Absence of integrin alpha 7 causes a novel form of muscular dystrophy.

Integrin alpha 7 beta 1 is a specific cellular receptor for the basement membrane protein laminin-1 (refs 1,2), as well as for the laminin isoforms -2 and -4 (ref. 3). The alpha 7 subunit is expressed mainly in skeletal and cardiac muscle and has been suggested to be involved in differentiation and migration processes during myogenesis. Three cytoplasmic and two extracellular splice variants that have been described are developmentally regulated and expressed in different sites in the muscle. In adult muscle, the alpha 7A and alpha 7B subunits are concentrated in myotendinous junctions but can also be detected in neuromuscular junctions and along the sarcolemmal membrane. To study the potential involvement of alpha 7 integrin, during myogenesis and its role in muscle integrity and function, we generated a null allele of the alpha 7 gene (Itga7) in the germline of mice by homologous recombination in embryonic stem (ES) cells. Surprisingly, mice homozygous for the mutation are viable and fertile, indicating that the alpha 7 beta 1 integrin is not essential for myogenesis. However, histological analysis of skeletal muscle revealed typical symptoms of a progressive muscular dystrophy starting soon after birth, but with a distinct variability in different muscle types. The observed histopathological changes strongly indicate an impairment of function of the myotendinous junctions. These findings demonstrate that alpha 7 beta 1 integrin represents an indispensable linkage between the muscle fibre and the extracellular matrix that is independent of the dystrophin-dystroglycan complex-mediated interaction of the cytoskeleton with the muscle basement membrane.

Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

Genetic analyses of integrin function in mice.

Recent mutations of most integrin genes in the mouse have provided new exciting insights into the role of these integrins in cell-extracellular matrix interactions during development. The embryonic lethal phenotypes obtained by ablating integrins which are predominantly expressed in the mesenchyme confirmed the essential function of those integrins in morphogenesis. In contrast, null alleles for several epithelial integrins which bind components of basement membranes showed milder phenotypes, suggesting the presence of novel and unexpected redundant and compensatory mechanisms.

Plasma fibronectin supports neuronal survival and reduces brain injury following transient focal cerebral ischemia but is not essential for skin-wound healing and hemostasis.

Fibronectin performs essential roles in embryonic development and is prominently expressed during tissue repair. Two forms of fibronectin have been identified: plasma fibronectin (pFn), which is expressed by hepatocytes and secreted in soluble form into plasma; and cellular fibronectin (cFn), an insoluble form expressed locally by fibroblasts and other cell types and deposited and assembled into the extracellular matrix. To investigate the role of pFn in vivo, we generated pFn-deficient adult mice using Cre-loxP conditional gene-knockout technology. Here we show that pFn-deficient mice show increased neuronal apoptosis and larger infarction areas following transient focal cerebral ischemia. However, pFn is dispensable for skin-wound healing and hemostasis.

B lymphocytes producing demyelinating autoantibodies: development and function in gene-targeted transgenic mice.

We studied the cellular basis of self tolerance of B cells specific for brain autoantigens using transgenic mice engineered to produce high titers of autoantibodies against the myelin oligodendrocyte glycoprotein (MOG), a surface component of central nervous system myelin. We generated "knock-in" mice by replacing the germline JH locus with the rearranged immunoglobulin (Ig) H chain variable (V) gene of a pathogenic MOG-specific monoclonal antibody. In the transgenic mice, conventional B cells reach normal numbers in bone marrow and periphery and express exclusively transgenic H chains, resulting in high titers of MOG-specific serum Igs. Additionally, about one third of transgenic B cells bind MOG, thus demonstrating the absence of active tolerization. Furthermore, peritoneal B-1 lymphocytes are strongly depleted. Upon immunization with MOG, the mature transgenic B cell population undergoes normal differentiation to plasma cells secreting MOG-specific IgG antibodies, during which both Ig isotype switching and somatic mutation occur. In naive transgenic mice, the presence of this substantial autoreactive B cell population is benign, and the mice fail to develop either spontaneous neurological disease or pathological evidence of demyelination. However, the presence of the transgene both accelerates and exacerbates experimental autoimmune encephalitis, irrespective of the identity of the initial autoimmune insult.

Increased adhesion and aggregation of platelets lacking cyclic guanosine 3',5'-monophosphate kinase I.

Atherosclerotic vascular lesions are considered to be a major cause of ischemic diseases, including myocardial infarction and stroke. Platelet adhesion and aggregation during ischemia-reperfusion are thought to be the initial steps leading to remodeling and reocclusion of the postischemic vasculature. Nitric oxide (NO) inhibits platelet aggregation and smooth muscle proliferation. A major downstream target of NO is cyclic guanosine 3', 5'-monophosphate kinase I (cGKI). To test the intravascular significance of the NO/cGKI signaling pathway in vivo, we have studied platelet-endothelial cell and platelet-platelet interactions during ischemia/reperfusion using cGKI-deficient (cGKI-/-) mice. Platelet cGKI but not endothelial or smooth muscle cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. The defect in platelet cGKI is not compensated by the cAMP/cAMP kinase pathway supporting the essential role of cGKI in prevention of ischemia-induced platelet adhesion and aggregation.

Modulation of beta1A integrin functions by tyrosine residues in the beta1 cytoplasmic domain.

beta1A integrin subunits with point mutations of the cytoplasmic domain were expressed in fibroblasts derived from beta1-null stem cells. beta1A in which one or both of the tyrosines of the two NPXY motifs (Y783, Y795) were changed to phenylalanines formed active alpha5 beta1 and alpha6 beta1 integrins that mediated cell adhesion and supported assembly of fibronectin. Mutation of the proline in either motif (P781, P793) to an alanine or of a threonine in the inter-motif sequence (T788) to a proline resulted in poorly expressed, inactive beta1A. Y783,795F cells developed numerous fine focal contacts and exhibited motility on a surface. When compared with cells expressing wild-type beta1A or beta1A with the D759A activating mutation of a conserved membrane-proximal aspartate, Y783, 795F cells had impaired ability to transverse filters in chemotaxis assays. Analysis of cells expressing beta1A with single Tyr to Phe substitutions indicated that both Y783 and Y795 are important for directed migration. Actin-containing microfilaments of Y783,795F cells were shorter and more peripheral than microfilaments of cells expressing wild-type beta1A. These results indicate that change of the phenol side chains in the NPXY motifs to phenyl groups (which cannot be phosphorylated) has major effects on the organization of focal contacts and cytoskeleton and on directed cell motility.

Beta 1 integrin-dependent and -independent polymerization of fibronectin.

The mouse cell line GD25, which lacks expression of the beta 1 family of integrin heterodimers due to disruption of the beta 1 integrin subunit gene, was used for expression of full-length cDNA coding for splice variant A of the mouse beta 1 integrin subunit. In a stably transformed clone (GD25-beta 1A), the expressed protein was found to form functional heterodimeric receptors together with the subunits alpha 3, alpha 5, and alpha 6. Both GD25 and GD25-beta 1A attached to fibronectin and formed focal contacts which contained alpha v beta 3, but no detectable alpha 5 beta 1A. The presence of GRGDS peptide allowed alpha 5 beta 1A to locate to focal contacts of GD25-beta 1A cultured on fibronectin, while the beta 1-null GD25 cells were unable to attach under these conditions. Affinity chromatography revealed that alpha 5 beta 1A and alpha v beta 3 could bind to a large cell-binding fragment of fibronectin. alpha 5 beta 1A strongly promoted polymerization of fibronectin into a fibrillar network on top of the cells. Whereas little alpha v beta 3 was colocalized with the fibronectin fibrils in GD25-beta 1A cells, this integrin was able to support fibronectin fibril polymerization in GD25 cells. However, the alpha v beta 3-induced polymerization was less efficient and occurred mainly in dense cultures of the GD25 cells. Thus, while both alpha 5 beta 1A and alpha v beta 3 are able to support adhesion to fibronectin, alpha v beta 3 dominates in the formation of focal contacts, and alpha 5 beta 1A has a prime function in fibronectin matrix assembly. This is the first report on fibronectin matrix assembly in the absence of beta 1 integrins.

Downregulation of tenascin expression by glucocorticoids in bone marrow stromal cells and in fibroblasts.

Tenascin, a predominantly mesenchymal extracellular matrix (ECM) glycoprotein has a rather restricted tissue distribution, but until now factors that inhibit its expression have not been identified. Glucocorticoids are known to be beneficial for establishment of myelopoiesis in long-term bone marrow cultures. Tenascin was found to be expressed in the bone marrow, and glucocorticoids were found to affect bone marrow tenascin expression. Both tenascin mRNAs and the mRNA of another ECM protein, laminin B1 chain, were drastically downregulated by glucocorticoids during initiation of bone marrow cultures. However, in already established long-term cultures glucocorticoids did not affect laminin B1 chain mRNA levels although tenascin mRNAs continued to be downregulated. Studies with a stromal cell line (MC3T3-G2/PA6) and fibroblasts (3T3) suggested that glucocorticoids act directly on the stromal cells that produce tenascin. In 3T3 cells this downregulation occurred within 12 h of glucocorticoid-treatment, suggesting that glucocorticoids acted through cis regulatory elements of the tenascin gene. We suggest that glucocorticoids in part regulate hematopoiesis by modifying the ECM. Furthermore, downregulation of tenascin expression by glucocorticoids may in part explain the restricted tissue distribution of tenascin in other tissues.

Collagen II is essential for the removal of the notochord and the formation of intervertebral discs.

Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II-deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice. Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that alpha1(XI) and alpha2 (XI) chains form unstable collagen XI molecules, demonstrating that the alpha3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.

Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding.

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115-residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.

Lack of beta 1 integrin gene in embryonic stem cells affects morphology, adhesion, and migration but not integration into the inner cell mass of blastocysts.

A gene trap-type targeting vector was designed to inactivate the beta 1 integrin gene in embryonic stem (ES) cells. Using this vector more than 50% of the ES cell clones acquired a disruption in the beta 1 integrin gene and a single clone was mutated in both alleles. The homozygous mutant did not produce beta 1 integrin mRNA or protein, while alpha 3, alpha 5, and alpha 6 integrin subunits were transcribed but not detectable on the cell surface. Heterozygous mutants showed reduced beta 1 expression and surface localization of alpha/beta 1 heterodimers. The alpha V subunit expression was not impaired on any of the mutants. Homozygous ES cell mutants lacked adhesiveness for laminin and fibronectin but not for vitronectin and showed a reduced association with a fibroblast feeder layer. Furthermore, they did not migrate towards chemoattractants in fibroblast medium. None of these functions were impaired in heterozygous mutants. Scanning electron microscopy revealed that homozygous cells showed fewer cell-cell junctions and had many microvilli not usually found on wild type and heterozygous cells. This profound change in cell shape is not associated with gross alterations in the expression and distribution of cytoskeletal components. Unexpectedly, microinjection into blastocysts demonstrated full integration of homozygous and heterozygous mutants into the inner cell mass. This will allow studies of the consequences of beta 1 integrin deficiency in several in vivo situations.

Beta 1 integrin is essential for teratoma growth and angiogenesis.

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of beta 1 integrin during teratoma formation, we compared teratomas induced by normal and beta1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, beta 1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of beta 1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in beta1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface. Normal teratomas had large blood vessels with a smooth inner surface, containing both host- and ES cell-derived endothelial cells. In contrast, beta 1-null teratomas had small vessels that were loosely embedded into the connective tissue. Furthermore, endothelial cells were always of host-derived origin and formed blood vessels with an irregular inner surface. Although beta 1- deficient endothelial cells were absent in teratomas, beta 1-null ES cells could differentiate in vitro into endothelial cells. The formation of a complex vasculature, however, was significantly delayed and of poor quality in beta1-null embryoid bodies. Moreover, while vascular endothelial growth factor induced proliferation of endothelial cells as well as an extensive branching of blood vessels in normal embryoid bodies, it had no effect in beta 1-null embryoid bodies.

Induction of cell scattering by expression of beta1 integrins in beta1-deficient epithelial cells requires activation of members of the rho family of GTPases and downregulation of cadherin and catenin function.

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and beta1 integrins influence each other using two different beta1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of beta1A or the cytoplasmic splice variant beta1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of beta1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of beta1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-beta1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM-cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length beta1A. This indicates that the disruption of cell-cell adhesion is not simply the consequence of the stimulated cell migration. Expression of beta1 integrins in GE11 cells resulted in a decrease in cadherin and alpha-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of alpha-catenin protein levels by beta1 integrins is likely to play a role in the morphological transition, since overexpression of alpha-catenin in GE11 cells before beta1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of beta1A, beta1D, or IL2R-beta1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell-cell adhesions when expressed before beta1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-beta1A cells. Our results indicate that beta1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of alpha-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

Perlecan maintains the integrity of cartilage and some basement membranes.

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading.

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.

Mouse ten-m/Odz is a new family of dimeric type II transmembrane proteins expressed in many tissues.

The Drosophila gene ten-m/odz is the only pair rule gene identified to date which is not a transcription factor. In an attempt to analyze the structure and the function of ten-m/odz in mouse, we isolated four murine ten-m cDNAs which code for proteins of 2,700-2, 800 amino acids. All four proteins (Ten-m1-4) lack signal peptides at the NH2 terminus, but contain a short hydrophobic domain characteristic of transmembrane proteins, 300-400 amino acids after the NH2 terminus. About 200 amino acids COOH-terminal to this hydrophobic region are eight consecutive EGF-like domains. Cell transfection, biochemical, and electronmicroscopic studies suggest that Ten-m1 is a dimeric type II transmembrane protein. Expression of fusion proteins composed of the NH2-terminal and hydrophobic domain of ten-m1 attached to the alkaline phosphatase reporter gene resulted in membrane-associated staining of the alkaline phosphatase. Electronmicroscopic and electrophoretic analysis of a secreted form of the extracellular domain of Ten-m1 showed that Ten-m1 is a disulfide-linked dimer and that the dimerization is mediated by EGF-like modules 2 and 5 which contain an odd number of cysteines. Northern blot and immunohistochemical analyses revealed widespread expression of mouse ten-m genes, with most prominent expression in brain. All four ten-m genes can be expressed in variously spliced mRNA isoforms. The extracellular domain of Ten-m1 fused to an alkaline phosphatase reporter bound to specific regions in many tissues which were partially overlapping with the Ten-m1 immunostaining. Far Western assays and electronmicroscopy demonstrated that Ten-m1 can bind to itself.

Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins.

beta1 integrins play a crucial role as cytoskeletal anchorage proteins. In this study, the coupling of the cytoskeleton and intracellular signaling pathways was investigated in beta1 integrin deficient (-/-) embryonic stem cells. Muscarinic inhibition of the L-type Ca2+ current (ICa) and activation of the acetylcholine-activated K+ current (IK,ACh) was found to be absent in beta1 integrin-/- cardiomyocytes. Conversely, beta adrenoceptor-mediated modulation of ICa was unaffected by the absence of beta1 integrins. This defect in muscarinic signaling was due to defective G protein coupling. This was supported by deconvolution microscopy, which demonstrated that Gi exhibited an atypical subcellular distribution in the beta1 integrin-/- cardiomyocytes. A critical role of the cytoskeleton was further demonstrated using cytochalasin D, which displaced Gi and impaired muscarinic signaling. We conclude that cytoskeletal integrity is required for correct localization and function of Gi-associated signaling microdomains.

Muscle beta1D integrin reinforces the cytoskeleton-matrix link: modulation of integrin adhesive function by alternative splicing.

Expression of muscle-specific beta1D integrin with an alternatively spliced cytoplasmic domain in CHO and GD25, beta1 integrin-minus cells leads to their phenotypic conversion. beta1D-transfected nonmuscle cells display rounded morphology, lack of pseudopodial activity, retarded spreading, reduced migration, and significantly enhanced contractility compared with their beta1A-expressing counterparts. The transfected beta1D is targeted to focal adhesions and efficiently displaces the endogenous beta1A and alphavbeta3 integrins from the sites of cell-matrix contact. This displacement is observed on several types of extracellular matrix substrata and leads to elevated stability of focal adhesions in beta1D transfectants. Whereas a significant part of cellular beta1A integrin is extractable in digitonin, the majority of the transfected beta1D is digitonin-insoluble and is strongly associated with the detergent-insoluble cytoskeleton. Increased interaction of beta1D integrin with the actin cytoskeleton is consistent with and might be mediated by its enhanced binding to talin. In contrast, beta1A interacts more strongly with alpha-actinin, than beta1D. Inside-out driven activation of the beta1D ectodomain increases ligand binding and fibronectin matrix assembly by beta1D transfectants. Phenotypic effects of beta1D integrin expression in nonmuscle cells are due to its enhanced interactions with both cytoskeletal and extracellular ligands. They parallel the transitions that muscle cells undergo during differentiation. Modulation of beta1 integrin adhesive function by alternative splicing serves as a physiological mechanism reinforcing the cytoskeleton- matrix link in muscle cells. This reflects the major role for beta1D integrin in muscle, where extremely stable association is required for contraction.

Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation.

The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.