin is associated with the acquisition of the invasive and metastatic phenotype.

Immunohistochemical distribution of laminin in squamous cell carcinoma of the head and neck--correlations between laminin staining and clinical and histological features

The distribution of the basement membrane glycoprotein laminin was investigated immunohistochemically in 66 patients with squamous cell carcinoma of the head and neck region. There were 18 patients with laryngeal cancer, 23 with pharyngeal cancer, 11 with maxillary cancer, and 14 with oral cancer. The specimens were taken from untreated primary lesions, and laminin was detected using the avidin-biotin-peroxidase complex method. In carcinoma tissue, staining was seen in border zone between the cancer cells and surrounding connective tissue, but discontinuity or disappearance of the staining was commonly observed. The patients were classified on the basis of laminin distribution into the following three groups: a normal group, in which staining was almost continuous around the cancer cells, a discontinuous group, in which staining was discontinuous but present in up to 50% of the cancer cells, and an absent group, in which staining was seen in 49% or less of cancer cells. These groups were matched against clinical and histological features. Laminin distribution was significantly correlated with metastasis, survival rate and keratinization. In the absent group, 12 (92%) of the 13 patients had lymph node metastasis, and 6 (46%) had distant metastasis. In the discontinuous group, 17 (94%) of the 18 patients had lymph node metastasis and 3 (17%) had distant metastasis. In contrast, in the normal group, 13 (37%) of the 25 patients had lymph node metastasis and 2 (6%) had distant metastasis. The 5 year survival rate was 31%, 52%, and 67% in the absent, discontinuous and normal group, respectively. Histologically, the ratios of poorly keratinized to highly keratinized carcinomas were 62%, 67%, and 34% in the absent, discontinuous, and normal group, respectively. These findings suggested that the absent and the discontinuous, group required intensive neck dissection and systemic anticancer therapy.

Role of laminin for axonal growth.

The basement membrane glycoprotein laminin has been shown in vitro to stimulate the growth or regeneration of neuronal processes [2], to act together with neurotrophic factors to promote neuronal survival [8] and to stimulate the proliferation of glial cells [23]. Furthermore, experiments after lesion in vivo indicate that laminin is required for the survival of neural cells [17] and is involved in the regeneration of axons peripheral nerves [27].

Immunocytochemistry of Skeletal Muscle Basal Lamina Grafts in Nerve Regeneration

The influence on nerve regeneration of the extracellular matrix glycoprotein laminin was studied after sciatic nerve transection in 90 outbred Sprague-Dawley rats. Nerve regeneration through basal lamina grafts was comparable with regeneration through traditional nerve grafts across gaps up to 2.0 cm in length. True axonal regeneration rather than axonal branching was demonstrated by retrograde horseradish peroxidase labeling of nerve cables. Pretreatment of basal lamina grafts with antilaminin antibodies reduced the total number of regenerated axons by 90 percent with a significant decrease of nerve conduction velocity and a significant impairment of walking track patterns. The basement membrane glycoprotein laminin serves a critical role in the regeneration of peripheral nerves through basal lamina grafts.

Genes Coding for Basement Membrane Glycoproteins Laminin, Nidogen, and Collagen IV Are Differentially Expressed in the Nervous System and by Epithelial, Endothelial, and Mesenchymal Cells of the Mouse Embryo

The pattern of laminin A, B1, B2, nidogen, and collagen alpha;1(IV) gene expression in the 12.5-day mouse embryo was determined by in situ hybridization. Laminin B1, B2, and collagen α1(IV) mRNAs were present in many epithelial and mesenchymal compartments. Laminin A mRNA had a more restricted distribution, being present in cells closely associated with basement membranes and also in the ependymal layer of the neural tube. Nidogen was not produced by any epithelium, but was abundant in mesenchymal and endothelial cells. These results demonstrate that mesenchymal cells contribute significantly to basement membrane production, and that many cells not associated with typical basement membranes produced high levels of mRNAs coding for basement membrane components. Very few cell types produced all five gene products, and some tissues preferentially expressed only one or two of the five genes. This study shows that basement membranes at the epithelial-mesenchymal interface in the majority of mouse embryonic tissues are assembled from components derived from both cell types, and that heterogeneous matrix structures containing different laminin subunits and/or nidogen are likely to be present in the central nervous system and other tissues of the midgestation mouse embryo.

Embryonal carcinoma and the basement membrane glycoproteins laminin and entactin.

The mouse embryonal carcinoma lines PCC4-F and F9 have played important roles in the isolation and characterization of the two ubiquitous basement membrane proteins, laminin and entactin. The contributions of these cells to our work on extracellular matrices are briefly summarized. The in vitro differentiation of PCC4-F gives rise to a multiplicity of cell types. Two of these cell types have been propagated as cell lines. One of these, M1536-B3, synthesizes and deposits copious quantities of extracellular matrix glycoproteins, which led to the initial discovery and characterization of laminin and entactin. In addition, M1536-B3 provides a model system for analyzing the assembly of laminin and the laminin-entactin complex and for manipulating extracellular matrix structure and composition. The other cell line, 4CQ, synthesizes a matrix consisting of fibronectin and entactin. F9 cells differentiate to endodermal cells in response to retinoic acid and dibutyryl cyclic AMP (Strickland and Mahdavi, Cell 15: 393-402, 1978). The differentiated cells synthesize basement membrane components and provided the probes for the cDNA cloning of entactin and the three chains of laminin. The F9 cells have been widely employed to examine the regulation of expression of the laminin and entactin genes.

Enhancement of Metastatic Potential of Murine and Human Melanoma Cells by Laminin Receptor Peptide G: Attachment of Cancer Cells to Subendothelial Matrix as a Pathway for Hematogenous Metastasis

Stable anchorage of circulating cancer cells to the vasculature is a critical step in the formation of hematogenous metastases. Although the basement membrane glycoprotein laminin clearly plays a crucial role in this event, the exact interactive pathways among cancer cells, laminin, and the vessel wall have not been elucidated. In a previous study, we identified synthetic peptide G, which contains the laminin-binding domain of the 67-kd laminin receptor and which inhibits tumor cell adhesion to endothelial cells. To assess the role of the interaction between laminin and the 67-kd laminin receptor in hematogenous metastasis formation, we studied the effect of peptide G on melanoma cell behavior in vivo and in vitro. The effect of peptide G and control peptides was studied in vivo on lung retention and colonizing potential of murine (B16BL6) and human (A2058) melanoma cells injected intravenously in C57BL/6 and nude mice, respectively. In addition, their effect on cell adhesion and chemotaxis to laminin and on binding of iodine 125-labeled laminin to cells was studied in vitro. In vivo, pretreatment of cells with peptide G resulted in a two- to 10-fold significant increase in the number of experimental lung metastases. A significant relative increase in lung retention of peptide G-treated tumor cells was observed 48 hours after injection, although after 4 hours a partial reduction was observed. In vitro, peptide G significantly increased laminin binding and cancer cell adhesion to laminin and subendothelial matrix, whereas chemotaxis to laminin was significantly inhibited. Peptide G differentially affected the biological response of cancer cells to laminin. In vitro, it increased laminin binding and cell adhesion to laminin and subendothelial matrix, whereas it inhibited cell chemotaxis to laminin. In vivo, the overall effect of peptide G was an augmentation of lung metastasis. Our findings suggest that direct adhesion of tumor cells to the subendothelial matrix is a main pathway for hematogenous metastases and that tumor cell-matrix interaction may be more relevant than tumor cell-endothelial cell attachment in this process.

Inverse modulation of steady-state messenger RNA levels of two non-integrin laminin-binding proteins in human colon carcinoma.

Interactions between cells and the basement membrane glycoprotein laminin are altered during colon cancer progression. Colon carcinoma and normal mucosa cells express a variety of laminin-binding proteins, including the 67-kd laminin receptor (67 LR) and a 31-kd human laminin-binding protein (HLBP31) homologous to the 31-kd human IgE-binding protein/galactoside-binding lectin. To investigate whether various laminin-binding proteins are differentially expressed in human colon carcinoma, we studied messenger RNA (mRNA) levels of the 67 LR and HLBP31 in matched tumor and adjacent normal mucosa samples from a series of 21 patients. Total cellular RNA from tumor and normal mucosa was isolated and analyzed by Northern and slot blot hybridization. In addition, HLBP31 protein levels were assessed by the immunoblot technique. Quantitative laminin affinity chromatography was also used to measure the synthesis of HLBP31 protein in five human cancer cell lines. The steady-state mRNA level of HLBP31 was downregulated (i.e., decreased) in 18 of 21 human colon carcinomas compared with the level in their corresponding normal colonic mucosa. On average, the level of HLBP31 mRNA was decreased 50% +/- 30% (+/- SD) in the colon cancers. The mean ratio of colon cancer HLBP31 mRNA to adjacent normal mucosa HLBP31 mRNA was twofold lower in primary tumors of patients with metastases (0.3 +/- 0.2 SD) than in primary tumors of patients free of metastatic lesions (0.6 +/- 0.2 SD). The differences between the two groups of patients were statistically significant (P less than .05, Wilcoxon-Mann-Whitney test). We have previously shown that the ratio of colon cancer 67 LR mRNA to corresponding normal mucosa 67 LR mRNA was increased in the same patient population. When the two ratios (ratio of cancer to normal HLBP31 mRNA and ratio of cancer to normal 67 LR mRNA) were compared, HLBP31 mRNA/67 LR mRNA was significantly lower (P less than .05) in primary tumors with metastases (mean +/- SD, 0.3 +/- 0.2) than in primary cancers without metastases (mean +/- SD, 0.7 +/- 0.5). The steady-state level of HLBP31 mRNA was directly correlated with the amount of HLBP31 protein in both colon tissue samples and human cancer cell lines. HLBP31 mRNA expression in colon cancer tissues is modulated inversely to that of 67 LR mRNA expression. The down-regulation of HLBP31 appears to be associated with the metastatic capabilities of colon cancer cells. Prospective studies on a large cohort should determine if the systematic detection of HLBP31 and 67 LR protein and/or mRNA can be a valuable adjunct in the prognostic evaluation of primary colon cancers.

Cadmium selenium interaction in the human placenta IN VITRO

Human placentation is a complex biological phenomenon that results from precisely regulated interactions between cells and the extracellular matrix. Galectin- 1 and galectin-3 belong to a newly defined family of galactose-binding lectins that can bind several glycoconjugates such as the basement membrane glycoprotein laminin, and are involved in many biological events including cell adhesion. In this study, the expression of these two galectins in first and third trimester normal human placenta was examined using single and double immunohistochemical staining and specific antibodies for galectins and cytokeratins. Galectin-3 was detected in all trophoblastic lineages including villous cytotrophoblasts and extravillous trophoblasts (trophoblastic cell columns, infiltrating trophoblasts, endovascular trophoblasts and placental bed giant cells). On the contrary, galectin-1 distribution was restricted to endometrium. A reduction of galectin-3 expression was observed from the villous trophoblasts to the trophoblastic cell columns. This pattern correlated with the switch from a proliferative to a migratory phenotype. Galectin-1 and galectin-3 were both detected in maternal decidual cells. Our data demonstrate a specific pattern of galectin-1 and galectin-3 expression in trophoblastic tissue, and suggest these lectins could contribute to cell-cell and cell-matrix interactions of trophoblast during placentation.

Laminin in the human embryo implantation: analogy to the invasion by malignant cells

To explore the possible similarities between the biochemical processes of embryo implantation and malignant invasion. The expression of a basement membrane (BM) glycoprotein laminin, a matrix binding cell surface receptor protein beta 1-integrin, and a BM collagen degrading metalloproteinase type IV collagenase, was studied in cultured human in vitro fertilized embryos. Eight healthy women suffering from tubal infertility were participating in the IVF program in the Department of Obstetrics and Gynecology in University Hospital of Oulu. Twenty oocytes and 110 pre-embryos that were not transferred for the fertilizations were used in this study. Fibronectin, laminin, beta 1-integrin, and type IV collagenase immunoreactive proteins were studied in embryos by immunoperoxidase staining, and type IV collagen degrading activity was measured from the culture media of the embryos. Laminin and beta 1-integrin were expressed in the early human embryos before the time of implantation. Type IV collagen degrading activity and the 72 kd-type IV collagenase immunoreactive protein were expressed at the time of implantation. Laminin supported the expression of type IV collagenase. The expression of laminin, beta 1-integrin, and type IV collagenase in vitro are temporally in good correlation with the time of the implantation in vivo. Laminin and beta 1-integrin can relate to the attachment of the embryos to the uterine BM and type IV collagenase to the degradation of the BM collagen during the implantation. Laminin can augment the process locally.

Laminin-induced retinoblastoma cell differentiation: possible involvement of a 100-kDa cell-surface laminin-binding protein.

Gene and protein expression of Y-79 retinoblastoma cells growing on poly(D-lysine) is switched from a photoreceptor-like to a conventional neuron-like pathway by the basement membrane glycoprotein laminin. Unlike other cell systems where laminin influences differentiation, Y-79 cells can neither attach to nor chemotactically respond to laminin. However, laminin increases attachment to poly(D-lysine). The laminin effects therefore seem to occur via an adhesion- and chemotaxis-independent mechanism. Moreover, these tumor cells do not exhibit high-affinity laminin binding, having only a single binding site of intermediate affinity. Laminin-Sepharose affinity chromatography of Y-79 cell surface proteins labeled with 125I revealed a single major radiolabeled 100-kDa protein eluted by 20 mM EDTA, with an electrophoretic behavior different from that of integrins. No other proteins were eluted under more stringent conditions. This material, which we call LBM-100 (100-kDa laminin-binding molecule), may be a "differentiative" laminin-binding protein through which laminin influences gene expression and development independently of attachment.

Helicobacter pylori and peptic ulcers: the present position.

The invasion of malignant cells through the basement membrane is a critical step in local infiltration and metastasis. Adhesion and invasion of malignant cells may be modulated by their receptor mediated binding to the basement membrane glycoprotein laminin. We studied the specific adhesion of human colon adenocarcinoma derived HT 29 cells to laminin and its proteolytic fragments. The major cell adhesion domain of laminin was localised in the central part of the cross shaped molecule. Immunoblotting experiments on separated HT 29 cell membranes using specific antibodies or radiolabelled laminin fragments revealed two major laminin-binding cell surface components with Mr of 67,000 and 69,000 D similar to the putative laminin receptor described for other tissues. Using a nitrocellulose filter disk assay, the specific interaction between cell surface binding proteins and proteolytic fragments originating from the central core of the laminin molecule could be further corroborated. In contrast, interaction of HT 29 cell membranes with the pentapeptide YIGSR (tyr-ile-gly-ser-arg), a sequence domain of the B1-chain of the laminin molecule, thought to be responsible for cell adhesion, was significantly weaker.

Composition and organization of cell–substratum contacts in normal and neoplastic renal epithelium

We have investigated the molecular basis of the organization of cell-substratum contact in normal and neoplastic renal epithelium. The several components of focal contacts and non-collagenous basement membrane glycoproteins have been identified by antibodies, detected by immunofluorescence and viewed by laser scanning microscopy. Tubular epithelial cells grown on glass coverslips expressed laminin at the cell periphery. Co-localized with laminin were receptors of the beta 1 integrin class, and the cytoplasmic plaque proteins vinculin and talin. When basement membrane glycoproteins laminin or fibronectin were exogenously supplied by growing cells on coated coverslips, the vinculin-, talin- and integrin-containing contacts became organized into linear arrays with intervening free cell membranes. Under these conditions the actin cytoskeleton became highly organized. By contrast some tumour cells showed a reduction in focal contact molecules and a failure to organize these structures in response to laminin or fibronectin. This loss of cell matrix interaction may be important during the progression of renal tumours.