Integrin function and regulation in development.

Integrins are a large family of membrane receptors, consisting of alpha and beta subunits, that play a pivotal role in the interaction of cells with the extracellular matrix. Such interaction regulates the organization of cells in organs and tissues during development as well as cell differentiation and proliferation. We have shown that unfertilized oocytes express integrins that might be important during fertilization. We also analyzed nervous system and muscle tissue development showing that integrin expression is precisely regulated during organization of these tissues. The results indicate that two distinct integrin alpha subunits mediate the outgrowth of processes in nerve and glial cells. Alpha1 integrin, a laminin receptor, is up-regulated by nerve growth factor and other differentiation stimuli and is involved in neurite extension by nerve cells. In contrast, process extension by glial cells is likely to involve the alphaV integrin. Moreover, the latter integrin subunit is also transiently expressed in muscle of the embryo body where it localizes predominantly at developing myotendinous junctions. After birth this integrin disappears and is substituted by the alpha7 subunit. At the same time, important changes also occur in the expression of the associated beta subunit. In fact, the beta1A isoform which is expressed in fetal muscles, is substituted by beta1D. These isoforms are generated by alternative splicing and differ in only a few amino acid residues at the COOH terminus of the protein. This region of the molecule is exposed at the cytoplasmic face of the plasma membrane and is connected to the actin filaments. Our results show that beta1D, which is expressed only in striated muscle tissues, binds to both cytoskeletal and extracellular matrix proteins with an affinity higher than beta1A. Thus, beta1D provides a stronger link between the cytoskeleton and extracellular matrix necessary to support mechanical tension during muscle contraction. These results indicate that cells can regulate their interactions with the extracellular matrix by changing their expression of alpha integrin subunits and thus ligand specificity, or by more subtle changes involving alternative usage of different cytoplasmic domains. The important role of both alpha and beta integrin subunit cytoplasmic domains during development is further illustrated by the analysis of targeted mutations which we have generated by homologous recombination in mice.

The muscle-specific laminin receptor alpha7 beta1 integrin negatively regulates alpha5 beta1 fibronectin receptor function.

alpha7 beta1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the alpha7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with alpha7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the alpha7 beta1. alpha7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of alpha7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the alpha5 beta1 fibronectin receptor. Although cell surface expression of alpha5 beta1 was reduced by a factor of 20-25% in alpha7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of 125I-fibronectin for its surface receptor was decreased by 50% in alpha7 transfectants, indicating that the alpha5 beta1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn2+, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn2+ restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in alpha7 transfectants. These data indicate that alpha7 expression leads to the functional down regulation of alpha5beta1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of a negative cooperativity between alpha7 and alpha5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.

beta1-integrin cytoplasmic subdomains involved in dominant negative function.

The beta1-integrin cytoplasmic domain consists of a membrane proximal subdomain common to the four known isoforms ("common" region) and a distal subdomain specific for each isoform ("variable" region). To investigate in detail the role of these subdomains in integrin-dependent cellular functions, we used beta1A and beta1B isoforms as well as four mutants lacking the entire cytoplasmic domain (beta1TR), the variable region (beta1COM), or the common region (beta1 deltaCOM-B and beta1 deltaCOM-A). By expressing these constructs in Chinese hamster ovary and beta1 integrin-deficient GD25 cells (Wennerberg et al., J Cell Biol 132, 227-238, 1996), we show that beta1B, beta1COM, beta1 deltaCOM-B, and beta1 deltaCOM-A molecules are unable to support efficient cell adhesion to matrix proteins. On exposure to Mn++ ions, however, beta1B, but none of the mutants, can mediate cell adhesion, indicating specific functional properties of this isoform. Analysis of adhesive functions of transfected cells shows that beta1B interferes in a dominant negative manner with beta1A and beta3/beta5 integrins in cell spreading, focal adhesion formation, focal adhesion kinase tyrosine phosphorylation, and fibronectin matrix assembly. None of the beta1 mutants tested shows this property, indicating that the dominant negative effect depends on the specific combination of common and B subdomains, rather than from the absence of the A subdomain in the beta1B isoform.

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.

Beta 1D integrin displaces the beta 1A isoform in striated muscles: localization at junctional structures and signaling potential in nonmuscle cells.

The cytoplasmic domains of integrins provide attachment of these extracellular matrix receptors to the cytoskeleton and play a critical role in integrin-mediated signal transduction. In this report we describe the identification, expression, localization, and initial functional characterization of a novel form of beta 1 integrin, termed beta 1D. This isoform contains a unique alternatively spliced cytoplasmic domain of 50 amino acids, with the last 24 amino acids encoded by an additional exon. Of these 24 amino acids, 11 are conserved when compared to the beta 1A isoform, but 13 are unique (Zhidkova, N. I., A. M. Belkin, and R. Mayne. 1995. Biochem. Biophys. Res. Commun. 214:279-285; van der Flier, A., I. Kuikman, C. Baudoin, R, van der Neuf, and A. Sonnenberg. 1995. FEBS Lett. 369:340-344). Using an anti-peptide antibody against the beta 1D integrin subunit, we demonstrated that the beta 1D isoform is synthesized only in skeletal and cardiac muscles, while very low amounts of beta 1A were detected by immunoblot in striated muscles. Whereas beta 1A could not be detected in adult skeletal muscle fibers and cardiomyocytes by immunofluorescence, beta 1D was localized to the sarcolemma of both cell types. In skeletal muscle, beta 1D was concentrated in costameres, myotendinous, and neuromuscular junctions. In cardiac muscle this beta 1 isoform was found in costamers and intercalated discs. beta 1D was associated with alpha 7A and alpha 7B in adult skeletal muscle. In cardiomyocytes of adult heart, alpha 7B was the major partner for the beta 1D isoform. beta 1D could not be detected in proliferating C2C12 myoblasts, but it appeared immediately after myoblast fusion and its amount continued to rise during myotube growth and maturation. In contrast, expression of the beta 1A isoform was downregulated during myodifferentiation in culture and it was completely displaced by beta 1D in mature differentiated myotubes. We also analyzed some functional properties of the beta 1D integrin subunit. Expression of human beta 1D in CHO cells led to its localization at focal adhesions. Clustering of this integrin isoform on the cell surface stimulated tyrosine phosphorylation of pp125FAK (focal adhesion kinase) and caused transient activation of mitogen-activated protein (MAP) kinases. These data indicate that beta 1D and beta 1A integrin isoforms are functionally similar with regard to integrin-mediated signaling.

Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility.

The integrin subunit beta 1B, a beta 1 isoform with a unique sequence at the cytoplasmic domain, forms heterodimers with integrin alpha chains and binds fibronectin, but it does not localize to focal adhesion sites (Balzac, F., A. Belkin, V. Koteliansky, Y. Balabanow, F. Altruda, L. Silengo, and G. Tarone. 1993. J. Cell Biol. 121:171-178). Here we analyze the functional properties of human beta 1B by expressing it in hamster CHO cells. When stimulated by specific antibodies, beta 1B does not trigger tyrosine phosphorylation of a 125-kD cytosolic protein, an intracellular signalling pathway that is activated both by the endogenous hamster or the transfected human beta 1A. Moreover, expression of beta 1B results in reduced spreading on fibronectin and laminin, but not on vitronectin. Expression of beta 1B also results in severe reduction of cell motility in the Boyden chamber assay. Reduced cell spreading and motility could not be accounted for by preferential association of beta 1B with a given integrin alpha subunit. These data, together with our previous results, indicate that beta 1B interferes with beta 1A function when expressed in CHO cells resulting in a dominant negative effect on cell adhesion and migration.

Alpha v integrin subunit is predominantly located in nervous tissue and skeletal muscle during mouse development.

Alpha v integrin subunit can dimerize with different beta subunits to form receptors for several matrix proteins. The function of these receptors in vivo is still largely unknown. We examined the localization of alpha v integrin during mouse development and showed that its distribution is dynamically regulated in the glia of the central nervous system and in skeletal muscle. Immunoreactivity in the neural tube was firstly localized at embryonic day 10.5 (E10.5) around cell bodies lining the lumen and along tiny fibres extending towards the outer margin. At E12.5 alpha v distribution follows the highly defined pattern of the radial glia: fascicles of immunoreactive fibres form parallel palisades, in particular along the hindbrain and the spinal cord. At E15.5, although with weaker intensity, alpha v was still detectable in radial glia fibres, and it codistributed with glial fibrillary acidic protein positive fascicles. After birth (P8) alpha v immunoreactivity in the brain and spinal cord decreased dramatically, but remained high in the radial glia of the cerebellum. In adult mice alpha v reactivity in the central nervous system disappeared. During myogenesis alpha v appears at E10.5 in myotomal cells and from E12.5 alpha v was evident in myoblasts and in myotubes. In the developing skeletal muscle of E15.5 embryos, immunoreactivity became more concentrated in the apical portion of the myotubes. In adult striated muscle the amount of alpha v subunit dramatically declined and immunostaining was no longer detectable. During development, alpha v was weakly evident in other sites including heart and endothelia of blood vessels, mesonephric tubula, smooth muscle of the digestive tract, and bronchia. Comparative analysis of the localization of alpha v, alpha 3, and alpha 5 integrin subunits indicated that alpha v has a unique and highly regulated distribution pattern. The distribution in the nervous system is consistent with a role of alpha v in neuron-glia interaction during the organization of the neuronal layers in the brain cortex and in the cerebellum. Moreover, alpha v is likely to be involved in the myotendinous junction during embryonic life, suggesting a dual functional role of this integrin in muscle and nervous tissue.

Internalization of the alpha 5 beta 1 integrin does not depend on "NPXY" signals.

The alpha 5 beta 1 integrin is a constitutively internalized fibronectin receptor. It contains in the cytoplasmic tail of its beta 1 subunit two NPXY sequences which have been proposed to mediate internalization. Indeed a NPXY motif constitutes the internalization signal for the Low Density Lipoprotein (LDL) and insulin receptors. To learn more about the putative role of the two NPXY sequences in internalization of the alpha 5 beta 1 receptor, we have made and expressed mutants of the human beta 1 subunit in Chinese Hamster Ovary (CHO) cells, in which the two tyrosines of the NPXY motifs were replaced by serine residues. A cytoplasmic variant beta 1B which does not contain any NPXY sequence was also analyzed. Our results indicate that the NPXY mutants and the cytoplasmic variant are still internalized. Thus in the alpha 5 beta 1 receptor, the highly conserved NPXY sequences do not function as internalization motifs.

The beta 1 integrin distal promoter is developmentally regulated in transgenic mice.

Transgenic mice harbouring 5' flanking sequences of the human beta 1 integrin gene linked to the Escherichia coli lacZ gene have been generated to examine spatial and temporal distribution of the promoter activity during development. Our previous data showed that this regulatory region is composed by two promoters, called distal and proximal, located closely on the human genome. To determine the role of each promoter region during development we generated transgenic mice using these two sequences linked to the lacZ reporter gene. Their analysis shows that these two sequences, as determined by in vitro studies, have different efficiencies in promoting transcription. Actually mice carrying the proximal promoter region exhibit a weak lacZ expression resulting in an undetectable beta-galactosidase activity in both embryonic and adult tissues. On the other hand, transgenic mice carrying the distal promoter express beta-galactosidase at high efficiency during embryonic development. The pattern of transgene expression is consistent with the localization of beta 1 protein on mouse embryos evidenced by immunohistochemistry. Moreover the distal promoter is subjected to a temporal modulation since in adult transgenic mice lacZ expression decreases to a level detected only by RT-PCR analysis. We have determined a similar down-regulation analysing by Northern blot beta 1 mRNA in adult and embryonic organs such as heart and gut.

Expression and functional analysis of a cytoplasmic domain variant of the beta 1 integrin subunit.

We have previously described a variant form of the integrin beta 1 subunit (beta 1B)1 characterized by an altered sequence at the cytoplasmic domain. Using polyclonal antibodies to a synthetic peptide corresponding to the unique sequence of the beta 1B, we analyzed the expression of this molecule in human tissues and cultured cells. Western blot analysis showed that the beta 1B is expressed in skin and liver and, in lower amounts, in skeletal and cardiac muscles. The protein was not detectable in brain, kidney, and smooth muscle. In vitro cultured keratinocytes and hepatoma cells are positive, but fibroblasts, endothelial cells, and smooth muscle cells are negative. An astrocytoma cell line derived from immortalized fetal astrocytes was found to express beta 1B. In these cells beta 1B represent integral of 30% of the beta 1 and form heterodimers with alpha 1 and alpha 5 subunits. To investigate the functional properties of beta 1B, the full-length cDNA coding for this molecule was transfected into CHO cells. Stable transfectants were selected and the beta 1B was identified by a mAb that discriminate between the transfected human protein and the endogenous hamster beta 1A. Immunoprecipitation experiments indicated that the beta 1B was exported at the cell surface in association with the endogenous hamster alpha subunits. The alpha 5/beta 1B complex bound to a fibronectin-affinity matrix and was specifically released by RGD-containing peptides. Thus beta 1B and beta 1A are similar as far as the alpha/beta association and fibronectin binding are concerned. The two proteins differ, however, in their subcellular localization. Immunofluorescence studies indicated, in fact, that beta 1B, in contrast to beta 1A, does not localize in focal adhesions. The restricted tissue distribution and the distinct subcellular localization, suggest that beta 1B has unique functional properties.

Expression, topography, and function of integrin receptors are severely altered in keratinocytes from involved and uninvolved psoriatic skin.

Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease.

A human integrin beta 1 subunit with a unique cytoplasmic domain generated by alternative mRNA processing.

The integrin subunit (beta 1) is common to a group of plasma membrane glycoprotein heterodimers that include the fibronectin, laminin and collagen receptors. These receptors span the plasma membrane, providing a transmembrane linkage between the extracellular matrix and the cytoskeleton. Here, we describe a variant of the human beta 1 differing from the previously described beta 1 in the cytoplasmic domain. The variant beta 1 transcript (beta 13'v) is present in different cell types and is synthesized at lower levels compared to the beta 1 mRNA. The cytoplasmic domain of the beta 13'v is characterized by a unique 12-amino acid C-terminal sequence. A Tyr residue present in this region, and known to be phosphorylated in the beta 1, is no longer part of a consensus sequence for phosphorylation by Tyr kinases. The integrin cytoplasmic domain anchors actin fibrils to the plasma membrane by interacting with cytoskeletal proteins such as talin and fibulin. The integrin beta 13'v with the variant cytoplasmic domain is likely to mediate a new type of membrane-cytoskeleton interaction during cell-cell and cell-matrix adhesion. Analysis of genomic clones showed that the new sequences of the variant mRNA are identical to an intron located between the last two exons of the beta 1 gene, indicating that the alternative message is generated either by premature transcription termination or by lack of splicing at this site.

Primary cultures of corticostriatal cells from newborn rats: a model to study muscarinic receptor subtypes regulation and function.

In the present work we characterized both the presynaptic and postsynaptic components of cholinergic transmission in a primary culture of corticostriatal neurons prepared from newborn rat brain. This culture preparation contains a small population of choline acetyltransferase (ChAT) immunoreactive neurons, corresponding to approximately 3% of the total cell number, and synthesizes increasing amounts of acetylcholine (ACh) from the third day in vitro (DIV), which reaches a plateau around the 10 day of culture. Muscarinic cholinergic receptors (mAChR), measured by the binding of the muscarinic antagonist [3H]quinuclidinyl benzilate ([3H]QNB), are detectable from the fifth DIV and increase linearly during the time of culture. At the twelfth DIV, the density of mAChRs (approximately 600 fmol/mg protein) is comparable to the density of mAChR in adult rat cortex. These receptors are coupled to second messenger systems, since muscarinic agonists inhibit adenylate cyclase activity and stimulate phosphoinositide breakdown with efficacies and potencies similar to those found in adult rat cortex. Moreover, by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, we were able to demonstrate the presence of the m1, m3, and m4 mAChR subtype mRNAs in this neuronal culture at 12 DIV. Our data suggest that corticostriatal neuronal cultures develop in vitro ACh-synthesizing neurons and functionally active cholinergic receptors. This therefore makes them ideally suited to study the development and properties of brain mAChR subtypes.