Role of integrin beta1-like protein in proliferation and differentiation of cultured stem cells from midgut of Heliothis virescens

Cultured midgut cells from Heliothis virescens larvae were incubated with anti-human integrin beta1 made in rabbit and then passed over a column of magnetic beads bound to anti-rabbit IgG (MACS, Miltenyi Bergisch Gladbach, Germany). Cells bound to integrin beta1 antibody also bound to the anti-rabbit IgG on the magnetic beads (MACS) and were retained in the column while it remained in the magnetic field. Non-bound cells were eluted at this time. They did not stain with anti-integrin antibody just after elution. Removing the column from the magnetic field allowed cells bound to the beads-integrin beta1 antibody to be eluted. All of these cells stained with human anti-integrin beta1 upon elution. Each cell fraction was cultured in medium for 3 days. During this time, the populations of cells tended to return to heterogeneous staining patterns characteristic of control populations. However, cells that did not stain immediately with anti-integrin beta1 antibody exhibited double the rate of multiplication and 8 times more differentiation than the integrin-antibody positive cells that eluted later, as well as the non-treated control cells. In a second experiment, midgut cells were incubated for 4 days with various titers of human anti-integrin beta1 to block surface integrin beta1-like reactive sites. Stem cells blocked with anti-integrin beta1 antibody during incubation exhibited double the rate of differentiation than non-treated control cells and those showing anti-integrin beta1-positive stain upon elution.     

Traction Forces Mediated by α6β4 Integrin: Implications for Basement Membrane Organization and Tumor Invasion

The integrin alpha6beta4, a laminin receptor that stabilizes epithelial cell adhesion to the basement membrane (BM) through its association with cytokeratins, can stimulate the formation and stabilization of actin-rich protrusions in carcinoma cells. An important, unresolved issue, however, is whether this integrin can transmit forces to the substrate generated by the acto-myosin system. Using a traction-force detection assay, we detected forces exerted through alpha6beta4 on either laminin-1 or on an anti-alpha6 antibody, demonstrating that this integrin can transmit forces without the need to engage other integrins. These alpha6beta4-dependent traction forces were organized into a compression machine localized to the base of lamellae. We hypothesized that the compression forces generated by alpha6beta4 result in the remodeling of BMs because this integrin plays a major role in the interaction of epithelial and carcinoma cells with such structures. Indeed, we observed that carcinoma cells are able to remodel a reconstituted BM through alpha6beta4-mediated compression forces by a process that involves the packing of BM material under the cells and the mechanical removal of BM from adjacent areas. The distinct signaling functions of alpha6beta4, which activate phosphoinositide 3-OH kinase and RhoA, also contribute to remodeling. Importantly, we demonstrate remodeling of a native BM by epithelial cells and the involvement of alpha6beta4 in this remodeling. Our findings have important implications for the mechanism of both BM organization and tumor invasion.     

Implications of Time Bomb model of ookinete invasion of midgut cells

In this review, we describe the experimental observations that led us to propose the Time Bomb model of ookinete midgut invasion and discuss potential implications of this model when considering malaria transmission-blocking strategies aimed at arresting parasite development within midgut cells. A detailed analysis of the molecular interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei parasites, as they migrate through midgut cells, revealed that ookinetes induce nitric oxide synthase (NOS) expression, remodeling of the actin cytoskeleton and characteristic morphological changes in the invaded epithelial cells. Parasites inflict extensive damage that ultimately leads to genome fragmentation and cell death. During their migration through the cytoplasm, ookinetes release a subtilisin-like protease (PbSub2) and the surface protein (Pbs21). The model proposes that ookinetes must escape rapidly from the invaded cells, as the responses mediating cell death could be potentially lethal to the parasites. In other words, the physical and/or chemical damage triggered by the parasite can be thought of as a 'lethal bomb'. Once this cascade of events is initiated, the parasite must leave the cellular compartment within a limited time to escape unharmed from the 'bomb' it has activated. The midgut epithelium has the ability to heal rapidly by 'budding off' the damaged cells to the midgut lumen without losing its integrity.     

Penetration of the mosquito (Aedes aegypti) midgut wall by the ookinetes of Plasmodium gallinaceum.

We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exists to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.     

Penetration of the Mosquito (Aedes aegypti) Midgut Wall by the Ookinetes of Plasmodium gallinaceum

ABSTRACT We observed Plasmodium gallinaceum ookinetes in both intracellular and intercellular positions in the midgut epithelium of the mosquito Aedes aegypti. After epithelial cell invasion intracellular ookinetes lacked a parasitophorous vacuolar membrane and were surrounded solely by their own pellicle. Thus, the ookinete in the midgut epithelium of the mosquito differs from erythrocytic and hepatic stages in that the parasite in the vertebrate host is surrounded by a vacuole. The midgut epithelial cytoplasm around the apical end of invading ookinetes was replaced by fine granular material deprived of normal organelles. Membranous structure was observed within the fine granular area. Most ookinetes were seen intracellularly on the luminal side and intercellularly on the haemocoel side of the midgut epithelial cells. These observations suggest that the ookinete first enters into the midgut epithelial cell, then exits to the space between the epithelial cells and moves to the basal lamina where the ookinete develops to the oocyst.     

Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis

Interaction between the major fimbriae of Porphyromonas gingivalis and gingival epithelial cells is important for bacterial adhesion and invasion. In this study, we identified integrins as an epithelial cell cognate receptor for P. gingivalis fimbriae. Immunoprecipitation and direct binding assays revealed a physical association between recombinant fimbrillin and beta1 integrins. In vitro adhesion and invasion assays demonstrated inhibition of binding and invasion of P. gingivalis by beta1 integrin antibodies. In contrast, invasion of a fimbriae-deficient mutant of P. gingivalis was not affected by integrin antibodies. Infection of gingival epithelial cells with wild-type P. gingivalis induced tyrosine phosphorylation of the 68 kDa focal adhesion protein paxillin, whereas the fimbriae-deficient mutant failed to evoke similar changes. Interestingly, activation of paxillin was not accompanied by an increase in the phosphorylation of focal adhesion kinase (FAK). These results provide evidence that P. gingivalis fimbriae promote adhesion to gingival epithelial cells through interaction with beta1 integrins, and this association represents a key step in the induction of the invasive process and subsequent cell responses to P. gingivalis infection.     

The parasite invasion marker SRPN6 reduces sporozoite numbers in salivary glands of Anopheles gambiae

For malaria transmission to occur, Plasmodium sporozoites must infect the salivary glands of their mosquito vectors. This study reports that Anopheles gambiae SRPN6 participates in a local salivary gland epithelial response against the rodent malaria parasite, Plasmodium berghei . We showed previously that SRPN6, an immune inducible midgut invasion marker, influences ookinete development. Here we report that SRPN6 is also specifically induced in salivary glands with the onset of sporozoite invasion. The protein is located in the basal region of epithelial cells in proximity to invading sporozoites. Knockdown of SRPN6 during the late phase of sporogony by RNAi has no effect on oocyst rupture but significantly increases the number of sporozoites present in salivary glands. Despite several differences between the passage of Plasmodium through the midgut and the salivary glands, this study identifies a striking overlap in the molecular responses of these two epithelia to parasite invasion.     

Integrin-mediated host cell invasion by type 1-piliated uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, typically express filamentous adhesive organelles called type 1 pili that mediate both bacterial attachment to and invasion of bladder urothelial cells. Several host proteins have previously been identified as receptors for type 1 pili, but none have been conclusively shown to promote UPEC entry into host bladder cells. Using overlay assays with FimH, the purified type 1 pilus adhesin, and mass spectroscopy, we have identified beta1 and alpha3 integrins as key host receptors for UPEC. FimH recognizes N-linked oligosaccharides on these receptors, which are expressed throughout the urothelium. In a bladder cell culture system, beta1 and alpha3 integrin receptors co-localize with invading type 1-piliated bacteria and F-actin. FimH-mediated bacterial invasion of host bladder cells is inhibited by beta1 and alpha3 integrin-specific antibodies and by disruption of the beta1 integrin gene in the GD25 fibroblast cell line. Phosphorylation site mutations within the cytoplasmic tail of beta1 integrin that alter integrin signaling also variably affect UPEC entry into host cells, by either attenuating or boosting invasion frequencies. Furthermore, focal adhesion and Src family kinases, which propagate integrin-linked signaling and downstream cytoskeletal rearrangements, are shown to be required for FimH-dependent bacterial invasion of target host cells. Cumulatively, these results indicate that beta1 and alpha3 integrins are functionally important receptors for type 1 pili-expressing bacteria within the urinary tract and possibly at other sites within the host.     

Proteolytic processing of endogenous and recombinant beta 4 integrin subunit

The alpha 6 beta 4 integrin is a receptor involved in the interaction of epithelial cells with basement membranes. This integrin is unique among the known integrins in that its beta 4 subunit has a large cytoplasmic domain. The function of this cytoplasmic domain is not known. In this paper we show that the beta 4 subunit undergoes proteolytic processing in cultured cells and provide evidence that this also happens in tissues. Immunoprecipitation experiments indicated that the cytoplasmic domain of beta 4 is susceptible to a calcium-dependent protease present in cellular extracts. In vitro assays with purified calpain showed that this enzyme can cleave beta 4 at two distinct sites in the cytoplasmic domain, generating truncated molecules of 165 and 130 kD. Immunoblotting experiments performed on cultured epithelial cells using an antibody to a peptide modeled after the COOH-terminus of the beta 4 subunit showed 70-kD fragments and several fragments of molecular masses between 185 and 115 kD. Similar fragments were detected in CHO cells transfected with the full-length beta 4 cDNA, but not in control transfected cells or in cells transfected with a mutant cDNA lacking the epitope of the cytoplasmic peptide antibody. The sizes of the fragments indicated that both the intracellular and extracellular domains of beta 4 are proteolytically processed. To examine the processing of the beta 4 subunit in epithelial tissues in vivo, human skin frozen sections were stained with antibodies to the ectodomain or the cytoplasmic domain of beta 4. The distinct staining patterns obtained with the two types of antibodies provided evidence that beta 4 is proteolytically processed in vivo in skin. Analogous experiments performed on sections of the cornea suggested that beta 4 is not proteolytically processed at a detectable level in this tissue. Thus, cleavage of the beta 4 subunit occurs in a tissue-specific fashion. These results suggest a potential mechanism of modulating the activities of the alpha 6 beta 4 integrin.     

Expression of the beta6 integrin subunit is associated with sites of neutrophil influx in lung epithelium.

Inhalation of ozone by Rhesus monkeys results in epithelial injury and granulocyte influx in both conducting airways and respiratory bronchioles. We have reported that ozone-induced neutrophil recruitment and subsequent epithelial repair can be inhibited in vivo with a CD18 antibody. The antibody-mediated effect is abrogated by local instillation of C5a (a CD18-independent neutrophil chemoattractant), thereby demonstrating a role for neutrophils in lung epithelial repair processes. As an extension of this study, we examined the effect of ozone and neutrophil influx on epithelial expression of the beta6 integrin, an adhesion molecule associated with proliferation and repair. Expression of beta6 integrin was determined by immunohistochemistry for ozone-exposed monkeys treated with either control immunoglobulins or a CD18 antibody. The tracheal epithelium of ozone-exposed monkeys treated with control immunglobulins expressed the beta6 integrin. In contrast, the tracheal epithelium of ozone-exposed monkeys treated with CD18 antibody exhibited very low to undetectable expression of beta6 integrin. In association with C5a instillation and neutrophil influx, beta6 integrin was also observed in respiratory bronchiolar epithelium from both control and ozone-exposed animals. These findings cumulatively suggest that lung epithelial cell expression of beta6 integrin is associated with sites of neutrophil recruitment.     

The alpha 6 beta 4 integrin and epithelial cell migration.

Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.     

Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus

Receptor-mediated endocytosis using a β1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

     

Role of the alpha(v)beta6 integrin in human oral squamous cell carcinoma growth in vivo and in vitro.

Expression of the alpha(v)beta6 integrin is strikingly upregulated in several types of carcinoma, including human oral squamous cell carcinoma (SCC). Employing a neutralizing monoclonal antibody to alpha(v)beta6, we investigated its role in cell adhesion, proliferation, migration, and in vivo growth of an invasive human SCC line, termed HSC-3. We found that alpha(v)beta6 is strictly required for HSC-3 cell growth in a three-dimensional collagen gel and also prominently contributes to cell migration in two different assay systems. In addition, the anti-alpha(v)beta6 antibody inhibited the invasive growth of HSC-3 cells transorally injected into nude mice. In the presence of the coinjected antibody, the average tumor size at 10 days was reduced by 59%. Histologically, antibody-treated tumors appeared less invasive than control tumors. Furthermore, intravenous application of a neutralizing antibody to the alpha(v) integrin subunit retarded HSC-3 tumor growth. These results point to a possible critical role of the alpha(v)beta6 integrin in controlling growth and invasion of human oral cancer cells.     

Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.     

Integrin alpha3beta1 engagement disrupts intercellular adhesion

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of beta1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell-cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell-cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand-collagen type I, fibronectin, or laminin 1-MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell-cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional beta1 integrin and specifically alpha3beta1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial-mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin-ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.     

Alterations in integrin receptor expression on chemically transformed human cells: specific enhancement of laminin and collagen receptor complexes

The abilities of malignant tumor cells to bind and migrate through basement membranes are important steps in invasion and metastasis. Malignant tumor cells would therefore be expected to express receptors on their surfaces for basement membrane and stromal components, such as collagens, laminin, and fibronectin, although the pattern of expression of these receptors on the malignant cells may be different from that on their normal progenitors. We report here that chemically transformed tumorigenic human cells express an altered pattern of integrin receptors on their cell surfaces as compared with their untransformed nontumorigenic counterparts. Specifically, N-methyl-N'-nitro-N-nitrosoguanidine transformation of HOS cells into highly tumorigenic cells results in a significant specific increase in the expression of (in descending order of level of cell surface expression) the integrins alpha 6/beta 1, alpha 2/beta 1, and alpha 1/beta 1, which are receptors for laminin, collagens, and collagen type IV and laminin, respectively. The level of expression of two fibronectin receptor integrins, alpha 5/beta 1 and alpha 3/beta 1, are, however, unaltered, whereas the level of expression of vitronectin receptor integrin, alpha v/beta 3, is drastically reduced on the transformed cells. Consistent with the increased expression of laminin and collagen receptors and the decreased expression of vitronectin receptors on the transformed cells, these cells attached three- to fivefold more strongly to laminin and collagen but attached very poorly to vitronectin. The MNNG-HOS cells were also found to have a greater potential for invasion through reconstituted basement membrane, matrigel, the major components of which are laminin and type IV collagen. The invasion of both the HOS and MNNG-HOS cells was inhibited 45-50% by a polyclonal anti-fibronectin receptor antibody. However, although the invasion of HOS cells could be inhibited up to 75% by an anti-alpha 6 monoclonal antibody, a similar concentration of this antibody had no effect on the alpha 6-overproducing MNNG-HOS cells. A fivefold higher concentration of this antibody did result in partial inhibition of MNNG-HOS invasion. These data indicate a critical role for the alpha 6/beta 1 laminin receptor in the invasion of these cells through basement membranes and demonstrate that chemical transformation of nontumorigenic human cells to highly tumorigenic cells is associated with an altered pattern of integrin expression which may play a direct role in the increased capacity of these cells to bind and invade through basement membranes.     

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.