RESUMO
Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of â¼300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation. Here, we report that hypoxia disrupts polarized epithelial morphogenesis of MDCK cells in a HIF1α-dependent manner by modulating the transforming growth factor-ß (TGFß) signaling pathway. Analysis of potential HIF1 targets in the TGFß pathway identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transmembrane glycoprotein related to the type I receptors of the TGFß family, whose expression was essentially lost in HIF1-depleted cells. Similar to what was observed in HIF1-deficient cells, BAMBI-depleted cells failed to efficiently activate TGFß signaling and retained epithelial polarity during hypoxia. Taken together, we show that hypoxic conditions promote TGFß signaling in a HIF1-dependent manner and BAMBI is identified in this pathway as a novel HIF1-regulated gene that contributes to hypoxia-induced loss of epithelial polarity.
Assuntos
Polaridade Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , Proteínas de Membrana/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Cães , Humanos , Hipóxia/genética , Hipóxia/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células Madin Darby de Rim Canino , Proteínas de Membrana/genética , Transdução de SinaisRESUMO
Laminin, a basement membrane protein discovered in 1979, was shortly thereafter implicated in the polarization of epithelial cells in both mammals and a variety of lower organisms. To transduce a spatial cue to the intrinsic polarization machinery, laminin must polymerize into a dense network that forms the foundation of the basement membrane. Evidence suggests that activation of the small GTPase Rac1 by ß1-integrins mobilizes laminin-binding integrins and dystroglycan to consolidate formation of the laminin network and initiate rearrangements of both the actin and microtubule cytoskeleton to help establish the apicobasal axis. A key coordinator of spatial signals from laminin is the serine-threonine kinase Par-1, which is known to affect dystroglycan availability, microtubule and actin organization, and lumen formation. The signaling protein integrin-linked kinase (ILK) may also play a role. Despite significant advances, knowledge of the mechanism by which assembled laminin produces a spatial signal remains fragmentary, and much more research into the complex functions of laminin in polarization and other cellular processes is needed.
Assuntos
Polaridade Celular , Células Epiteliais/fisiologia , Laminina/fisiologia , AnimaisRESUMO
The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2ß1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2ß1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.
Assuntos
Células Epiteliais/metabolismo , Integrina alfaV/metabolismo , Mecanotransdução Celular , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Colágeno Tipo I/farmacologia , Cães , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Técnicas de Silenciamento de Genes , Integrina beta1/metabolismo , Laminina/metabolismo , Células Madin Darby de Rim Canino , Mecanotransdução Celular/efeitos dos fármacos , Camundongos , Oligopeptídeos/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front-rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin-Darby canine kidney cells by suppressing expression of their α subunits using RNA interference. We determined that knockdown of LM-511 inhibits directional migration and destabilizes cell-cell contacts, in part by disturbing the localization and activity of the polarization machinery. Suppression of integrin α3, a laminin receptor subunit, in cells synthesizing normal amounts of both laminins has a similar effect as knockdown of LM-511. Surprisingly, simultaneous suppression of both laminin α5 and laminin α3 restores directional migration and cell-cell contact stability, suggesting that cells recognize a haptotactic gradient formed by a combination of laminins.
Assuntos
Movimento Celular , Células Epiteliais/fisiologia , Rim/citologia , Laminina/metabolismo , Animais , Adesão Celular , Polaridade Celular , Células Cultivadas , Cães , Células Epiteliais/metabolismo , Expressão Gênica , Técnicas de Silenciamento de Genes , Integrina alfa3beta1/metabolismo , Integrina alfa6beta4/metabolismo , Laminina/genética , Microscopia de Vídeo , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Transporte Proteico , Interferência de RNA , Imagem com Lapso de TempoRESUMO
Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-ß1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-ß type I receptor (TßR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-ß1 secretion and activation mediated by integrin αVß3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-ß1 is secreted apically, whereas TßR-I and integrin αVß3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-ß1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.
Assuntos
Moléculas de Adesão Celular/biossíntese , Integrina alfaVbeta3/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Moléculas de Adesão Celular/genética , Linhagem Celular , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Integrina alfaVbeta3/genética , Rim/citologia , Rim/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad4/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta1/farmacologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , CalininaRESUMO
The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ε) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by â¼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCε- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCε-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCε, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.
Assuntos
Cloretos/química , AMP Cíclico/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteína Quinase C-delta/metabolismo , Proteína Quinase C-épsilon/metabolismo , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Adenoviridae/metabolismo , Biotinilação , Endocitose , Humanos , Íons/química , Isoformas de Proteínas , Transporte Proteico , Interferência de RNA , Membro 2 da Família 12 de Carreador de Soluto , Acetato de Tetradecanoilforbol/químicaRESUMO
Hg(2+) is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg(2+) affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg(2+) decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg(2+)-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg(2+) treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg(2+)-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren's syndrome, and possibly other disorders of fluid transport.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Mercúrio/toxicidade , Junções Íntimas/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Ocludina , Fosforilação/efeitos dos fármacos , Ratos , Glândulas Salivares/citologia , Glândulas Salivares/efeitos dos fármacos , Glândulas Salivares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Glândula Submandibular/citologia , Glândula Submandibular/efeitos dos fármacos , Glândula Submandibular/metabolismo , Junções Íntimas/efeitos dos fármacosRESUMO
Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.
Assuntos
Colágeno Tipo II/metabolismo , Colágeno Tipo I/metabolismo , Expressão Gênica , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Animais , Células Cultivadas , Colágeno Tipo I/química , Elasticidade , Feminino , Expressão Gênica/fisiologia , Técnicas In Vitro , Ligamento Patelar/lesões , Ligamento Patelar/patologia , Ligamento Patelar/cirurgia , Estimulação Física , Coelhos , Estresse Mecânico , Resistência à TraçãoRESUMO
Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical-basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 alpha3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical-basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin alpha5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium.
Assuntos
Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/metabolismo , Polaridade Celular , Células Epiteliais/citologia , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/química , Movimento Celular , Proliferação de Células , Células Cultivadas , Cães , Integrinas/metabolismo , Laminina/deficiência , Laminina/metabolismo , Dados de Sequência Molecular , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno , Ratos , CalininaRESUMO
In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl(-) secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl(-) secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the epsilon-isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na(+)-K(+)-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC-epsilon-dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC-epsilon-dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl(-) secretion.
Assuntos
Cloretos/metabolismo , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína Quinase C/fisiologia , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Carbacol/farmacologia , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Humanos , Mucosa Intestinal/citologia , Proteína Quinase C-épsilon , Transporte Proteico , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling. The PKC family of proteins has been implicated in ischemic stress in nonepithelial systems. The role of PKC isoforms in chemical ischemia in intestinal epithelial cells is evaluated in this study. Additionally, the phosphorylation of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS) is also studied. Chemical ischemia resulted in the transient activation of only the isoform PKC-epsilon as detected by translocation employing the subcellular fractionation technique. The pharmacological agonists phorbol 12-myristate 13-acetate and carbachol also led to the translocation of PKC-epsilon. By immunofluoresence, MARCKS is noted to be located at the lateral membrane under control conditions. In response to carbachol, MARCKS translocates to the cytosol, indicating its phosphorylation, which is additionally confirmed biochemically. Consistent with this observation, carbachol induces the translocation of PKC-epsilon to proximity with MARCKS at the lateral membrane. In response to chemical ischemia, MARCKS fails to translocate and phosphorylation does not increase. Additionally, the translocation of PKC-epsilon is not to the lateral membrane but rather basally. The data suggest that the differential translocation of PKC-epsilon in response to pharmacological agonists versus ischemic stress may lead to different effects on downstream targets.
Assuntos
Intestinos/citologia , Intestinos/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/farmacologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Células Epiteliais/fisiologia , Humanos , Hipóxia , Substrato Quinase C Rico em Alanina Miristoilada , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C-épsilon , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.
Assuntos
Polaridade Celular , Células Epiteliais/fisiologia , Integrinas/metabolismo , Laminina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Adesão Celular , Técnicas de Cultura de Células , Linhagem Celular , Colágeno Tipo I/metabolismo , Cães , Ativação Enzimática , Células Epiteliais/ultraestruturaRESUMO
The attachment of epithelial cells to the extracellular matrix substratum is essential for their differentiation and polarization. Despite this, the precise adhesion mechanism and its regulation are poorly understood. In the kidney, an ischemic insult causes renal tubular epithelial cells to detach from the basement membrane, even though they remain viable. To understand this phenomenon, and to probe the regulation of epithelial cell attachment, we used a model system consisting of newly adherent Madin-Darby canine kidney (MDCK) cells subjected to ATP depletion to mimic ischemic injury. We found that MDCK cells detach from collagen I after 60 min of ATP depletion but reattach when resupplied with glucose. Detachment is not caused by degradation or endocytosis of beta(1)-integrins, which mediate attachment to collagen I. Basal actin filaments and paxillin-containing adhesion complexes are disrupted by ATP depletion and quickly reform on glucose repletion. However, partial preservation of basal actin by overexpression of constitutively active RhoA does not significantly affect cell detachment. Furthermore, Y-27632, an inhibitor of the RhoA effector Rho-kinase, does not prevent reattachment of cells on glucose addition, even though reformation of central stress fibers and large adhesion complexes is blocked. In contrast, reattachment of ATP-depleted cells and detachment of cells not previously subjected to ATP depletion are prevented by ML-7, an inhibitor of myosin light chain kinase (MLCK). We conclude that initial adherence of MDCK cells to a collagen I substratum is mediated by peripheral actin filaments and adhesion complexes regulated by MLCK but not by stress fibers and adhesion complexes controlled by RhoA.
Assuntos
Células Epiteliais/metabolismo , Adesões Focais/fisiologia , Cadeias Leves de Miosina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular , Células Epiteliais/citologia , Integrina beta1/metabolismo , Rim/citologia , Peptídeos/metabolismo , Fosforilação , Fibras de Estresse/metabolismoRESUMO
Epithelial cells polarize in response to cell-substratum and cell-cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin. Investigation of the roles of integrins in epithelial cell polarization has depended on the use of function-blocking antibodies both to determine ligand specificity of individual integrins and to disrupt and redirect normal morphogenesis. In this article we describe techniques for employing function-blocking anti-integrin antibodies in adhesion assays of the polarized Madin-Darby canine kidney (MDCK) cell line and to demonstrate the involvement of beta1 integrins in collagen-induced tubulocyst formation. These techniques can be easily expanded to other antibodies and epithelial cell lines to characterize specific functions of individual integrins in epithelial morphogenesis.
Assuntos
Anticorpos , Polaridade Celular/fisiologia , Células Epiteliais/citologia , Imunoensaio/métodos , Integrinas/imunologia , Animais , Adesão Celular/fisiologia , Células Epiteliais/fisiologiaRESUMO
Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes. Of the various laminin isoforms, laminin-5 is of particular interest because of its proposed role in the healing of skin wounds. In this study, we investigate the expression of laminin-5 in rat kidney after unilateral ischemia. Using a polyclonal antibody generated against laminin-5, we find that immunostaining is confined to the basement membranes of collecting ducts in the papilla and the major and minor calyces in normal kidney. With injury and regeneration, however, immunostaining becomes much more intense and widespread in basement membranes along the nephron. Immunoblotting of ischemic kidney extracts reveals significantly increased expression of a polypeptide of approximately 220 kDa, possibly corresponding to a precursor of one of the three laminin-5 chains. Immunoblotting and immunostaining also demonstrate significantly increased expression and altered localization of the alpha(3)-integrin subunit, a receptor for laminin-5. These results indicate that there is induction of a laminin isoform, possibly laminin-5, and alpha(3)beta(1)-integrin in the ischemic kidney and may implicate this receptor-ligand combination in the pathogenesis of acute renal failure and/or repair of the injured kidney epithelium.