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Results for “Integrin alpha6beta1”

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PubMed1998

Divalent cations and ligands induce conformational changes that are highly divergent among beta1 integrins.

Here we show striking differences in conformational regulation among beta1 integrins. Upon manganese stimulation, a beta1 epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on alpha4beta1), moderately (on alpha5beta1), weakly (on alpha2beta1), or was scarcely detectable (on alpha6beta1 and alpha3beta1). Comparable results were seen for the beta1 epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (alpha4beta1), moderate (alpha5beta1), weak (alpha2beta1, alpha6beta1), or minimal (alpha3beta1) induction of the 9EG7 epitope. Exchange or deletion of alpha chain cytoplasmic tails did not alter Mn2+-induced 9EG7 epitope levels. Upon removal of calcium by EGTA or EDTA, the hierarchy of 9EG7 epitope induction was similar (alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), except that EGTA reduced rather than induced 9EG7 expression on alpha4beta1. Thus in contrast to other beta1 integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on alpha4beta1. Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on alpha4beta1, whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on alpha4beta1 was eliminated by a D698E mutation in alpha4, suggesting that Asp-698 may play a key role in maintaining atypical alpha4beta1 response to calcium. In conclusion, our results (i) demonstrate that mAb such as 9EG7 and 15/7 have limited diagnostic utility as reporters of ligand or Mn2+ occupancy for beta1 integrins, (ii) indicate pronounced differences in conformational flexibilities (alpha4beta1 > alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), (iii) allow us to hypothesize that beta1 integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical alpha4beta1 response to calcium that requires alpha4 Asp-698.

Amino Acid Sequence↗

PubMed1997

Immunolocalization of integrins in the normal lung and in pulmonary carcinomas.

Cryosections of normal adult lung (n = 7) and pulmonary epithelial tumors, including squamous (n = 8), adeno (n = 8), bronchioloalveolar (n = 5), and large cell (n = 4) carcinomas (SCC, ACC, BAC, LCC), carcinoids (Cd, n = 7), and neuroendocrine carcinomas (NEC) of variable grades (n = 14) were immunostained by the avidin-biotin peroxidase (ABC) method with monoclonal antibodies to the alpha1-6 and alpha(v) and the beta1-4 integrin subunits. Normal adult alveolar septae showed variably intense immunoreactivity for alpha1,3,6 and beta1, whereas reactions for alpha5 and alpha(v) were weaker and uneven; the remaining integrin subunits were not detected. Bronchial and bronchiolar epithelium showed variably intense staining for alpha2.3,6,v and beta1,4. Reactions were often, though not invariably, basally polarized. SCC, ADC, and LCC showed variably intense reactions for alpha2.3,6,v and beta1,4. BAC were strongly and uniformly stained for alpha1.3 and beta1. In Cd, alpha1,2,3,v and beta1 reactions were noted, whereas in NEC, weak alpha1,3 and beta1 staining was detected with only traces of alpha6 and alpha(v). We conclude that alveolar epithelial cells do not express the hemidesmosome-associated, laminin-binding integrin alpha6beta4 of the bronchial epithelium but rather the alpha1beta1 and alpha3beta1, collagen IV, and laminin receptors, respectively. SCC, ADC, and sampled LCC express an integrin repertory qualitatively similar to that of the bronchial epithelium. Distinct from the latter, the integrin repertory of BAC parallels that of the alveolar epithelium by its strong expression of the multipotential alpha1beta1 and alpha3beta1 integrins. NEC tumors do not display the laminin receptors alpha6beta4 and alpha6beta1 shown by SCC and ADC but express instead alpha1beta1, a collagen IV-laminin receptor rarely found in epithelial neoplasms except for BAC. In NEC tumors, integrins, especially alpha2, decrease with dedifferentiation. Notably distinct from epithelial mesotheliomas, the major fibronectin-binding integrin alpha5beta1 was not found in any type of lung carcinoma.

Adenocarcinoma↗

PubMed1998

Neuronal receptors mediating responses to antibodyactivated laminin-1.

Embryonic retinal neurons lose the ability to extend neurites on laminin-1 (LN-1) with increasing developmental age yet still do so on other laminin isoforms. However, after treatment of LN-1 with antibodies to "short-arm" regions or removal of the short arms proteolytically, LN-1 supports attachment and extension of neurites even by late embryonic retinal neurons. We have mapped a domain for antibody-mediated "activation" of LN-1 to the N-terminal end of the alpha1 chain. Furthermore, we show that the primary receptors used in the retinal neuron response to "activated" LN-1 are integrins alpha3 beta1 and alpha6 beta1; these are the same receptors used by these neurons for outgrowth on other LN isoforms. Interestingly, alpha3 beta1 is preferentially involved in neurite outgrowth, whereas alpha6beta1 preferentially mediates attachment and spreading. However, in cultures from alpha3 integrin-deficient mice, alpha6 beta1 mediates retinal ganglion cell neurite outgrowth and compensates for the absence of alpha3 beta1. Finally, we show that key features of the retinal neuron response to LN-1 also characterize neurons of the hippocampus, thalamus, and cerebral cortex; these include poor response to untreated LN-1, extensive neurite outgrowth on antibody-activated LN-1 or on fragment E8, and dependence of this response on integrin alpha6 beta1 and at least one other long arm-binding beta1 integrin. These data suggest that regulation of LN-1 function via the process of activation could have important consequences for axonal regeneration. Curiously, the data also imply that the mechanism of laminin activation involves enhanced function at sites that cannot be considered cryptic.

Animals↗

PubMed2003

Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells.

The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the alpha6beta4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the alpha6beta4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced alpha6beta4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of beta4 expression in these cells augmented the formation of alpha6beta1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the alpha6beta4 integrin in invasion and migration that has been demonstrated previously by expression of the beta4 subunit in beta4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the alpha6beta4 integrin may be a useful approach to prevent carcinoma cell progression.

Biotinylation↗

PubMed2002

Differential expression of alpha 3 and alpha 6 integrins in the developing mouse inner ear.

The development of the mammalian inner ear involves a complex series of cell-cell and cell-extracellular matrix interactions. These interactions are likely to be mediated by families of adhesion molecules, including the integrins. We have studied the expression of three integrin subunits known to be expressed on epithelia in a number of tissues (namely, alpha3, alpha6, and beta4) during the development of the murine inner ear. At E10.5, both alpha3 and alpha6 were expressed in the epithelial layers of the otocyst. The expression of alpha6 was concentrated in an anterioventral region of the epithelium and in a proportion of the cells forming the cochlear-vestibular and facial ganglia. By E12.5, alpha6 showed a more restricted expression, confined mainly to the pro-sensory epithelia and the neural processes from the cochlear-vestibular ganglion. In contrast, alpha3 was expressed in epithelia adjacent to the pro-sensory areas. This reciprocal expression pattern was maintained until birth. Between birth and P6, a switch in expression occurred such that alpha3 was upregulated and alpha6 was downregulated in the sensory epithelia of both the auditory and vestibular systems. At this stage, alpha3 was expressed in all the epithelia lining the scala media, thus defining the endolymph compartment. The expression of beta4 was restricted to epithelial/mesenchymal borders throughout the developmental stages studied, suggesting that alpha6 expression observed within the epithelium and neuronal tissue was alpha6beta1. The early expression and changing pattern of alpha3 and alpha6 integrins during development of the mammalian inner ear suggests that they may be involved in the molecular processes that define epithelial boundaries and guide sensory innervation.

Animals↗

PubMed2005

Integrin repertoire on myogenic cells changes during the course of primary myogenesis in the mouse.

Cells interact with the extracellular matrix through receptors, most commonly of the integrin family. We (Cachaco et al. [2003] Development 130:1659-1671) and others (Schwander et al. [2003] Dev. Cell 4:673-685) have demonstrated a role for beta1 integrins in mouse primary myogenesis. However, it is unclear what alpha subunits pair with beta1 during this process in vivo. Here, we determined alpha subunit expression patterns at embryonic day (E) 11.5-E14.5. Differentiated myotomal myocytes express all alpha subunits studied. As the muscle masses form both in trunk (E12.5) and limbs (E11.5-E12.5), laminin receptors alpha6beta1 and alpha7beta1 are undetectable, and an assembled laminin matrix is absent. Instead alpha1beta1, alpha4beta1, alpha5beta1, and an alpha v-containing integrin are expressed and unassembled laminin and fibronectin are abundant around myogenic cells. At E13.5-E14.5, alpha6beta1 and alpha7beta1 are expressed, and a laminin matrix forms around individual myotubes. Thus, myogenic cells change their integrin expression pattern during the course of primary myogenesis in the mouse, suggesting different roles for fibronectin- and laminin-containing matrices in this process.

Animals↗

PubMedDate not supplied

beta 1-Integrin expression and function in human bladder cancer cells: modulation by TNF alpha.

Cytofluorimetric and biochemical analysis in two different grade human bladder cancer cell lines showed that G3 EJ cells exhibited higher levels of alpha5beta1 and alpha6beta1 heterodimers, and the G2 RT112 cell line higher levels of alpha2beta1. Alpha6/beta4 receptor was detected only in RT112 cells. Adhesion assays with extracellular matrix proteins indicated that both cells bound to fibronectin, laminin and collagen 1, the adhesive properties being related to the integrin profile. Inhibition tests revealed that alpha5beta1 mediated adhesion to fibronectin, alpha3beta1 and alpha6beta1 to laminin, and that alpha2beta1 was the main mediator of adhesion to collagen I in both cell lines. In EJ but not in RT112 cells, tumor necrosis factor-alpha induced the upregulation of alpha2, which mediated increased adhesion to collagen I. The different effects of TNFalpha on the two cell lines were not attributable to differences in tumor necrosis factor responsiveness, as both cells expressed comparable levels of tumor necrosis factor receptor-1 and the tumor necrosis factor-inducible intercellular adhesion molecule-1.

Carcinoma, Transitional Cell↗

PubMedDate not supplied

Differential modulation of integrin expression in chondrocytes during expansion for tissue engineering.

Cartilage tissue engineering has an important role to play in the generation of graft material for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. Dedifferentiated cells produce matrix of less quality. Normal cartilage is composed of chondrocytes, which are embedded within an extracellular matrix (ECM). The ECM plays a key role in controlling cellular characteristics and contains the integrins as a large family of heterodimeric cell adhesion receptors involved in cell-cell and cell-matrix interactions. In this study, the characteristic changes of integrin expression and expression of matrix proteins during the course of dedifferentiation of chondrocytes in cell culture for 1, 6 and 21 days, analyzed at the mRNA level by microarray analysis and at the protein level by immunohistochemistry, are described. The components of the fibronectin receptor, integrin beta1,alpha5, in conjunction with the ligand fibronectin, were up-regulated during dedifferentiation. Integrin beta3 was expressed in the grey area. The components of the vitronectin-receptor, integrin alpha2b, alpha v, as well as integrin beta5, were activated on day 21, but neither vitronectin nor osteopontin were expressed by the cells. With ongoing dedifferentiation, activation of the GPIIb/GPIIIa receptor was found. The integrins beta2, beta4, beta6, beta8 and alpha2, alpha4, alpha6, alpha7 and alpha11 were never expressed. ILK, CD47 and ICAP1, as components of the intracellular signalling cascade of several integrins, were activated with ongoing dedifferentiation. In conclusion, a candidate for signal transmission during dedifferentiation is the fibronectin receptor (integrin alpha5beta1) in conjunction with its ligand fibronectin. Other receptors, e.g. for vitronectin and osteopontin (alphaVbeta3) or laminin (alpha6beta1) or their ligands, do not seem to be involved in signal transmission for dedifferentiation. In addition, the GPIIb/IIIa-receptor seems to assist the process of dedifferentiation. Intracellularly, ILK, ICAP1 and CD47 might assist the transduction of the integrin-dependent signals.

Cell Culture Techniques↗

PubMed2002

The role of the specificity-determining loop of the integrin beta subunit I-like domain in autonomous expression, association with the alpha subunit, and ligand binding.

Integrin beta subunits contain a highly conserved I-like domain that is known to be important for ligand binding. Unlike integrin I domains, the I-like domain requires integrin alpha and beta subunit association for optimal folding. Pactolus is a novel gene product that is highly homologous to integrin beta subunits but lacks associating alpha subunits [Chen, Y., Garrison, S., Weis, J. J., and Weis, J. H. (1998) J. Biol. Chem. 273, 8711-8718] and a approximately 30 amino acid segment corresponding to the specificity-determining loop (SDL) in the I-like domain. We find that the SDL is responsible for the defects in integrin beta subunit expression and folding in the absence of alpha subunits. When transfected in the absence of alpha subunits into cells, extracellular domains of mutant beta subunits lacking SDL, but not wild-type beta subunits, were well secreted and contained immunoreactive I-like domains. The purified recombinant soluble beta1 subunit with the SDL deletion showed an elongated shape in electron microscopy, consistent with its structure in alphabeta complexes. The SDL segment is not required for formation of alpha5beta1, alpha4beta1, alphaVbeta3, and alpha6beta4 heterodimers, but is essential for fomation of alpha6beta1, alphaVbeta1, and alphaLbeta2 heterodimers, suggesting that usage of subunit interface residues is variable among integrins. The beta1 SDL is required for ligand binding and for the formation of the epitope for the alpha5 monoclonal antibody 16 that maps to loop segments connecting blades 2 and 3 of beta-propeller domain of alpha5, but is not essential for nearby beta-propeller epitopes.

Amino Acid Sequence↗

PubMed1998

Expression of alpha5 (CD49e) and alpha6 (CD49f) integrin subunits on T cells in the circulation and the lamina propria of normal and inflammatory bowel disease colonic mucosa.

Intestinal lamina propria T cells are believed to be derived, via the systemic circulation, from gut-associated lymphoid tissue. After migration into the lamina propria, T cells are capable of luminally directed migration following the loss of surface epithelial cells. For adhesion and migration within the extracellular matrix, T cells are likely to utilize the integrin family of adhesion molecules. The aim of this study was to quantitatively and qualitatively investigate the expression of alpha5 and alpha6 integrin subunits on the surface of human T cells that: (a) migrated out of the lamina propria, (b) remained resident within the matrix and (c) were present in the circulation. In both subpopulations of CD4 and CD8-positive T cells, from both normal and inflamed (inflammatory bowel disease) colonic mucosa, there were significantly fewer alpha5 and alpha6-positive cells than in the peripheral blood. In addition, there were significantly fewer alpha6 integrin molecules on the surface of CD4 and CD8-positive lamina propria T-cell subpopulations, compared with those in the circulation. Our studies suggest that, following migration into the lamina propria, there is down-regulation of alpha5 and alpha6 integrin-subunit expression on the surface of T cells. Molecules other than members of very late activation antigen-5 (VLA-5) (alpha5beta1) and VLA-6 (alpha6beta1) families of adhesion molecules are likely to be important in interactions with extracellular components in the lamina propria of normal and inflamed human colonic mucosa.

Antigens, CD↗

PubMed1999

Laminin receptors in differentiated thyroid tumors: restricted expression of the 67-kilodalton laminin receptor in follicular carcinoma cells.

The expression of integrin laminin receptors was investigated in normal thyroid primary cultures; immortalized normal thyroid cells (TAD-2); papillary (NPA), follicular (WRO), and anaplastic (ARO) thyroid tumor cell lines; seven thyroid tumors (four papillary and three follicular carcinomas); and normal thyroid glands. The expression of alpha1beta1, alpha2beta1, alpha3beta1, alpha6beta1, and alpha6beta4 was found in all tumor specimens and in tumor cell lines, whereas normal thyroid cells and TAD-2 cells lacked the expression of alpha6beta4. Despite the presence of several integrin laminin receptors, adhesion of TAD-2, NPA, and ARO cells to immobilized laminin-1 was poor, whereas WRO cells and follicular carcinoma-derived cells displayed a strong adhesion. Indeed, WRO and follicular carcinoma-derived cells showed expression of a nonintegrin laminin receptor, the 67-kDa high affinity laminin receptor (67LR). TAD-2, NPA, and ARO cells as well as nodular goiter, toxic adenoma, follicular adenoma, and papillary carcinoma-derived cells did not express the 67LR. Adhesion of WRO and follicular carcinoma-derived cells to laminin-1 was specifically inhibited by a recombinant polypeptide containing laminin-binding domains of 67LR, demonstrating that this receptor confers to follicular carcinoma cells attachment capacity to laminin. Moreover, tissue specimens from follicular carcinomas expressed the 67LR, whereas follicular adenomas and normal thyroid tissues were negative. In thyroid tumors, integrin receptors, although abundant, participate weakly in adhesion to laminin. The expression in follicular carcinoma cells of a functional, high affinity 67LR together with nonfunctional integrin LM receptors could be responsible for the tendency of follicular carcinoma cells to metastasize by mediating stable contacts with basal membranes.

Adenocarcinoma, Follicular↗

PubMed1997

The two phenylalanines in the GFFKR motif of the integrin alpha6A subunit are essential for heterodimerization.

The membrane-proximal domain of the integrin alpha subunit contains a conserved motif of five amino acid residues, GFFKR. We deleted this motif from the human alpha6A subunit and found that in COS-7 cells this mutant cannot associate with the beta1 subunit and is retained in the endoplasmic reticulum. Point mutations in the GFFKR motif of the glycine residue or the two highly charged amino acids, or deletion of the lysine and arginine residues, had no effect on the ability of alpha6 to interact with beta1 and to be expressed at the cell surface. In contrast, by replacing either of the two phenylalanines with alanine, or by deletion of both of these residues, alpha6 was incapable of associating with beta1. The alpha6 point mutants that associated with beta1 were expressed in K562 cells and their responsiveness to integrin-activating factors was determined. None of these transfectants bound spontaneously to laminin-1, but binding could be induced by either PMA or the stimulating anti-beta1 antibody TS2/16 to the same extent as that of the wild-type transfectant. The ability of these mutants to initiate focal-contact formation in CHO cells plated on laminin-1 substrates also appeared to be unaltered. Thus the behaviour of alpha6 mutants involving the glycine, lysine or arginine residues was indistinguishable from that of wild-type alpha6 both in inside-out and outside-in signalling. In contrast, deletion of the cytoplasmic domain of alpha6 C-terminal of the GFFKR motif resulted in a loss of responsiveness of alpha6beta1 to PMA stimulation and formation of focal contacts on laminin-1. However, this mutant was targeted to focal contacts formed by other integrins, even when they had not bound ligand. Together, these results suggest that the two phenylalanine residues of the GFFKR motif provide a site for interaction of the alpha6A subunit with beta1, whereas the cytoplasmic domain C-terminal of this motif is involved in the regulation of bidirectional signalling via alpha6Abeta1.

Amino Acid Sequence↗

PubMed2001

Evaluation of endothelial cell integrins after in \ vitro contact with polyethylene terephthalate.

The aim of this research was to evaluate the effect of polyethylene terephthalate (Woven Dacron) on the expression of endothelial integrins. Human umbilical vein endothelial cells were cultured on the material for 24 h. The integrins VLA-2 (alpha2beta1-CD49b/CD29), receptor for laminin and collagen, VLA-5 (alpha5beta1-CD49e/CD29), receptor for fibronectin, VLA-6 (alpha6beta1-CD49f/CD29), receptor for laminin, and alphaVbeta3-CD51/CD61 (receptor for vitronectin) were evaluated by flow cytometry. After contact with polyethylene terephthalate, a slight but significant decrease in the percentage of both CD29 and CD49e positive cells was observed, which suggests a lower number of cells expressing the fibronectin receptor alpha5beta1. Moreover, a significant increase in the mean channel for CD49b and for the vitronectin receptor CD51/CD61 was observed. The reduction in the fibronectin receptor could account for the poor endothelialization observed in vivo on polyethylene terephthalate. The increased expression of the vitronectin receptor, favoring the migration of smooth muscle cells, could give some information about the pathogenesis of intimal hyperplasia, which is a complication of vascular grafts.

Journal Article↗

PubMed1998

Targeted disruption of the beta1 integrin gene in a lymphoma cell line greatly reduces metastatic capacity.

Integrins have been implicated in tumor metastasis. To investigate this, we generated beta1 integrin-negative double knockout (DKO) mutants of the highly metastatic ESb murine T-lymphoma cell line. The in vivo growth capacity of the mutants, which had lost alpha4beta1 and alpha6beta1 expression, was not altered, but their metastatic capacity was greatly reduced. Tail vein injection of 10(4) ESb and single-knockout cells led to death of all animals within 9-11 days. In contrast, only one-half of the animals injected with 10(4) DKO cells died, but much later, after 20-60 days. The other one-half remained disease-free for up to 100 days. Whereas ESb and single-knockout cells disseminated predominantly to liver and spleen, metastasis of DKO cells to these organs was rare, even after this prolonged period. Instead, skeletal muscles were invaded extensively. Metastatic capacity was largely restored in a DKO clone, which had been transfected with beta1 cDNA and expressed beta1 at similar levels as ESb cells. We conclude that beta1 integrins are essential for efficient liver and spleen colonization by the ESb lymphoma.

Alleles↗

PubMed1997

Generation of monoclonal antibodies to integrin-associated proteins. Evidence that alpha3beta1 complexes with EMMPRIN/basigin/OX47/M6.

The alpha3beta1 integrin forms complexes with other cell-surface proteins, including transmembrane-4 superfamily (TM4SF) proteins (e. g. CD9, CD53, CD63, CD81, and CD82). To identify additional cell-surface proteins associated with alpha3beta1 integrin, a monoclonal antibody selection protocol was developed. Mice were immunized with integrin alpha3beta1-containing complexes isolated from HT1080 fibrosarcoma cells, and then 712 hybridoma clones were produced, and 95 secreted antibodies that recognized the HT1080 cell surface. Among these, 12 antibodies directly recognizing integrin alpha3 or beta1 subunits were eliminated. Of the remaining 83, 16 co-immunoprecipitated proteins that resembled integrins under non-stringent detergent conditions. These 16 included 15 monoclonal antibodies recognizing EMMPRIN/basigin/OX-47/M6, a 45-55-kDa transmembrane protein with two immunoglobulin domains. The EMMPRIN protein associated with alpha3beta1 and alpha6beta1, but not alpha2beta1 or alpha5beta1, as shown by reciprocal immunoprecipitation experiments. Also, association with alpha3beta1 was confirmed by cell-surface cross-linking and immunofluorescence co-localization experiments. Importantly, EMMPRIN-alpha3beta1 complexes appear not to contain TM4SF proteins, suggesting that they are distinct from TM4SF protein-alpha3beta1 complexes.

Animals↗

PubMed1997

Integrin binding to immobilized collagen and fibronectin stimulates the proliferation of human thyroid cells in culture.

The expression of integrins of the beta1 family and their possible biological effects were investigated in normal human thyroid cells in monolayer culture. The expression of beta1 and alpha(1-6) integrin subunits was determined by flow cytofluorometry with specific antibodies. Follicular cells of subconfluent monolayer cultures expressed alpha2beta1 and alpha3beta1 at high levels, while alpha1beta1 was only slightly expressed, and alpha4beta1, alpha5beta1, and alpha6beta1 were never detected. Cell attachment assays were performed in fibronectin-, type I collagen-, and laminin-coated microtiter plates. Thyroid cells, while adherent to collagen and fibronectin, showed poor attachment to laminin despite the abundance of their putative receptors alpha2beta1 and alpha3beta1. In serum-free medium, collagen and fibronectin induced cytoskeletal organization, change of cell shape from round to flat, and cell spreading. [3H]Thymidine incorporation and proliferation assays were used to evaluate the effects of collagen and fibronectin on DNA synthesis and cell growth in the absence of a change in spreading or cell shape. Both substrates, in low serum-containing medium, induced a concentration-dependent increase in [3H]thymidine incorporation partially inhibited by RGD-containing peptides that blocked the cell attachment. Thyrocytes cultured in low serum-containing medium on immobilized fibronectin or collagen showed a dose-dependent stimulation of proliferation. These data indicate that fibronectin and collagen can regulate the cytoskeletal organization and cell shape and stimulate the proliferation of normal human thyroid cells in culture and that integrins mediate these effects of extracellular matrix proteins.

Cell Adhesion↗

PubMed2003

Fer kinase/FerT and adherens junction dynamics in the testis: an in vitro and in vivo study.

Fer kinase is a 94-kDa cytoplasmic cell-cell actin-based adherens junction (AJ)-associated nonreceptor protein tyrosine kinase (PTK) found in multiple epithelia including the testis, whereas FerT kinase (51 kDa) is the truncated testis-specific form of Fer kinase, lacking the Fps/Fes/Fer/CIP4 (products of oncogenes identified in avian and feline sarcoma, encoding tyrosine protein kinases) and the three coiled-coil domains versus Fer kinase. Yet the role(s) of Fer kinase in AJ dynamics in the testis remains largely unexplored. We have used an in vitro model of AJ assembly with Sertoli-germ cell cocultures and an in vivo model of AJ disassembly in which adult rats were treated with 1-(2,4-dichlorobenzyl)-indazole-3-carbohydrazide (AF-2364) to study changes in the expression and/or localization of Fer kinase during AJ restructuring. Fer kinase/FerT was expressed by Sertoli and germ cells when cultured in vitro. Using an antibody prepared against a synthetic peptide, NH2-SAPQNCPEEIFTIMMKCWDYK-COOH, corresponding to residues 779-799 of Fer kinase in the rat, which failed to cross-react with FerT kinase, for immunohistochemistry, Fer kinase was detected in the seminiferous epithelium in virtually all stages of the epithelial cycle. At stages XIII-VI, Fer kinase was associated largely with round and elongating spermatids. At stages VII-VIII, Fer kinase associated almost exclusively with round spermatids with very weak staining associated with elongated spermatids. This stage-specific localization of Fer kinase in the epithelium was confirmed by using staged tubules for semiquantitative reverse transcription-polymerase chain reaction. Studies by immunoprecipitation revealed that Fer kinase associated with N-cadherin, gamma-catenin, p120ctn, c-Src (a putative PTK and the product of the transforming, sarcoma-inducing gene of Rous sarcoma virus), Rab 8 (a GTPase), actin, vimentin, but not E-cadherin, afadin, nectin-3, and integrin beta1, suggesting Fer kinase associates not only with the actin-based cell-cell AJ structures, such as the N-cadherin/catenin complex (but not the alpha6beta1 integrin/laminin and the afadin/nectin complex), but also with intermediate filament-based cell-cell desmosomes. An induction in Fer kinase expression was detected during Sertoli-germ cell AJ assembly in vitro but not during AF-2364-induced AJ disruption in vivo. Yet this AF-2364-induced Fer kinase plummeting associated with an induction in N-cadherin, beta-catenin, and p120ctn, particularly at the base of the seminiferous epithelium. In summary, Fer kinase structurally associates with the N-cadherin/catenin protein complex in the testis and can possibly be used to mediate signaling function via the cadherin/catenin protein complex.

Adherens Junctions↗

PubMed2004

Integrins in the mouse myotome: developmental changes and differences between the epaxial and hypaxial lineage.

Integrins are cellular adhesion receptors that mediate signaling and play key roles in the development of multicellular organisms. However, their role in the cellular events leading to myotome formation is completely unknown. Here, we describe the expression patterns of the alpha1, alpha4, alpha5, alpha6, and alpha7 integrin subunits in the mouse myotome and correlate them with the expression of several differentiation markers. Our results indicate that these integrin subunits may be differentially involved in the various phases of myogenic determination and differentiation. A detailed characterization of the myogenic cell types expressing the alpha4 and alpha6 subunits showed a regionalization of the myotome and dermomyotome based on cell-adhesion properties. We conclude that alpha6beta1 may be an early marker of epaxial myogenic progenitor cells. In contrast, alpha4beta1 is up-regulated in the intercalated myotome after myocyte differentiation. Furthermore, alpha4beta1 is expressed in the hypaxial dermomyotome and is maintained by early hypaxial myogenic progenitor cells colonizing the myotome.

Animals↗