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The main laminin-binding integrins α3ß1, α6ß1 and α6ß4 are co-expressed in the developing kidney collecting duct system. We previously showed that deleting the integrin α3 or α6 subunit in the ureteric bud, which gives rise to the kidney collecting system, caused either a mild or no branching morphogenesis phenotype, respectively. To determine whether these two integrin subunits cooperate in kidney collecting duct development, we deleted α3 and α6 in the developing ureteric bud. The collecting system of the double knockout phenocopied the α3 integrin conditional knockout. However, with age, the mice developed severe inflammation and fibrosis around the collecting ducts, resulting in kidney failure. Integrin α3α6-null collecting duct epithelial cells showed increased secretion of pro-inflammatory cytokines and displayed mesenchymal characteristics, causing loss of barrier function. These features resulted from increased nuclear factor kappa-B (NF-kB) activity, which regulated the Snail and Slug (also known as Snai1 and Snai2, respectively) transcription factors and their downstream targets. These data suggest that laminin-binding integrins play a key role in the maintenance of kidney tubule epithelial cell polarity and decrease pro-inflammatory cytokine secretion by regulating NF-kB-dependent signaling. [ABSTRACT FROM AUTHOR]

Kidney collecting system development requires integrin-dependent cell-extracellular matrix interactions. Integrins are heterodimeric transmembrane receptors consisting of α and β subunits; crucial integrins in the kidney collecting systemexpress the β1 subunit. The β1 cytoplasmic tail has two NPxYmotifs that mediate functions by binding to cytoplasmic signaling and scaffolding molecules. Talins, scaffolding proteins that bind to the membrane proximal NPxY motif, are proposed to activate integrins and to link them to the actin cytoskeleton. We have defined the role of talin binding to the β1 proximal NPxY motif in the developing kidney collecting system in mice that selectively express a Y-to-A mutation in this motif. The mice developed a hypoplastic dysplastic collecting system. Collecting duct cells expressing this mutation had moderate abnormalities in cell adhesion, migration, proliferation and growth factor-dependent signaling. In contrast, mice lacking talins in the developing ureteric bud developed kidney agenesis and collecting duct cells had severe cytoskeletal, adhesion and polarity defects. Thus, talins are essential for kidney collecting duct development through mechanisms that extend beyond those requiring binding to the β1 integrin subunit NPxY motif. [ABSTRACT FROM AUTHOR]

The Rho GTPase Cdc42 regulates key signaling pathways required for multiple cell functions, including maintenance of shape, polarity, proliferation, migration, differentiation and morphogenesis. Although previous studies have shown that Cdc42 is required for proper epithelial development andmaintenance, its exactmolecular function in kidney development is not well understood. In this study, we define the specific role of Cdc42 during murine kidney epithelial tubulogenesis by deleting it selectively at the initiation of ureteric bud or metanephric mesenchyme development. Deletion in either lineage results in abnormal tubulogenesis, with profound defects in polarity, lumen formation and the actin cytoskeleton. Ultimately, these defects lead to renal failure. Additionally, in vitro analysis of Cdc42-null collecting duct cells shows that Cdc42 controls these processes by regulating the polarity Par complex (Par3-Par6-aPKC-Cdc42) and the cytoskeletal proteins N-Wasp and ezrin. Thus, we conclude that the principal role of Cdc42 in ureteric bud and metanephric mesenchyme development is to regulate epithelial cell polarity and the actin cytoskeleton. [ABSTRACT FROM AUTHOR]

PINCH-1 is a LIM-only domain protein that forms a ternary complex with integrin-linked kinase (ILK) and parvin (to form the IPP complex) downstream of integrins. Here, we demonstrate that PINCH-1 (also known as Limsl) gene ablation in the epidermis of mice caused epidermal detachment from the basement membrane, epidermal hyperthickening and progressive hair loss. PINCH-1-deficient keratinocytes also displayed profound adhesion, spreading and migration defects in vitro that were substantially more severe than those of ILK-deficient keratinocytes indicating that PINCH-1 also exerts functions in an ILK-independent manner. By isolating the PINCH-1 interactome, the LIM-domain-containing and actin-binding protein epithelial protein lost in neoplasm (EPLIN, also known as LIMA1) was identified as a new PINCH-1-associated protein. EPLIN localized, in a PINCH-1-dependent manner, to integrin adhesion sites of keratinocytes in vivo and in vitro and its depletion severely attenuated keratinocyte spreading and migration on collagen and fibronectin without affecting PINCH-1 levels in focal adhesions. Given that the low PINCH-1 levels in ILK-deficient keratinocytes were sufficient to recruit EPLIN to integrin adhesions, our findings suggest that PINCH-1 regulates integrin-mediated adhesion of keratinocytes through the interactions with ILK as well as EPLIN. [ABSTRACT FROM AUTHOR]

Integrin-dependent interactions between cells and extracellular matrix regulate lung development; however, specific roles for β1-containing integrins in individual cell types, including epithelial cells, remain incompletely understood. In this study, the functional importance of β1 integrin in lung epithelium during mouse lung development was investigated by deleting the integrin from E10.5 onwards using surfactant protein C promoter-driven Cre. Thesemutant mice appeared normal at birth but failed to gain weight appropriately and died by 4 months of age with severe hypoxemia. Defects in airway branching morphogenesis in association with impaired epithelial cell adhesion and migration, aswell asalveolarizationdefectsandpersistentmacrophagemediated inflammation were identified. Using an inducible system to delete β1 integrin after completion of airway branching, we showed that alveolarization defects, characterizedby disrupted secondary septation, abnormal alveolar epithelial cell differentiation, excessive collagen I and elastin deposition, and hypercellularity of the mesenchyme occurred independently of airway branching defects. By depleting macrophages using liposomal clodronate, we found that alveolarization defects were secondary to persistent alveolar inflammation. β1 integrin-deficient alveolar epithelial cells produced excessive monocyte chemoattractant protein 1 and reactive oxygen species, suggesting a direct role for β1 integrin in regulating alveolar homeostasis. Taken together, these studies define distinct functions of epithelial β1 integrin during both early and late lung development that affect airway branchingmorphogenesis, epithelial cell differentiation, alveolar septation and regulation of alveolar homeostasis. [ABSTRACT FROM AUTHOR]

The type II transmembrane protein CD98, best known as the heavy chain of the heterodimeric amino acid transporters (HAT), is required for the surface expression and basolateral localization of this transporter complex in polarized epithelial cells. CD98 also interacts with β1 integrins resulting in an increase in their affinity for ligand. In this study we explored the role of the transmembrane and cytoplasmic domains of CD98 on integrin-dependent cell adhesion and migration in polarized renal epithelial cells. We demonstrate that the transmembrane domain of CD98 was sufficient, whereas the five N-terminal amino acids of this domain were required for CD98 interactions with β1 integrins. Overexpression of either full-length CD98 or CD98 lacking its cytoplasmic tail increased cell adhesion and migration, whereas deletion of the five N-terminal amino acids of the trans membrane domain of CD98 abrogated this effect. CD98 and mutants that interacted with β1 integrins increased both focal adhesion formation and FAK and AKT phosphorylation. CD98- induced cell adhesion and migration was inhibited by addition of phosphoinositol 3-OH kinase (PI3-K) inhibitors suggesting these cell functions are PI3-K-dependent. Finally, CD98 and mutants that interacted with β1, induced marked changes in polarized renal epithelial cell branching morphogenesis in collagen gels. Thus, in polarized renal epithelial cells, CD98 might be viewed as a scaffolding protein that interacts with basolaterally expressed amino acid transporters and β1 integrins and can alter diverse cell War functions such as amino acid transport as well as cell adhesion, migration and branching morphogenesis. [ABSTRACT FROM AUTHOR]

Although integrin engagement initiates signaling events such as focal-adhesion kinase (FAK) and Src kinase activation, the role of phosphoinositide turnover in cell adhesion is less clear. To assess PLC-γ1 function in this process, Plcg1-/- fibroblasts (Null) were compared with the same fibroblasts in which PLC-γ1 was re-expressed (Null+). Following plating on fibronectin, Null cells displayed a significantly impaired rate of adhesion compared with Null+ cells. This defect was detected at low concentrations of fibronectin; at high fibronectin concentrations, the Null and Null+ cells displayed equivalent adhesion characteristics. The differences were not due to PLC-γ1-dependent changes in integrin subunit expression, nor was integrin receptor clustering impaired with the absence of PLC-γ1. Experiments with site-specific antibodies and PLC-γ1 mutants showed that fibronectin selectively increased phosphorylation of Tyr783 and that mutagenesis of this residue, but not Tyr771 or Tyr1253, abrogated fibronectin-dependent adhesion. The SH2 domains of PLCγ1 were also required for maximal adhesion on fibronectin. Adhesion to fibronectin induced PLC-γ1 tyrosine phosphorylation that was inhibited by a Src-kinase inhibitor, but not an epidermal-growth-factor-receptor kinase inhibitor. Moreover, in cells null for Src family members, but not in cells null for FAK family members, integrin-dependent PLC-γ1 tyrosine phosphorylation was greatly reduced. Finally, the data demonstrated that PLCκ1 co-immunoprecipitated with Src following fibronectin-induced integrin activation, and this association did not depend on FAK expression. [ABSTRACT FROM AUTHOR]

In MDCK cells, hepatocyte growth factor/scatter factor (HGF/SF) induces epithelial cell dissociation, scattering, migration, growth and formation of branched tubular structures. By contrast, these cells neither scatter nor form tubular structures in response to the epidermal growth factor (EGF) family of growth factors. Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors and is synthesized as a membraneassociated precursor molecule (proHB-EGF). ProHB-EGF is proteolytically cleaved to release a soluble ligand (sHBEGF) that activates the EGF receptor. Although recent studies suggest possible physiological functions, the role of proHB-EGF remains largely undefined. Using MDCK cells stably expressing proHB-EGF, a noncleavable deletion mutant of proHB-EGF or soluble HB-EGF, we show that epithelial cell functions differ depending on the form of HB-EGF being expressed. Expression of noncleavable membrane-anchored HB-EGF promoted cell-matrix and cell-cell interactions and decreased cell migration, HGF/SF-induced cell scattering and formation of tubular structures. By contrast, expression of soluble HB-EGF induced increased cell migration, decreased cell-matrix and cell-cell interactions and promoted the development of long unbranched tubular structures in response to HGF/SF. These findings suggest that HB-EGF can not only modulate HGF/SF-induced cellular responses in MDCK cells but also that membrane-bound HB-EGF and soluble HB-EGF give rise to distinctly different effects on cell-cell and cellextracellular matrix interactions. [ABSTRACT FROM AUTHOR]

Synthetic hydrogels with controlled physicochemical matrix properties serve as powerful in vitro tools to dissect cell–extracellular matrix (ECM) interactions that regulate epithelial morphogenesis in 3D microenvironments. In addition, these fully defined matrices overcome the lot-to-lot variability of naturally derived materials and have provided insights into the formation of rudimentary epithelial organs. Therefore, we engineered a fully defined synthetic hydrogel with independent control over proteolytic degradation, mechanical properties, and adhesive ligand type and density to study the impact of ECM properties on epithelial tubulogenesis for inner medullary collecting duct (IMCD) cells. Protease sensitivity of the synthetic material for membrane-type matrix metalloproteinase-1 (MT1-MMP, also known as MMP14) was required for tubulogenesis. Additionally, a defined range of matrix elasticity and presentation of RGD adhesive peptide at a threshold level of 2 mM ligand density were required for epithelial tubulogenesis. Finally, we demonstrated that the engineered hydrogel supported organization of epithelial tubules with a lumen and secreted laminin. This synthetic hydrogel serves as a platform that supports epithelial tubular morphogenetic programs and can be tuned to identify ECM biophysical and biochemical properties required for epithelial tubulogenesis. [ABSTRACT FROM AUTHOR]

An abstract of the article related to the regulation of p38 mitogen-activated protein kinase (MAPK) dependent cell cycle arrest by Integrin-linked kinase during ureteric bud development, is presented.

The kidney collecting system develops from branching morphogenesis of the ureteric bud (UB). This process requires signaling by growth factors such as glial cell line derived neurotrophic factor (GDNF) and fibroblast growth factors (FGFs) as well as cell extracellular matrix interactions mediated by integrins. The importance of integrin signaling in UB development was investigated by deleting integrin β1 at initiation (E10.5) and late (E18.5) stages of development. Deletion at E10.5 resulted in a severe branching morphogenesis phenotype. Deletion at E18.5 did not alter renal development but predisposed the collecting system to severe injury following ureteric obstruction. β1 integrin was required for renal tubular epithelial cells to mediate GDNF- and FGF-dependent signaling despite normal receptor localization and activation in vitro. Aberrations in the same signaling molecules were present in the β1-null UBs in vivo. Thus β1 integrins can regulate organ branching morphogenesis during development by mediating growth-factor-dependent signaling in addition to their well-defined role as adhesion receptors. [ABSTRACT FROM AUTHOR]