Loss of alpha6 integrins in keratinocytes leads to an increase in TGFbeta and AP1 signaling and in expression of differentiation genes

Mice lacking the alpha6 integrin chain die at birth with severe skin blistering. To further study the function of alpha6 integrin in skin, we generated conditionally immortalized cell lines from the epidermis of wild-type and alpha6 deficient mouse embryos. Mutant cells presented a decreased adhesion on laminin 5, the major component of the basement membrane in the skin, and on laminins 10/11 and 2. A DNA array analysis revealed alterations in the expression of extracellular matrix (ECM) components including laminin 5, cytoskeletal elements, but also membrane receptors like the hemidesmosomal components integrin beta4 and collagen XVII, or growth factors and signaling molecules of the TGFbeta, EGF, and Wnt pathways. Finally, an increase of several epidermal differentiation markers was observed in cells and tissue at the protein level. Further examination of the mutant tissue revealed alterations in the filaggrin signal. These differences may be linked to an upregulation of the TGFbeta and the Jun/Fos pathways in mutant keratinocytes. These results are in favor of a role for integrin alpha6beta4 in the maintenance of basal keratinocyte properties and epidermal homeostasis.     

Laminin alpha5 is necessary for submandibular gland epithelial morphogenesis and influences FGFR expression through beta1 integrin signaling

Laminin alpha chains have unique spatiotemporal expression patterns during development and defining their function is necessary to understand the regulation of epithelial morphogenesis. We investigated the function of laminin alpha5 in mouse submandibular glands (SMGs). Lama5(-/-) SMGs have a striking phenotype: epithelial clefting is delayed, although proliferation occurs; there is decreased FGFR1b and FGFR2b, but no difference in Lama1 expression; later in development, epithelial cell organization and lumen formation are disrupted. In wild-type SMGs alpha5 and alpha1 are present in epithelial clefts but as branching begins alpha5 expression increases while alpha1 decreases. Lama5 siRNA decreased branching, p42 MAPK phosphorylation, and FGFR expression, and branching was rescued by FGF10. FGFR siRNA decreased Lama5 suggesting that FGFR signaling provides positive feedback for Lama5 expression. Anti-beta1 integrin antibodies decreased FGFR and Lama5 expression, suggesting that beta1 integrin signaling provides positive feedback for Lama5 and FGFR expression. Interestingly, the Itga3(-/-):Itga6(-/-) SMGs have a similar phenotype to Lama5(-/-). Our findings suggest that laminin alpha5 controls SMG epithelial morphogenesis through beta1 integrin signaling by regulating FGFR expression, which also reciprocally regulates the expression of Lama5. These data link changes in basement membrane composition during branching morphogenesis with FGFR expression and signaling.     

Implications of protease activation in cardiac dysfunction and development of genetic cardiomyopathy in hamsters

It has become evident that protein degradation by proteolytic enzymes, known as proteases, is partly responsible for cardiovascular dysfunction in various types of heart disease. Both extracellular and intracellular alterations in proteolytic activities are invariably seen in heart failure associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, hypertensive cardiomyopathy, diabetic cardiomyopathy, and ischemic cardiomyopathy. Genetic cardiomyopathy displayed in different strains of hamsters provides a useful model for studying heart failure due to either cardiac hypertrophy or cardiac dilation. Alterations in the function of several myocardial organelles such as sarcolemma, sarcoplasmic reticulum, myofibrils, mitochondria, as well as extracellular matrix have been shown to be due to subcellular remodeling as a consequence of changes in gene expression and protein content in failing hearts from cardiomyopathic hamsters. In view of the increased activities of various proteases, including calpains and matrix metalloproteinases in the hearts of genetically determined hamsters, it is proposed that the activation of different proteases may also represent an important determinant of subcellular remodeling and cardiac dysfunction associated with genetic cardiomyopathy.     

Cellular localization of integrin isoforms in phenylephrine-induced hypertrophic cardiac myocytes

Cardiac hypertrophy is characterized by remodeling of the extracellular matrix (ECM). Integrins are cell-surface molecules that link the ECM to the cellular cytoskeleton where they play roles as signaling molecules and transducers of mechanical force. To clarify the possible roles of integrins in cardiac myocyte hypertrophy, we investigated the cellular localization and expression of ECM proteins and integrins in both normal cardiac myocytes and phenylephrine-induced hypertrophic myocytes. Addition of phenylephrine (PE) to cultured neonatal cardiac myocytes induced sarcomeric organization, increase in cell size, and synthesis of the hypertrophic marker, atrial natriuretic factor (ANF). In particular, fibronectin and collagen underwent dramatic localization changes during PE-induced cardiac hypertrophy. Significant changes were noted in the cellular localization of the respective collagen and fibronectin receptors, integrin alpha1 and alpha5, from diffuse to a sarcomeric banding pattern. Expression levels of integrins were also increased during hypertrophy. Treatment with okadaic acid (OA), an inhibitor of protein phosphatase 2A (PP2A), resulted in inhibition of hypertrophic response. These results suggest that dephosphorylation of integrin beta1 may be important in the induction of cardiac hypertrophy.     

Reduced cGMP signaling activates NF-kappaB in hypertrophied hearts of mice lacking natriuretic peptide receptor-A

Mice lacking natriuretic peptide receptor-A (NPRA) develop progressive cardiac hypertrophy and congestive heart failure. However, the mechanisms responsible for cardiac hypertrophic growth in the absence of NPRA signaling are not yet known. We sought to determine the activation of nuclear factor-kappaB (NF-kappaB) in Npr1 (coding for NPRA) gene-knockout (Npr1-/-) mice exhibiting cardiac hypertrophy and fibrosis. NF-kappaB binding activity was 4-fold greater in the nuclear extract of Npr1-/- mutant mice hearts as compared with wild-type (Npr1+/+) mice hearts. In parallel, inhibitory kappaB kinase-beta activity and IkappaB-alpha protein phosphorylation were also increased 3- and 4-fold, respectively, in hypertrophied hearts of mutant mice. cGMP levels were significantly reduced 5-fold in plasma and 10-fold in ventricular tissues of mutant mice hearts relative to wild-type controls. The present findings provide direct evidence that ablation of NPRA/cGMP signaling activates NF-kappaB binding activity associated with hypertrophic growth of mutant mice hearts.     

Laminin alpha5 is required for dental epithelium growth and polarity and the development of tooth bud and shape

In tooth development, the oral ectoderm and mesenchyme coordinately and reciprocally interact through the basement membrane for their growth and differentiation to form the proper shape and size of the tooth. Laminin alpha5 subunit-containing laminin-10/11 (LM-511/521) is the major laminin in the tooth germ basement membrane. Here, we have examined the role of laminin alpha5 (Lama5) in tooth development using laminin alpha5-null mouse primary dental epithelium and tooth germ organ cultures. Lama5-null mice develop a small tooth germ with defective cusp formation and have reduced proliferation of dental epithelium. Also, cell polarity and formation of the monolayer of the inner dental epithelium are disturbed. The enamel knot, a signaling center for tooth germ development, is defective, and there is a significant reduction of Shh and Fgf4 expression in the dental epithelium. In the absence of laminin alpha5, the basement membrane in the inner dental epithelium becomes discontinuous. In normal mice, integrin alpha6beta4, a receptor for laminin alpha5, is strongly localized at the basal layer of the epithelium, whereas in mutant mice, integrin alpha6beta4 is expressed around the cell surface. In primary dental epithelium culture, laminin-10/11 promotes cell growth, spreading, and filopodia-like microspike formation. This promotion is inhibited by anti-integrin alpha6 and beta4 antibodies and by phosphatidylinositol 3-kinase inhibitors and dominant negative Rho-GTPase family proteins Cdc42 and Rac. In organ culture, anti-integrin alpha6 antibody and wortmannin reduce tooth germ size and shape. Our studies demonstrate that laminin alpha5 is required for the proliferation and polarity of basal epithelial cells and suggest that the interaction between laminin-10/11-integrin alpha6beta4 and the phosphatidylinositol 3-kinase-Cdc42/Rac pathways play an important role in determining the size and shape of tooth germ.     

Differential Expression of Dicer, miRNAs, and Inflammatory Markers in Diabetic Ins2+/− Akita Hearts

Diabetic cardiomyopathy is a leading cause of morbidity and mortality, and Insulin2 mutant (Ins2+/?) Akita is a genetic mice model of diabetes relevant to humans. Dicer, miRNAs, and inflammatory cytokines are associated with heart failure. However, the differential expression of miRNAs, dicer, and inflammatory molecules are not clear in diabetic cardiomyopathy of Akita. We measured the levels of miRNAs, dicer, pro-inflammatory tumor necrosis factor alpha (TNFα), and anti-inflammatory interleukin 10 (IL-10) in C57BL/6J (WT) and Akita hearts. The results revealed increased heart to body weight ratio and robust expression of brain natriuretic peptide (BNP: a hypertrophy marker) suggesting cardiac hypertrophy in Akita. The multiplex RT-PCR, qPCR, and immunoblotting showed up regulation of dicer, whereas miRNA array elicited spread down regulation of miRNAs in Akita including dramatic down regulation of let-7a, miR-130, miR-142-3p, miR-148, miR-338, miR-345-3p, miR-384-3p, miR-433, miR-450, miR-451, miR-455, miR-494, miR-499, miR-500, miR-542-3p, miR-744, and miR-872. Conversely, miR-295 is induced in Akita. Cardiac TNFα is upregulated at mRNA (RT-PCR and qPCR), protein (immunoblotting), and cellular (immunohistochemistry and confocal microscopy) levels, and is robust in hypertrophic cardiomyocytes suggesting direct association of TNFα with hypertrophy. Contrary to TNFα, cardiac IL-10 is downregulated in Akita. In conclusion, induction of dicer and TNFα, and attenuation of IL-10 and majority of miRNA are associated with cardiomyopathy in Akita and could be used for putative therapeutic target for heart failure in diabetics.     

Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the glomerular basement membrane

In developing glomeruli, laminin alpha5 replaces laminin alpha1 in the glomerular basement membrane (GBM) at the capillary loop stage, a transition required for glomerulogenesis. To investigate domain-specific functions of laminin alpha5 during glomerulogenesis, we produced transgenic mice that express a chimeric laminin composed of laminin alpha5 domains VI through I fused to the human laminin alpha1 globular (G) domain, designated Mr51. Transgene-derived protein accumulated in many basement membranes, including the developing GBM. When bred onto the Lama5 -/- background, Mr51 supported GBM formation, preventing the breakdown that normally occurs in Lama5 -/- glomeruli. In addition, podocytes exhibited their typical arrangement in a single cell layer epithelium adjacent to the GBM, but convolution of glomerular capillaries did not occur. Instead, capillaries were distended and exhibited a ballooned appearance, a phenotype similar to that observed in the total absence of mesangial cells. However, here the phenotype could be attributed to the lack of mesangial cell adhesion to the GBM, suggesting that the G domain of laminin alpha5 is essential for this adhesion. Analysis of an additional chimeric transgene allowed us to narrow the region of the alpha5 G domain essential for mesangial cell adhesion to alpha5LG3-5. Finally, in vitro studies showed that integrin alpha3beta1 and the Lutheran glycoprotein mediate adhesion of mesangial cells to laminin alpha5. Our results elucidate a mechanism whereby mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the GBM.     

wing blister, a new Drosophila laminin alpha chain required for cell adhesion and migration during embryonic and imaginal development

We report the molecular and functional characterization of a new alpha chain of laminin in Drosophila. The new laminin chain appears to be the Drosophila counterpart of both vertebrate alpha2 (also called merosin) and alpha1 chains, with a slightly higher degree of homology to alpha2, suggesting that this chain is an ancestral version of both alpha1 and alpha2 chains. During embryogenesis, the protein is associated with basement membranes of the digestive system and muscle attachment sites, and during larval stage it is found in a specific pattern in wing and eye discs. The gene is assigned to a locus called wing blister (wb), which is essential for embryonic viability. Embryonic phenotypes include twisted germbands and fewer pericardial cells, resulting in gaps in the presumptive heart and tracheal trunks, and myotubes detached from their target muscle attachment sites. Most phenotypes are in common with those observed in Drosophila laminin alpha3, 5 mutant embryos and many are in common with those observed in integrin mutations. Adult phenotypes show blisters in the wings in viable allelic combinations, similar to phenotypes observed in integrin genes. Mutation analysis in the eye demonstrates a function in rhabdomere organization. In summary, this new laminin alpha chain is essential for embryonic viability and is involved in processes requiring cell migration and cell adhesion.     

Human bronchial epithelial cells secrete laminin 5, express hemidesmosomal proteins, and assemble hemidesmosomes.

Epithelial cells attach to the basement membrane through adhesive contacts between the basal cells of the epithelium and the proteins of the extracellular matrix (ECM). The hemidesmosome (HD) is a specialized cell-ECM contact, that mediates the attachment of the epithelial cell basal surface to the ECM. In bronchial epithelial cells, the protein components that constitute the HD have not been demonstrated. Using immunohistochemical techniques, we determined that normal human bronchial epithelial (NHBE) cells express the HD cell surface integrin alpha6beta4 and produce laminin 5, the ECM protein associated with HDs. Furthermore, expression of the HD-associated structural proteins, bullous pemphigoid antigens 1 (BPAG 1) and 2 (BPAG 2), was demonstrated in NHBE cells by immunofluorescence microscopy and immunoblot analyses. In addition, we confirmed the presence of laminin 5 in the basement membrane (BM) of bronchial epithelial biopsy specimens and of BP230, BP180, and the alpha6beta4 integrin heterodimer at the site of bronchial epithelial cell-ECM interaction in vivo. Finally, using electron microscopy, we were able to demonstrate intact HDs in a glutaraldehyde-fixed NHBE cell monolayer. These findings suggest that bronchial epithelium forms HDs and that the laminin 5-alpha6beta4 integrin interaction may be important in stabilizing epithelial cell adhesion to the BM in the lung.     

Defective glomerulogenesis in the absence of laminin alpha5 demonstrates a developmental role for the kidney glomerular basement membrane

Laminins are major components of all basement membranes. They are a diverse group of alpha/beta/gamma heterotrimers formed from five alpha, three beta, and three gamma chains. Laminin alpha5 is a widely expressed chain found in many embryonic and adult basement membranes. During embryogenesis, alpha5 has a role in disparate developmental processes, including neural tube closure, digit septation, and placentation. Here, we analyzed kidney development in Lama5 mutant embryos and found a striking defect in glomerulogenesis associated with an abnormal glomerular basement membrane (GBM). This correlates with failure of the developmental switch in laminin alpha chain deposition in which alpha5 replaces alpha1 in the GBM at the capillary loop stage of glomerulogenesis. In the absence of a normal GBM, glomerular epithelial cells were in disarray, and endothelial and mesangial cells were extruded from within the constricting glomerulus, leading to a complete absence of vascularized glomeruli. In addition, a minority of Lama5 mutant mice lacked one or both kidneys, indicating that laminin alpha5 is also important in earlier kidney development. Our results demonstrate a dual role for laminin alpha5 in kidney development, illustrate a novel defect in glomerulogenesis, and indicate a heretofore unappreciated developmental role for the GBM in influencing the behavior of epithelial and endothelial cells.     

Laminin alpha1 chain LG4 module promotes cell attachment through syndecans and cell spreading through integrin alpha2beta1

The laminin alpha1 chain is a subunit of laminin-1, a heterotrimeric basement membrane protein. The LG4-5 module at the C terminus of laminin alpha1 contains major binding sites for heparin, sulfatide, and alpha-dystroglycan and plays a critical role in early embryonic development. We previously identified active synthetic peptides AG73 and EF-1 from the sequence of laminin alpha1 LG4 for binding to syndecan and integrin alpha2beta1, respectively. However, their activity and functional relationship within the laminin-1 and LG4 as well as the functional relation between these sites and alpha-dystroglycan binding sites in LG4 are not clear. To address these questions, we created mutant recombinant LG4 proteins containing alanine substitutions within the AG73 (M1), EF-1 (M2, M3), and alpha-dystroglycan binding sites (M4, M5) and analyzed their activities. We found that recombinant proteins rec-M1 and rec-M5, containing mutations within M1 and M5, respectively, did not bind heparin or lymphoid cell lines expressing syndecans. These results suggest that LG4 binds to heparin and syndecans through M1 and M5. Rec-M1 and rec-M5 reduced fibroblast attachment, whereas mutant rec-M2 and rec-M3 retained cell attachment activity but did not promote cell spreading. Fibroblast attachment to rec-LG4 was inhibited by heparin but not by integrin antibodies. Spreading of fibroblasts on rec-LG4 was inhibited by anti-integrin alpha2 and beta1 but not by anti-integrin alpha1 and alpha6. These results suggest that the M1 and M5 sites are necessary for cell attachment on LG4 through syndecans and that the EF-1 site is for cell spreading activity through integrin alpha2beta1. In contrast, laminin-1-mediated fibroblast attachment and spreading were not inhibited by heparin or anti-integrin alpha2. Our findings indicate that LG4 has a unique function distinct from laminin-1 and suggest that laminin alpha1 LG4-5 may also be produced by a proteolytic cleavage in certain tissues where it exerts its activity.     

Cardiac overexpression of myotrophin triggers myocardial hypertrophy and heart failure in transgenic mice

Cardiac hypertrophy and heart failure remain leading causes of death in the United States. Many studies have suggested that, under stress, myocardium releases factors triggering protein synthesis and stimulating myocyte growth. We identified and cloned myotrophin, a 12-kDa protein from hypertrophied human and rat hearts. Myotrophin (whose gene is localized on human chromosome 7q33) stimulates myocyte growth and participates in cellular interaction that initiates cardiac hypertrophy in vitro. In this report, we present data on the pathophysiological significance of myotrophin in vivo, showing the effects of overexpression of cardio-specific myotrophin in transgenic mice in which cardiac hypertrophy occurred by 4 weeks of age and progressed to heart failure by 9-12 months. This hypertrophy was associated with increased expression of proto-oncogenes, hypertrophy marker genes, growth factors, and cytokines, with symptoms that mimicked those of human cardiomyopathy, functionally and morphologically. This model provided a unique opportunity to analyze gene clusters that are differentially up-regulated during initiation of hypertrophy versus transition of hypertrophy to heart failure. Importantly, changes in gene expression observed during initiation of hypertrophy were significantly different from those seen during its transition to heart failure. Our data show that overexpression of myotrophin results in initiation of cardiac hypertrophy that progresses to heart failure, similar to changes in human heart failure. Knowledge of the changes that take place as a result of overexpression of myotrophin at both the cellular and molecular levels will suggest novel strategies for treatment to prevent hypertrophy and its progression to heart failure.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5(-/-) mice up to E16.5 was examined. We observed normal lung branching morphogenesis and vasculogenesis, but incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

Generation of a conditionally null allele of the laminin alpha1 gene

Laminins are heterotrimeric glycoproteins of the basement membranes. Laminin 1 (alpha1, beta1, gamma1) is the major laminin expressed during early mouse embryogenesis. To gain access to the physiological function of laminin alpha1 chain, we developed a conditionally null allele of its encoding gene (Lama1) using the cre/loxP system. Floxed-allele-carrying mice (Lama1(flox/flox)) display no overt phenotype. Lama1(flox/flox) mice were crossed with transgenic deleter mice (CMV-Cre) to generate Lama1-deficient mice (Lama1(Delta/Delta)). Lama1(Delta/Delta) embryos die during the early postimplantation period after embryonic day 6.5. They lack Reichert's membrane, an extraembryonic basement membrane in which laminin alpha1 is normally highly expressed. In parallel, Lama1(Delta/Delta) embryos display 1) parietal and visceral endoderm differentiation defects with altered expression of cytokeratin 19 and GATA4, respectively, and 2) an induction of apoptosis. This new mouse model is of particular interest as it will allow time- and tissue-specific inactivation of the Lama1 gene in various organs.