Overexpression of integrin α11 induces cardiac fibrosis in mice.

AIM: To understand the role of the collagen-binding integrin α11 in vivo, we have used a classical approach of creating a mouse strain overexpressing integrin α11. A transgenic mouse strain overexpressing α11 in muscle tissues was analysed in the current study with special reference to the heart tissue. METHODS: We generated and phenotyped integrin α11 transgenic (TG) mice by echocardiography, magnetic resonance imaging and histology. Wild-type (WT) mice were subjected to aortic banding (AB) and the expression of integrin α11 was measured in flow cytometry-sorted cardiomyocytes and non-myocytes. RESULTS: TG mice developed left ventricular concentric hypertrophy by 6 months, with increased collagen deposition and reactivation of mRNA encoding foetal genes associated with cardiovascular pathological remodelling compared to WT mice. Masson's trichrome staining revealed interstitial fibrosis, confirmed additionally by magnetic resonance imaging and was found to be most prominent in the cardiac septum of TG but not WT mice. TG hearts expressed increased levels of transforming growth factor-ß2 and transforming growth factor-ß3 and upregulated smooth muscle actin. Macrophage infiltration coincided with increased NF-κB signalling in TG but not WT hearts. Integrin α11 expression was increased in both cardiomyocytes and non-myocyte cells from WT AB hearts compared to sham-operated animals. CONCLUSION: We report for the first time that overexpression of integrin α11 induces cardiac fibrosis and left ventricular hypertrophy. This is a result of changes in intracellular hypertrophic signalling and secretion of soluble factors that increase collagen production in the heart.

Integrin α11ß1 regulates cancer stromal stiffness and promotes tumorigenicity and metastasis in non-small cell lung cancer.

Integrin α11ß1 is a stromal cell-specific receptor for fibrillar collagens and is overexpressed in carcinoma-associated fibroblasts (CAFs). We have investigated its direct role in cancer progression by generating severe combined immune deficient (SCID) mice deficient in integrin α11 (α11) expression. The growth of A549 lung adenocarcinoma cells and two patient-derived non-small cell lung carcinoma (NSCLC) xenografts in these α11 knockout (α11(-/-)) mice was significantly impeded, as compared with wild-type (α11(+/+)) SCID mice. Orthotopic implantation of a spontaneously metastatic NCI-H460SM cell line into the lungs of α11(-/-) and α11(+/+) mice showed significant reduction in the metastatic potential of these cells in the α11(-/-) mice. We identified that collagen cross-linking is associated with stromal α11 expression, and the loss of tumor stromal α11 expression was correlated with decreased collagen reorganization and stiffness. This study shows the role of integrin α11ß1, a receptor for fibrillar collagen in differentiation of fibroblasts into CAFs. Furthermore, our data support an important role for α11 signaling pathway in CAFs, promoting tumor growth and metastatic potential of NSCLC cells and being closely associated with collagen cross-linking and the organization and stiffness of fibrillar collagen matrices.

Integrins α2ß1 and α11ß1 regulate the survival of mesenchymal stem cells on collagen I.

Although mesenchymal stem cells (MSCs) are the natural source for bone regeneration, the exact mechanisms governing MSC crosstalk with collagen I have not yet been uncovered. Cell adhesion to collagen I is mostly mediated by three integrin receptors - α1ß1, α2ß1 and α11ß1. Using human MSC (hMSC), we show that α11 subunit exhibited the highest basal expression levels but on osteogenic stimulation, both α2 and α11 integrins were significantly upregulated. To elucidate the possible roles of collagen-binding integrins, we applied short hairpin RNA (shRNA)-mediated knockdown in hMSC and found that α2 or α11 deficiency, but not α1, results in a tremendous reduction of hMSC numbers owing to mitochondrial leakage accompanied by Bcl-2-associated X protein upregulation. In order to clarify the signaling conveyed by the collagen-binding integrins in hMSC, we analyzed the activation of focal adhesion kinase, extracellular signal-regulated protein kinase and serine/threonine protein kinase B (PKB/Akt) kinases and detected significantly reduced Akt phosphorylation only in α2- and α11-shRNA hMSC. Finally, experiments with hMSC from osteoporotic patients revealed a significant downregulation of α2 integrin concomitant with an augmented mitochondrial permeability. In conclusion, our study describes for the first time that disturbance of α2ß1- or α11ß1-mediated interactions to collagen I results in the cell death of MSCs and urges for further investigations examining the impact of MSCs in bone conditions with abnormal collagen I.

The alpha11beta1 integrin has a mechanistic role in control of interstitial fluid pressure and edema formation in inflammation.

OBJECTIVE: Collagen-binding integrins may be involved in controlling interstitial fluid pressure (Pif), transcapillary fluid flux, and tissue fluid volume. Our aim was to explore whether the newly discovered collagen binding alpha11beta1 integrin has a mechanistic role in inflammatory edema formation. METHODS AND RESULTS: In collagen matrices seeded with a mixture of mast cells and fibroblasts, fibroblasts lacking the alpha11 integrin subunit (alpha11(-/-)) contracted collagen gels less efficiently than control fibroblasts, suggesting that the alpha11beta1 integrin is able to mediate tensile force in connective tissues. In alpha11(-/-) mice, control Pif in skin did not differ from the pressure found in wild-type mice. Whereas a reduction in Pif was found in control mice after inducing inflammation, thereby contributing to fluid extravasation and edema formation, such a reduction was not seen in alpha11(-/-) mice. That this effect is mediated through the extracellular compartment is suggested by a similar plasma protein extravasation ratio in alpha11(-/-) and wild-type mice. CONCLUSIONS: Our data suggest that alpha11beta1 integrins on dermal fibroblasts mediate collagen lattice remodeling and have a mechanistic role in controlling Pif in inflammation and thereby fluid extravasation and edema formation in vivo.

A role for alpha11beta1 integrin in the human periodontal ligament.

We previously demonstrated a role for alpha11beta1 integrin in periodontal ligament (PDL)-driven tooth eruption in the mouse. To explore a possible role for alpha11beta1 in the human periodontium, we have characterized the expression and function of alpha11 in human PDL tissue, in human PDL fibroblasts (hPDLF), and in human gingival fibroblasts (hGF). alpha11 expression was detected in PDL tissue, in hPDLF, and in hGF cells. Platelet-derived growth factor-BB and insulin-like growth factor II stimulated contraction of collagen lattices by both types of fibroblasts. alpha2 integrin blocking antibodies and the use of alpha11 siRNA demonstrated a role for both alpha2beta1 and alpha11beta1 in collagen lattice remodeling. Analysis of the proximal ITGA11 promoter from persons with chronic periodontal disease failed to reveal any polymorphism. Analysis of our data shows that alpha11beta1 is a major collagen receptor on cultured human PDL cells and implies that it is also functionally important in the PDL in vivo.

Physiology and pathology of collagen receptors.

Just before the transition from pre-genomic to the post-genomic era, the two latest members of the mammalian integrin family were identified. These integrins, which were named alpha10beta1 and alpha11beta1, are both collagen receptors and are related. Rather than being twins, they can be regarded as close cousins. They both belong to the subfamily of integrins that contain an I-domain in the alpha subunit. This domain is also the part that endows these integrins with the capacity to bind the GFOGER sequence in collagens. In the current review, we summarize and update the current knowledge about the in vitro and in vivo functions of these integrins.

Glucocorticoids down-regulate the extracellular matrix proteins fibronectin, fibulin-1 and fibulin-2 in bone marrow stroma.

Glucocorticoids regulate hematopoietic cell interactions with the bone marrow microenvironment, but the molecules involved in the regulation are still largely unknown. We have studied the effect of glucocorticoids on mRNA expression and protein synthesis of the major extracellular matrix adhesion protein fibronectin and three other extracellular proteins, fibulin-1, fibulin-2 and nidogen-1, in mouse bone marrow cultures and in a hematopoiesis supporting the stromal MC3T3-G2/PA6 cell line. Glucocorticoids suppressed mRNA expression and protein synthesis of fibronectin, fibulin-1 and fibulin-2, but not nidogen-1, in adherent cells of bone marrow cultures, as shown by Northern blot analysis and immunoprecipitation. mRNA levels of all four proteins were down-regulated by dexamethasone in MC3T3-G2/PA6 cells, indicating a direct glucocorticoid effect on cells synthesizing extracellular matrix proteins. Dexamethasone down-regulated fibronectin mRNA rapidly, within 2 h of treatment, in the stromal cells. This effect did not require mRNA or protein synthesis, as shown by Northern blot analysis after treatment by actinomycin D and cycloheximide. Interferon-alpha, which also has been reported to modulate haematopoietic cell-matrix interactions, did not affect mRNA expression of the proteins in MC3T3-G2/PA6 cells. Our results indicate that glucocorticoids down-regulate expression of several mesenchymal-type extracellular matrix molecules in bone marrow, but with a variable effect on different proteins. Thus one mechanism by which glucocorticoids regulate haematopoiesis may be by altering the relative proportions of extracellular matrix proteins.

alpha11beta1 integrin is a receptor for interstitial collagens involved in cell migration and collagen reorganization on mesenchymal nonmuscle cells.

alpha11beta1 integrin constitutes a recent addition to the integrin family. Here, we present the first in vivo analysis of alpha11 protein and mRNA distribution during human embryonic development. alpha11 protein and mRNA were present in various mesenchymal cells around the cartilage anlage in the developing skeleton in a pattern similar to that described for the transcription factor scleraxis. alpha11 was also expressed by mesenchymal cells in intervertebral discs and in keratocytes in cornea, two sites with highly organized collagen networks. Neither alpha11 mRNA nor alpha11 protein could be detected in myogenic cells in human embryos. The described expression pattern is compatible with alpha11beta1 functioning as a receptor for interstitial collagens in vivo. To test this hypothesis in vitro, full-length human alpha11 cDNA was stably transfected into the mouse satellite cell line C2C12, lacking endogenous collagen receptors. alpha11beta1 mediated cell adhesion to collagens I and IV (with a preference for collagen I) and formed focal contacts on collagens. In addition, alpha11beta1 mediated contraction of fibrillar collagen gels in a manner similar to alpha2beta1, and supported migration on collagen I in response to chemotactic stimuli. Our data support a role for alpha11beta1 as a receptor for interstitial collagens on mesenchymally derived cells and suggest a multifunctional role of alpha11beta1 in the recognition and organization of interstitial collagen matrices during development.

Assembly of laminin polymers is dependent on beta1-integrins.

Recent reports suggest that laminin deposition is controlled by the cell via specific receptors, one of which is dystroglycan. In this study, the involvement of beta1-integrins in this process was investigated by comparing beta1-integrin-deficient cells of different phenotypes with their normal counterparts. Normal embryonic stem (ES) cells and embryoid bodies (EBs) derived from them were found to deposit cell-associated laminin into fibrillar networks, and in the EBs a basement membrane was assembled under the primitive endoderm. beta1-deficient ES cells and their EBs formed only small amounts of dot-like laminin deposits. Skeletal myotubes formed after prolonged differentiation in EBs were found to be surrounded by laminin, nidogen, and perlecan by immunofluorescent staining irrespective of the presence of beta1-integrins on the myotubes. However, at the electron microscope level only very thin sheet-like structures were detected close to the beta1-deficient myotubes, while the wt myotubes formed thick basement membranes. An epithelial cell line, GE11, derived from the beta1-integrin-deficient ES cells was also unable to assemble laminin on the cell surface, while transfection of the cells with the integrin beta1 subunit resulted in formation of a dense laminin network. Taken together, these results suggest that dystroglycan and beta1-integrins can both contribute to the recruitment of laminin to cell surfaces and that integrins are required at a subsequent step in the formation of basement membranes.

Posttranslational modifications and beta/gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin-3 from placenta.

Laminins assemble into trimers composed of alpha, beta, and gamma chains which posttranslationally are glycosylated and sometimes proteolytically cleaved. In the current paper we set out to characterize posttranslational modifications and the laminin isoforms formed by laminin alpha1 and alpha5 chains. Comparative pulse-chase experiments and deglycosylation studies in JAR cells established that the M(r) 360,000 laminin alpha1 chain is glycosylated into a mature M(r) 400,000 band while the M(r) 370,000 laminin alpha5 chain is glycosylated into a M(r) 390,000 form that upon secretion is further processed into a M(r) 380,000 form. Hence, despite the shorter peptide length of alpha1 chain in comparison with the alpha5 chain, secreted alpha1 assumes a larger size in SDS-PAGE due to a higher degree of N-linked glycosylation and due to the lack of proteolytic processing. Immunoprecipitations and Western blotting of JAR laminins identified laminin alpha1 and laminin alpha5 chains in laminin-1 and laminin-10. In placenta laminin alpha1 chain (M(r) 400,000) and laminin alpha5 chain (M(r) 380, 000/370,000 doublet) were found in laminin-1/-3 and laminin-10/-11. Immunohistochemically we could establish that the laminin alpha1 chain in placenta is deposited in the developing villous and trophoblast basement membrane, also found to contain laminin beta2 chains. Surprisingly, a fraction of the laminin alpha1 chain from JAR cells and placenta could not be precipitated by antibodies to laminin beta1-beta3 chains, possibly pointing to an unexpected complexity in the chain composition of alpha1-containing laminin isoforms.

Laminin alpha1-chain shows a restricted distribution in epithelial basement membranes of fetal and adult human tissues.

Two novel monoclonal antibodies were raised and used to study the expression of laminin (Ln) alpha1-chain in developing and adult human tissues. In both fetal and adult kidney, a distinct immunoreactivity was seen in basement membranes (BM) of most proximal tubules but not in the distal tubular or glomerular BM or in the basal laminae of blood vessels. Immunoprecipitation of metabolically labeled cultured human renal proximal tubular cells showed an abundant production and deposition of Ln alpha1-chain to the extracellular matrix, suggestive of an epithelial origin of kidney Ln-1. Quantitative cell adhesion experiments with JAR choriocarcinoma cells showed that purified human Ln-1 is a good substrate for cell adhesion that it is differently recognized by integrin receptors when compared to mouse Ln-1. In fetal and adult testes immunoreactivity was solely confined to BM of the seminiferous epithelium. In the airways BM-confined reaction was only seen in fetal budding bronchial tubules (16-19 weeks) at the pseudoglandular stage of development. In the skin a distinct immunoreactivity was confined to BM of developing hair buds but not in epithelial BMs of adult epidermis or of epidermal appendages. In other adult tissues, immunoreactivity was found in BMs of thyroid, salivary, and mammary glands as well as in BMs of endometrium and endocervix, but not of ectocervix or vagina. No immunoreactivity was found in BMs of most of the digestive tract, including the liver and pancreas, except for BMs of esophageal submucosal glands and duodenal Brunner's glands. In fetal specimens, BMs of the bottoms of the intestinal and gastric glands were positive. Basal laminae of blood vessels were generally negative for Ln alpha1 chain with the exception of specimens of both fetal and adult central nervous system in which immunoreactivity for Ln alpha1 chain was prominently confined to capillary walls. The results suggest that outside the central nervous system, Ln alpha1 chain shows a restricted and developmentally regulated expression in BMs of distinct epithelial tissues.

Laminin chains in developing and adult human myotendinous junctions.

In addition to being the specialized site for transmission of force from the muscle to the tendon, the myotendinous junction (MTJ) also plays an important role in muscle splitting during morphogenesis. An early event in the formation of the MTJ is a regional deposition of basement membranes. We used immunocytochemistry to investigate the distribution of laminin chains during the development of MTJs in human limb muscle at 8-22 weeks of gestation (wg) and in adult MTJs. We used polyclonal antibodies and a new monoclonal antibody (MAb) against the human laminin alpha1 G4/G5 domains. At 8-10 wg, laminin alpha1 and laminin alpha5 chains were specifically localized to the MTJ. Laminin alpha1 chain remained restricted to the MTJ at 22 wg as the laminin beta2 chain had appeared, whereas the laminin alpha5 chain became deposited along the entire length of the myotubes from 12 wg. In the adult MTJ, only vestigial amounts of laminin alpha1 and laminin alpha5 chains could be detected. On the basis of co-distribution data, we speculate that laminin alpha1 chain in the forming MTJ undergoes an isoform switch from laminin 1 to laminin 3. Our data indicate a potentially important role for laminin alpha1 chain in skeletal muscle formation. (J Histochem Cytochem 48:201-209, 2000)

cDNA cloning and chromosomal localization of human alpha(11) integrin. A collagen-binding, I domain-containing, beta(1)-associated integrin alpha-chain present in muscle tissues.

We previously identified a novel integrin alpha-chain in human fetal muscle cells (Gullberg, D., Velling, T., Sjöberg, G., and Sejersen, T. (1995) Dev. Dyn. 204, 57-65). We have now isolated the full-length cDNA for this integrin subunit, alpha(11). The open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains seven conserved FG-GAP repeats, an I domain with a metal ion-dependent adhesion site motif, a short transmembrane region, and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS. alpha(11), like other I domain integrins, lacks a dibasic cleavage site for generation of a heavy chain and a light chain, and it contains three potential divalent cation binding sites in repeats 5-7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804-826) distinguishes the alpha(11) integrin sequence from other integrin alpha-chains. Amino acid sequence comparisons reveal the highest identity of 42% with the alpha(10) integrin chain. Immunoprecipitation with antibodies to alpha(11) integrin captures a 145-kDa protein distinctly larger than the 140-kDa alpha(2) integrin chain when analyzed by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. Fluorescence in situ hybridization maps the integrin alpha(11) gene to chromosome 15q23, in the vicinity of an identified locus for Bardet-Biedl syndrome. Based on Northern blotting, integrin alpha(11) mRNA levels are high in the adult human uterus and in the heart and intermediate in skeletal muscle and some other tissues tested. During in vitro myogenic differentiation, alpha(11) mRNA and protein are up-regulated. Studies of ligand binding properties show that alpha(11)beta(1) binds collagen type I-Sepharose, and cultured muscle cells localize alpha(11)beta(1) into focal contacts on collagen type I. Future studies will reveal the importance of alpha(11)beta(1) for muscle development and integrity in adult muscle and other tissues.

Laminin alpha chains in colon carcinoma cell lines: detection of a truncated laminin alpha1 mRNA in Caco-2 cells.

Changes in basement membrane structure are known to accompany carcinoma formation. We analyzed laminin alpha1 and alpha5 chains in colon carcinoma cell lines. Variable levels of the Mr 380,000 alpha5 laminin protein and 11-kb alpha5 laminin mRNA were noted. In contrast, laminin alpha1 protein was not synthesized by any of the colon carcinoma cell lines tested. Northern blotting revealed expression of a 10-kb laminin alpha1 mRNA only in control cells. Unexpectedly, expression of a truncated laminin alpha1 message of approximately 8 kb was found in one cell line, the adenocarcinoma cell line Caco-2. By RT-PCR and Northern blotting a deletion in the laminin alpha1 mRNA was mapped to the 5' end, spanning nucleotides 41-1835. The deletion spans the translation start site, explaining the complete lack of the protein. Southern blotting of genomic Caco-2 DNA did not reveal any larger truncation, suggesting a point mutation manifested at the posttranscriptional level. The identified truncation is the first genetic defect described for the laminin alpha1 chain and suggests that mutations in the LAM A1 gene might underlie the observed lack of the laminin alpha1 chain in some colon carcinomas.

Laminins during muscle development and in muscular dystrophies.

Cellular interactions with the extracellular matrix during muscle formation and in muscular dystrophy have received increased interest during the past years. Laminins constitute a growing family of proteins with complex expression patterns in forming basement membranes during muscle development. In skeletal muscle, laminins constitute major ligands for cell surface receptors involved in the transmission of force from the cell interior, but laminins might also influence signal transmission events during muscle formation and in muscle regeneration. During myogenesis the laminin alpha1 chain is present around the epithelial somite; but later, in forming muscle, the laminin alpha1 chain is restricted to the myotendinous junction. The laminin alpha2, alpha4 and alpha5 chains are major laminin chains in the muscle basement membrane during muscle formation, but laminin alpha4 and alpha5 chains are absent in adult muscle. The importance of laminins for muscle integrity is manifested in congenital muscular dystrophies with defects in the laminin alpha2 chain. There is no good evidence for the presence of laminin alpha1 chain in dystrophic muscle, but some other fetal muscle laminins can be detected in dystrophic muscle. Characterization of laminin expression patterns in muscular dystrophies might be of diagnostic and therapeutic value. In this paper, we review the recent publications on the biological functions of muscle laminins and discuss their roles in skeletal muscle.

Integrins during muscle development and in muscular dystrophies.

Cellular interactions with the extracellular matrix (ECM) have been shown to be important for a number of developmental events from the time of fertilization up till the maturation of the organism. In the following review we will discuss what is currently known about these interactions with special emphasis on the role of integrins during the formation of skeletal muscle. The importance of cell-ECM interactions will also be illustrated by a discussion of what happens when these interactions go awry, as happens in muscular dystrophies.

Human corneal epithelial basement membrane and integrin alterations in diabetes and diabetic retinopathy.

Corneas of diabetic patients have abnormal healing and epithelial adhesion, which may be due to alterations of the corneal extracellular matrix (ECM) and basement membrane (BM). To identify such alterations, various ECM and BM components and integrin receptors were studied by immunofluorescence on sections of normal and diabetic human corneas. Age-matched corneas from 15 normal subjects, 10 diabetics without diabetic retinopathy (DR), and 12 diabetics with DR were used. In DR corneas, the composition of the central epithelial BM was markedly altered, compared to normal or non-DR diabetic corneas. In most cases the staining for entactin/nidogen and for chains of laminin-1 (alpha1beta1gamma1) and laminin-10 (alpha5beta1gamma1 was very weak, discontinuous, or absent over large areas. Other BM components displayed less frequent changes. The staining for alpha3beta1 (VLA-3) laminin binding integrin was also weak and discontinuous in DR corneal epithelium. Components of stromal ECM remained unchanged even in DR corneas. Therefore, distinct changes were identified in the composition of the epithelial BM in DR corneas. They may be due to increased degradation or decreased synthesis of BM components and related integrins. These alterations may directly contribute to the epithelial adhesion and wound healing abnormalities found in diabetic corneas.