Intestinal restitution: progression of actin cytoskeleton rearrangements and integrin function in a model of epithelial wound healing.

Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (alpha6beta4, alpha6beta1, and alpha3beta1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. alpha6beta4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the alpha6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha6 subunit inhibit lamellae formation, indicating that redistribution of the alpha6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha3beta1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The alpha6 integrins, particularly alpha6beta4, help contain retraction and both the alpha6 integrins and alpha3beta1 integrin contribute to lamellae formation.

Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium.

Disruptions in the mucosal lining of the gastrointestinal tract reseal by epithelial cell migration, a process termed restitution. We examined the involvement of laminin isoforms and their integrin receptors in restitution using the intestinal epithelial cell line T84. T84 cells express primarily laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced in cells adjoining wounds. Because T84 cells stained faintly with MAbs 4C7 (laminin alpha 1 subunit) and with MAbs 4F11 and 1B4 (laminin alpha 2 subunit), we suggest that expression of laminins 6 and 7 is enhanced in response to wounding. The alpha 3 beta 1 integrin and the alpha 6-containing integrins function in wound closure because MAbs specific for the beta 1 integrin subunit (MAb13), the alpha 3 subunit (IVA5), and the alpha 6 subunit (2B7) potently inhibited T84 migration into wounds. Immunofluorescence using UMA9, a beta 4-integrin-specific MAb, revealed that alpha 6 beta 4 integrin exists in a Triton-X-100-insoluble structure at the basal surface and that the staining of this structure is enhanced in cells adjoining wounds. In addition, a Triton-X-100-soluble pool of alpha 6 beta 4, as well as alpha 3 beta 1 and presumably alpha 6 beta 1, was found along lateral surfaces of T84 cells. On flattened cells adjoining wounds, staining for these integrins was distributed diffusely, suggesting a redistribution that accompanies cell migration. Taken together, these data suggest that wound-induced epithelial cell migration is a finely tuned process that is dependent upon the regulated function and localization of specific laminins and their integrin receptors.

A function for the integrin alpha6beta4 in the invasive properties of colorectal carcinoma cells.

Expression of the integrin alpha6beta4, a receptor for the laminin family of matrix proteins, has been correlated with the progression and metastatic potential of several different tumors, including colorectal carcinoma. For this reason, defining the mechanistic contribution of alpha6beta4 to the aggressive behavior of colorectal and other carcinoma cells is an issue of timely importance for cancer biology. In the present study, we sought to gain insight into the function of alpha6beta4 in colorectal carcinoma cells by studying the behavior of clone A cells, which express high surface levels of this integrin, and by restoring alpha6beta4 expression in RKO cells, a beta4-deficient rectal carcinoma cell line. The data obtained reveal that alpha6beta4 expression increases the adhesive strength of these cells on laminin-1 matrices, although it does not increase their ability to migrate on such matrices. The RKO/beta4 transfectants were considerably more spread on Matrigel, laminin-1, and collagen I than the mock transfectants and displayed numerous extensions suggestive of pseudopodia. More importantly, we discovered that expression of alpha6beta4 facilitates the ability of colorectal carcinoma cells to invade both Matrigel and collagen I matrices. The alpha6beta4-dependent increases in adhesion and invasion, as well as the observed morphological changes, required an intact beta4 cytoplasmic domain. These data argue for a ligand-independent role for alpha6beta4 in promoting cell invasion, and they have important implications for the involvement of this integrin in colorectal carcinoma progression.

A brief period of retrograde hyperthermic perfusion enhances myocardial protection from global ischemia: association with accumulation of Hsp 70 mRNA and protein.

The induction of heat shock proteins in the myocardium has been suggested as a possible intervention to allow for enhanced cardioprotection. We examined the cardioprotective effects of Hsp 70 induction in a clinically relevant model, in which a brief period of retrograde hyperthermic perfusion (42 degrees C) was applied for 15 min, only 5 min prior to global ischemia and reperfusion in the isolated perfused rat heart. Our results indicate that in retrograde hyperthermic perfused hearts (n = 17) there was enhanced dP/dT, end diastolic pressure, and peak developed pressure during normothermic reperfusion following 15 min of global ischemia when compared to control hearts perfused at 37 degrees C (n = 18). Northern analysis indicated Hsp 70 mRNA, in retrograde hyperthermic perfused hearts, was increased 9.0 +/- 0./70-fold (P < 0.001) by 30 min and 9.1 +/- 0.45-fold (P < 0.001) by 60 min of normothermic reperfusion. Western analysis revealed that the Hsp, heat inducible 72 kD protein was increased 1.74 +/- 0.35-fold (P < 0.001) by 30 min and 1.79 +/- 0.31-fold at 60 min of normothermic reperfusion when compared to no ischemia hearts. Our results demonstrate that the use of 15 min of retrograde hyperthermic perfusion, only 5 min prior to global ischemia and reperfusion, provide for enhanced myocardial functional recovery. Enhanced myocardial functional recovery was associated with the accumulation of Hsp 70 mRNA and the Hsp 72 kD protein.

Activation of the p21 pathway of growth arrest and apoptosis by the beta 4 integrin cytoplasmic domain.

The integrin alpha 6 beta 4, a receptor for members of the laminin family of basement membrane components, contributes to the function of epithelial cells and their oncogenically transformed derivatives. In our efforts to study alpha 6 beta 4-mediated functions in more detail and to assess the contribution of the beta 4 cytoplasmic domain in such functions, we identified a rectal carcinoma cell line that lacks expression of the beta 4 integrin subunit. This cell line, termed RKO, expresses alpha 6 beta 1 but not alpha 6 beta 4, and it interacts with laminin-1 less avidly than similar cell lines that express alpha 6 beta 4. We expressed a full-length beta 4 cDNA, as well as a mutant cDNA that lacks the beta 4 cytoplasmic domain, in RKO cells and isolated stable subclones of these transfectants. In this study, we report that subclones that expressed the full-length beta 4 cDNA in association with endogenous alpha 6 exhibited partial G1 arrest and apoptosis, properties that were not evident in RKO cells transfected with either the cytoplasmic domain mutant or the expression vector alone. In an effort to define a mechanism for these observed changes in growth, we observed that expression of the alpha 6 beta 4 integrin induced expression of the p21 (WAF1; CiP1) protein, an inhibitor of cyclin-dependent kinases. These data suggest that the beta 4 integrin cytoplasmic domain is linked to a signaling pathway involved in cell cycle regulation in the beta 4 transfected RKO cells.

The rapid expression of myocardial hsp 70 mRNA and the heat shock 70 kDa protein can be achieved after only a brief period of retrograde hyperthermic perfusion.

The induction of heat shock proteins in the myocardium has been suggested as a possible intervention to allow for enhanced cardioprotection. We have postulated that a brief period of retrograde hyperthermic perfusion would be sufficient to induce Hsp 70 mRNA and protein accumulation. To investigate this hypothesis, rat hearts (n = 45) were perfused at 37 degrees C for 30 min, then perfused for 15 min at 42 degrees C, and allowed to recover at 37 degrees C for 120 min. Control hearts (n = 23) were perfused at 37 degrees C continuously. Northern analysis indicated that in hearts treated with a brief period of retrograde hyperthermic perfusion Hsp 70 mRNA levels were increased 2.85 +/- 0.02-fold (P < 0.01) by 10 min, 4.88 +/- 0.94-fold (P < 0.01) by 15 min, 9.19 +/- 0.62-fold (P < 0.001) by 30 min, 9.4 +/- 0.52-fold (P < 0.001) by 60 min, 9.45 +/- 0.57-fold (P < 0.001) by 90 min and 9.66 +/- 0.99-fold (P < 0.001) by 120 min of normothermic recovery as compared to control hearts. Western analysis revealed that the heat inducible Hsp 72 kDa protein was increased 2.37 +/- 0.45-fold (P < 0.01) at 60 min, 2.53 +/- 0.25-fold (P < 0.01) at 90 min, and 2.7 +/- 0.6&-fold (P < 0.01) at 120 min when compared to control hearts. Our results indicate that the induction of the heat shock protein Hsp 70 can be rapidly achieved through retrograde hyperthermic perfusion of the myocardium.

Quantitative switch in integrin expression accompanies differentiation of F9 cells treated with retinoic acid.

F9 embryonal carcinoma cells resemble epithelial cells when in monolayer culture. After treatment with retinoic acid these cells differentiate into fibroblastic-like cells in a sequence that has been modeled as the mammalian equivalent of the differentiation from stem cells of the inner cell mass to parietal endoderm. This study examined the changes in integrin subtypes that accompany retinoic acid-induced differentiation of F9 cells. Although several integrins were found to be present on the surface of F9 cells and retinoic acid-induced (RA) cells, the two dominant integrins were alpha 3 beta 1 and alpha 5 beta 1. Differentiation of F9 cells resulted in about 10- to 25-fold increase in the amount of alpha 3 beta 1 integrin protein as measured by immunoprecipitation of cell surface labeled material. There was a corresponding several-fold reduction of alpha 5 beta 1 protein. The concentration of alpha 3 mRNA was about the same in F9 and RA cells while the concentration of alpha 5 mRNA dropped several-fold after retinoic acid treatment. Thus alpha 3 regulation appeared to be largely posttranscriptional while the drop in alpha 5 protein may have been a result of transcriptional down-regulation. Quantitative measurement of adhesion suggested that most of the F9 and RA cell-substrate adhesion to fibronectin or laminin is mediated by these integrins. They are the dominant integrins present, and antibodies to either these integrins or to the substrate blocked the adhesion. Despite the large switch in integrin subtype protein expression there was little difference between the two cell types in initial cell interactions when adhesive affinities were measured quantitatively. Also there was no difference between the two phenotypes in rate of initial adhesive strengthening. The phenotypic difference was first observed with later events in the attachment and spreading of the RA-treated cells to the substrate. These results show that retinoic acid treatment alters the amounts of alpha 5 and alpha 3 integrin subunits during the F9 to RA phenotypic switch. The data show that these integrins are important in the cell-substrate adhesion to fibronectin and laminin. They show, however, that the phenotypic changes observed with differentiation are not associated with the initial preferential adhesions to the substrate, but rather with consequences that alter the cytoskeletal architecture of the cell.

A novel structural variant of the human beta 4 integrin cDNA.

The ability of the alpha 6 beta 4 integrin to function as a laminin receptor appears to be cell-type dependent. We reported that this integrin functions as a laminin receptor on clone A cells, a colon carcinoma cell line (Lee et al., J. Cell Biol., 117:671-678), but this integrin may not function as a laminin receptor on all cell types in which it is expressed. One potential mode of alpha 6 beta 4 regulation resides in the beta 4 cytoplasmic domain because structural variants of this domain exist. We isolated beta 4 clones from a clone A cDNA library and identified a 21 bp (7aa), in-frame deletion not previously reported. This 7aa variant is located within a region that exhibits a relatively high degree of homology (42%) with the 70aa insert previously reported by Tamura et al. (J. Cell Biol., 111:1593-1604). One major difference between these two regions is that the region we have highlighted does not contain the four potential serine/threonine phosphorylation sites that are present in the 210 bp (70aa) insert. PCR analysis revealed that the 7aa variant is also expressed in RNA obtained from normal colon and placenta.

Inside-out integrin signaling in macrophages. Analysis of the role of the alpha 6A beta 1 and alpha 6B beta 1 integrin variants in laminin adhesion by cDNA expression in an alpha 6 integrin-deficient macrophage cell line.

Leukocytes use the alpha 6 beta 1 integrin to adhere to laminin based on mAb inhibition and affinity chromatography studies. This adhesion requires leukocyte stimulation with either PMA or specific cytokines, a process that has been termed "inside-out" integrin signaling. In the present study, the involvement of alpha 6 integrin structural variants in this regulated adhesion was examined using mouse macrophages. The two known alpha 6 structural variants, alpha 6A and alpha 6B, differ only in their cytoplasmic domain sequences. Using reverse transcriptase-polymerase chain reaction, we observed that macrophages express only the alpha 6A structural variant, in contrast to most cell types which express both alpha 6A and alpha 6B variants. The role of this integrin subunit in macrophage adhesion was assessed by cDNA transfection of P388D1 cells. We found that this mouse macrophage cell line does not adhere to laminin even in response to phorbol 12-myristate 13-acetate (PMA) stimulation, though it does adhere normally to fibronectin and tissue culture plastic. Subsequent analysis employing reverse transcriptase-polymerase chain reaction and immunoprecipitation of surface labeled cells revealed that this cell line expresses neither the alpha 6A nor alpha 6B integrin subunits. Stable transfection of either the chick or human alpha 6A cDNAs into P388D1 cells resulted in chimeric alpha 6A beta 1 surface expression. The alpha 6A transfectants exhibited inside-out integrin signaling because PMA stimulation markedly increased their ability to adhere to laminin but it did not increase alpha 6A beta 1 surface expression. Similar results were obtained after transfection of the human alpha 6B cDNA. Analysis of the human transfectants was facilitated by the generation of a monoclonal antibody, 2B7, that is specific for the human alpha 6 integrin subunit. These observations demonstrate that both alpha 6A beta 1 and alpha 6B beta 1 can be regulated by inside-out signaling pathways in macrophages, even though this cell type expresses only alpha 6A beta 1. The data presented also demonstrate clearly that the alpha 6A and alpha 6B cytoplasmic domains do not differ in their ability to be regulated by PMA.

Decreased expression of Mac-2 (carbohydrate binding protein 35) and loss of its nuclear localization are associated with the neoplastic progression of colon carcinoma.

The Mac-2 lectin (carbohydrate binding protein 35) is a soluble, 32- to 35-kDa phosphoprotein that binds galactose-containing glycoconjugates. We report here that the colonic epithelium is a major site of Mac-2 expression in vivo based on immunohistochemistry of human tissue specimens. In this epithelium, proliferating cells at the base of the crypts do not express Mac-2 but its expression increases with differentiation along the crypt-to-surface axis. Mac-2 expression is concentrated in the nuclei of these differentiated epithelial cells. The progression from normal mucosa to adenoma to carcinoma is associated with significant changes in Mac-2 nuclear localization and expression. In all adenomas (9/9) and carcinomas (13/13) examined, Mac-2 was not present in the nucleus but was localized in the cytoplasm. Sequencing of Mac-2 cDNAs from normal mucosa and carcinoma revealed no specific mutations that could account for this loss of nuclear localization. We also observed a 5- to 10-fold decrease in Mac-2 mRNA levels in cancer compared to normal mucosa as well as a significant reduction in the amount of Mac-2 protein expressed. These observations suggest that Mac-2 exclusion from the nucleus and its decreased expression may be related to the neoplastic progression of colon cancer.

The integrin alpha 6 beta 4 is a laminin receptor.

In this study, the putative laminin receptor function of the alpha 6 beta 4 integrin was assessed. For this purpose, we used a human cell line, referred to as clone A, that was derived from a highly invasive, colon adenocarcinoma. This cell line, which expresses the alpha 6 beta 4 integrin, adheres to the E8 and not to the P1 fragment of laminin. The adhesion of clone A cells to laminin is extremely rapid with half-maximal adhesion observed at 5 min after plating. Adhesion to laminin is blocked by GoH3, and alpha 6 specific antibody (60% inhibition), as well as by A9, a beta 4 specific antibody (30% inhibition). Most importantly, we demonstrate that alpha 6 beta 4 binds specifically to laminin-Sepharose columns in the presence of either Mg2+ or Mn2+ and it is eluted from these columns with EDTA but not with NaCl. The alpha 6 beta 4 integrin does not bind to collagen-Sepharose, but the alpha 2 beta 1 integrin does bind. Clone A cells do not express alpha 6 beta 1 as evidenced by the following observations: (a) no beta 1 integrin is detected in beta 1 immunoblots of GoH3 immunoprecipitates; and (b) no alpha 6 beta 1 integrin is seen in GoH3 immunoprecipitates of clone A extracts that had been immunodepleted of all beta 4 containing integrin using the A9 antibody. These data establish that laminin is a ligand for the alpha 6 beta 4 integrin and that this integrin can function as a laminin receptor independently of alpha 6 beta 1.

Carbohydrate-binding protein 35 (Mac-2), a laminin-binding lectin, forms functional dimers using cysteine 186.

Carbohydrate-binding protein 35 (CBP35), also known as the macrophage surface antigen Mac-2, is a lactosamine-specific lectin whose extracellular properties include the ability to agglutinate cells and to bind avidly to the basement membrane glycoprotein laminin. Although these and other properties would be facilitated by dimerization of this lectin, previous studies have argued against multimeric forms of this protein. We report here that macrophage CBP35, purified by laminin affinity chromatography, exists as several distinct species (Mr 35,000, 67,000, and 80,000) when analyzed under non-reducing conditions. This unexpected finding prompted us to study the biochemistry of multimerization using recombinant CBP35 (rCBP35). rCBP35 expressed in Escherichia coli forms disulfide-linked homodimers (Mr 67,000). The dimeric form of CBP35 binds to laminin with higher affinity than does monomer and by a lactosamine-dependent mechanism. Site-directed mutagenesis indicated that cysteine 186, the single cysteine residue in CBP35, is required for dimerization. These results raise the possibility that homo- and heterodimeric forms of CBP35 contribute to its postulated functions in cell-matrix interactions and growth regulation.

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.

Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response.

Cell-substratum adhesion strengths have been quantified using fibroblasts and glioma cells binding to two extracellular matrix proteins, fibronectin and tenascin. A centrifugal force-based adhesion assay was used for the adhesive strength measurements, and the corresponding morphology of the adhesions was visualized by interference reflection microscopy. The initial adhesions as measured at 4 degrees C were on the order of 10(-5)dynes/cell and did not involve the cytoskeleton. Adhesion to fibronectin after 15 min at 37 degrees C were more than an order of magnitude stronger; the strengthening response required cytoskeletal involvement. By contrast to the marked strengthening of adhesion to FN, adhesion to TN was unchanged or weakened after 15 min at 37 degrees C. The absolute strength of adhesion achieved varied according to protein and cell type. When a mixed substratum of fibronectin and tenascin was tested, the presence of tenascin was found to reduce the level of the strengthening of cell adhesion normally observed at 37 degrees C on a substratum of fibronectin alone. Parallel analysis of corresponding interference reflection micrographs showed that differences in the area of cell surface within 10-15 nm of the substratum correlated closely with each of the changes in adhesion observed: after incubation for 15 min on fibronectin at 37 degrees C, glioma cells increased their surface area within close contact to the substrate by integral to 125-fold. Cells on tenascin did not increase their surface area of contact. The increased surface area of contact and the inhibitory activity of cytochalasin b suggest that the adhesive "strengthening" in the 15 min after initial binding brings additional adhesion molecules into the adhesive site and couples the actin cytoskeleton to the adhesion complex.