ABSTRACT
Laminins are a major constituent of the basement membranes of the kidney collecting system. Integrins, transmembrane receptors formed by non-covalently bound α and ß subunits, serve as laminin receptors, but their role in development and homeostasis of the kidney collecting system is poorly defined. Integrin α3ß1, one of the major laminin receptors, plays a minor role in kidney collecting system development, while the role of α6 containing integrins (α6ß1 and α6ß4), the other major laminin receptors, is unknown. Patients with mutations in α6 containing integrins not only develop epidermolysis bullosa, but also have abnormalities in the kidney collecting system. In this study, we show that selectively deleting the α6 or ß4 integrin subunits at the initiation of ureteric bud development in mice does not affect morphogenesis. However, the collecting system becomes dilated and dysmorphic as the mice age. The collecting system in both null genotypes was also highly susceptible to unilateral ureteric obstruction injury with evidence of excessive tubule dilatation and epithelial cell apoptosis. Mechanistically, integrin α6-null collecting duct cells are unable to withstand high mechanical force when adhered to laminin. Thus, we conclude that α6 integrins are important for maintaining the integrity of the kidney collecting system by enhancing tight adhesion of the epithelial cells to the basement membrane. These data give a mechanistic explanation for the association between kidney collecting system abnormalities in patients and epidermolysis bullosa.
Subject(s)
Basement Membrane/metabolism , Integrin alpha6beta1/genetics , Integrin alpha6beta4/genetics , Kidney Tubules, Collecting/metabolism , Laminin/genetics , Ureteral Obstruction/metabolism , Animals , Apoptosis , Basement Membrane/pathology , Cell Adhesion , Cell Movement , Cell Proliferation , Epithelial Cells/metabolism , Epithelial Cells/pathology , Fibrosis , Gene Expression Regulation , Humans , Integrin alpha6beta1/deficiency , Integrin alpha6beta4/deficiency , Kidney Tubules, Collecting/pathology , Laminin/metabolism , Mice , Mice, Knockout , Protein Binding , Signal Transduction , Ureter/surgery , Ureteral Obstruction/pathology , Ureteral Obstruction/surgeryABSTRACT
Human papillomaviruses (HPV) are small DNA tumor viruses. HPV infection requires entry of virions into epithelial host cells that support the viral life cycle. Here, we used an in vivo mouse model, in which HPV pseudoviruses (PVs) are scored for their ability to transduce reporter genes, to test the role of various cellular proteins in entry. We initially investigated the role of integrin α(6)ß(4) in mediating early steps of HPV infection. Deficiency of integrin α(6)ß(4) is modestly but significantly suppressed reporter-gene transduction by PVs in conditional integrin ß(4) knockout mice. We also investigated the role of syndecan 1, a heparin sulfate proteoglycan (HSPG) for its role in HPV infection. We did not see a significant reduction in reporter-gene transduction by PVs in syndecan-1 null mice. This indicates that this HSPG is not essential for early steps in HPV infection, but does not discount a need of other HSPGs in mediating HPV infection.
Subject(s)
Integrin alpha6beta4/metabolism , Papillomavirus Infections/etiology , Syndecan-1/metabolism , Animals , Disease Models, Animal , Female , Genes, Viral , Human papillomavirus 16/genetics , Human papillomavirus 16/pathogenicity , Humans , Integrin alpha6beta4/deficiency , Integrin alpha6beta4/genetics , Mice , Mice, Knockout , Papillomavirus Infections/immunology , Papillomavirus Infections/metabolism , Papillomavirus Infections/virology , Receptors, Virus/metabolism , Syndecan-1/deficiency , Syndecan-1/genetics , Transduction, GeneticABSTRACT
An increased expression of the integrin alpha6beta4 is correlated with a poor prognosis in patients with squamous cell carcinomas. However, little is known about the role of alpha6beta4 in the early stages of tumor development. We have isolated cells from mouse skin (mouse tumor-initiating cells [mTICs]) that are deficient in both p53 and Smad4 and carry conditional alleles of the beta4 gene (Itgb4). The mTICs display many features of multipotent epidermal stem cells and produce well-differentiated tumors after subcutaneous injection into nude mice. Deletion of Itgb4 led to enhanced tumor growth, indicating that alpha6beta4 mediates a tumor-suppressive effect. Reconstitution experiments with beta4-chimeras showed that this effect is not dependent on ligation of alpha6beta4 to laminin-5, but on the recruitment by this integrin of the cytoskeletal linker protein plectin to the plasma membrane. Depletion of plectin, like that of beta4, led to increased tumor growth. In contrast, when mTICs had been further transformed with oncogenic Ras, alpha6beta4 stimulated tumor growth, as previously observed in human squamous neoplasms. Expression of different effector-loop mutants of Ras(V12) suggests that this effect depends on a strong activation of the Erk pathway. Together, these data show that depending on the mutations involved, alpha6beta4 can either mediate an adhesion-independent tumor-suppressive effect or act as a tumor promotor.
Subject(s)
Epidermis/metabolism , Epidermis/pathology , Integrin alpha6beta4/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Alleles , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Cell Transformation, Neoplastic , Desmosomes/metabolism , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation, Neoplastic , Integrin alpha6beta4/deficiency , Integrin alpha6beta4/genetics , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Plectin/genetics , Plectin/metabolism , Protein Binding , RNA Interference , Stem Cells/cytology , Stem Cells/metabolism , ras Proteins/geneticsABSTRACT
ErbB2 (HER2, Neu) and Ras play key roles in tumor invasion and metastasis. We identified a novel mechanism by which integrin alpha(6)beta(4) regulates ErbB2 expression, Ras activation, and the invasion of breast carcinoma cells. Here we show that integrin alpha(6)beta(4) regulates Ras activity especially in serum-depleted condition. Down-regulation of beta(4) integrin by beta(4) short hairpin RNA (shRNA) decreased Ras activity and carcinoma invasion whereas reexpression of this integrin restored Ras activity. ErbB2, a binding partner of epidermal growth factor receptor (EGFR), and EGFR modulated Ras activity, and integrin alpha(6)beta(4) regulated phospho-EGFR level without affecting EGFR expression. We also found that integrin alpha(6)beta(4) is involved in ErbB2 expression. Depletion of beta(4) by shRNA reduced ErbB2 protein level without affecting ErbB2 mRNA level and reexpression of beta(4) increased ErbB2 protein level. Reduction of eukaryotic initiation factor 4E, a rate-limiting factor for cap-dependent translation, decreased ErbB2 protein level, and beta(4) shRNA cells exhibited a shift in ErbB2 mRNA to light polysomes compared with control cells. These results show that integrin alpha(6)beta(4) regulates ErbB2 through translational control. In summary, we propose a novel mechanism for ErbB2 up-regulation and Ras activation in serum-depleted breast cancer cells; integrin alpha(6)beta(4) regulates the expression of ErbB2 and the subsequent phosphorylation of EGFR and activation of Ras. These findings provide a mechanism that substantiates the reported role of alpha(6)beta(4) in carcinoma invasion.
Subject(s)
Breast Neoplasms/metabolism , ErbB Receptors/metabolism , Integrin alpha6beta4/physiology , Receptor, ErbB-2/metabolism , ras Proteins/metabolism , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/biosynthesis , ErbB Receptors/genetics , Eukaryotic Initiation Factor-4E/metabolism , Humans , Integrin alpha6beta4/biosynthesis , Integrin alpha6beta4/deficiency , Integrin alpha6beta4/genetics , Neoplasm Invasiveness , Phosphorylation , Protein Biosynthesis , Quinazolines , RNA, Small Interfering/genetics , Receptor, ErbB-2/biosynthesis , Receptor, ErbB-2/genetics , Signal Transduction , Transcriptional Activation , Transfection , Tyrphostins/pharmacologyABSTRACT
The alpha6beta4 integrin has been widely implicated in carcinoma function in vitro; however, in vivo data are scarce. To determine the importance of alpha6beta4 in tumor progression, a SUM-159 breast carcinoma cell line that is essentially devoid of alpha6beta4 expression was generated using an RNA interference strategy. Loss of alpha6beta4 expression inhibits colony formation in soft agar assays, suggesting a vital role for alpha6beta4 in survival signaling and anchorage-independent growth. Orthotopic injection of the beta4-deficient cell line into the mammary fat pad of immunocompromised mice yielded significantly fewer and smaller tumors than the control cell line, revealing a role for the alpha6beta4 integrin in tumor formation. Under conditions that mimicked the in vivo environment, decreased expression of the alpha6beta4 integrin led to enhanced apoptosis as determined by the percentage of Annexin V-FITC+, PI- cells and the presence of caspase-3 cleavage products. Recombinant vascular endothelial growth factor (VEGF) significantly inhibited the cell death observed in the beta4-deficient cell line, demonstrating the importance of VEGF expression in this survival pathway. Furthermore, loss of alpha6beta4 expression leads to enhanced apoptosis and reduced expression of VEGF in breast carcinoma cells in vivo. Importantly, the specificity of alpha6beta4 in both the in vitro and in vivo assays showed that reexpression of the beta4 subunit into the beta4-deficient cell line could rescue the functional phenotype. Taken together, these data implicate the alpha6beta4 integrin in tumor formation by regulating tumor cell survival in a VEGF-dependent manner.