YAP Activation in Renal Proximal Tubule Cells Drives Diabetic Renal Interstitial Fibrogenesis.

An increasing number of studies suggest that the renal proximal tubule is a site of injury in diabetic nephropathy (DN), and progressive renal tubulointerstitial fibrosis is an important mediator of progressive kidney dysfunction in DN. In this study, we observed increased expression and activation of YAP (yes-associated protein) in renal proximal tubule epithelial cells (RPTC) in patients with diabetes and in mouse kidneys. Inducible deletion of Yap specifically in RPTC or administration of the YAP inhibitor verteporfin significantly attenuated diabetic tubulointerstitial fibrosis. EGFR-dependent activation of RhoA/Rock and PI3K-Akt signals and their reciprocal interaction were upstream of proximal tubule YAP activation in diabetic kidneys. Production and release of CTGF in culture medium were significantly augmented in human embryonic kidney (HEK)-293 cells transfected with a constitutively active YAP mutant, and the conditioned medium collected from these cells activated and transduced fibroblasts into myofibroblasts. This study demonstrates that proximal tubule YAP-dependent paracrine mechanisms play an important role in diabetic interstitial fibrogenesis; therefore, targeting Hippo signaling may be a therapeutic strategy to prevent the development and progression of diabetic interstitial fibrogenesis.

Non-visual arrestins regulate the focal adhesion formation via small GTPases RhoA and Rac1 independently of GPCRs.

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation.

Arrestins regulate cell spreading and motility via focal adhesion dynamics.

Focal adhesions (FAs) play a key role in cell attachment, and their timely disassembly is required for cell motility. Both microtubule-dependent targeting and recruitment of clathrin are critical for FA disassembly. Here we identify nonvisual arrestins as molecular links between microtubules and clathrin. Cells lacking both nonvisual arrestins showed excessive spreading on fibronectin and poly-d-lysine, increased adhesion, and reduced motility. The absence of arrestins greatly increases the size and lifespan of FAs, indicating that arrestins are necessary for rapid FA turnover. In nocodazole washout assays, FAs in arrestin-deficient cells were unresponsive to disassociation or regrowth of microtubules, suggesting that arrestins are necessary for microtubule targeting-dependent FA disassembly. Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells. In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype. Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.

Epithelial ß1 integrin is required for lung branching morphogenesis and alveolarization.

Integrin-dependent interactions between cells and extracellular matrix regulate lung development; however, specific roles for ß1-containing integrins in individual cell types, including epithelial cells, remain incompletely understood. In this study, the functional importance of ß1 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. These mutant 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, as well as alveolarization defects and persistent macrophage-mediated inflammation were identified. Using an inducible system to delete ß1 integrin after completion of airway branching, we showed that alveolarization defects, characterized by 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. ß1 integrin-deficient alveolar epithelial cells produced excessive monocyte chemoattractant protein 1 and reactive oxygen species, suggesting a direct role for ß1 integrin in regulating alveolar homeostasis. Taken together, these studies define distinct functions of epithelial ß1 integrin during both early and late lung development that affect airway branching morphogenesis, epithelial cell differentiation, alveolar septation and regulation of alveolar homeostasis.

The integrin ß1 subunit regulates paracellular permeability of kidney proximal tubule cells.

Epithelial cells lining the gastrointestinal tract and kidney have different abilities to facilitate paracellular and transcellular transport of water and solutes. In the kidney, the proximal tubule allows both transcellular and paracellular transport, while the collecting duct primarily facilitates transcellular transport. The claudins and E-cadherin are major structural and functional components regulating paracellular transport. In this study we present the novel finding that the transmembrane matrix receptors, integrins, play a role in regulating paracellular transport of renal proximal tubule cells. Deleting the integrin ß1 subunit in these cells converts them from a "loose" epithelium, characterized by low expression of E-cadherin and claudin-7 and high expression of claudin-2, to a "tight" epithelium with increased E-cadherin and claudin-7 expression and decreased claudin-2 expression. This effect is mediated by the integrin ß1 cytoplasmic tail and does not entail ß1 heterodimerization with an α-subunit or its localization to the cell surface. In addition, we demonstrate that deleting the ß1 subunit in the proximal tubule of the kidney results in a major urine-concentrating defect. Thus, the integrin ß1 tail plays a key role in regulating the composition and function of tight and adherens junctions that define paracellular transport properties of terminally differentiated renal proximal tubule epithelial cells.

Mutations in the paxillin-binding site of integrin-linked kinase (ILK) destabilize the pseudokinase domain and cause embryonic lethality in mice.

Integrin-linked kinase (ILK) localizes to focal adhesions (FAs) where it regulates cell spreading, migration, and growth factor receptor signaling. Previous reports showed that overexpressed ILK in which Val(386) and Thr(387) were substituted with glycine residues (ILK-VT/GG) could neither interact with paxillin nor localize to FA in cells expressing endogenous wild-type ILK, implying that paxillin binding to ILK is required for its localization to FAs. Here, we show that introducing this mutation into the germ line of mice (ILK-VT/GG) caused vasculogenesis defects, resulting in a general developmental delay and death at around embryonic day 12.5. Fibroblasts isolated from ILK-VT/GG mice contained mutant ILK in FAs, showed normal adhesion to and spreading on extracellular matrix substrates but displayed impaired migration. Biochemical analysis revealed that VT/GG substitutions decreased ILK protein stability leading to decreased ILK levels and reduced binding to paxillin and α-parvin. Because paxillin depletion did not affect ILK localization to FAs, the embryonic lethality and the in vitro migration defects are likely due to the reduced levels of ILK-VT/GG and diminished binding to parvins.

ß1 integrin NPXY motifs regulate kidney collecting-duct development and maintenance by induced-fit interactions with cytosolic proteins.

Loss of ß1 integrin expression inhibits renal collecting-system development. Two highly conserved NPXY motifs in the distal ß1 tail regulate integrin function by associating with phosphtyrosine binding (PTB) proteins, such as talin and kindlin. Here, we define the roles of these two tyrosines in collecting-system development and delineate the structural determinants of the distal ß1 tail using nuclear magnetic resonance (NMR). Mice carrying alanine mutations have moderate renal collecting-system developmental abnormalities relative to ß1-null mice. Phenylalanine mutations did not affect renal collecting-system development but increased susceptibility to renal injury. NMR spectra in bicelles showed the distal ß1 tail is disordered and does not interact with the model membrane surface. Alanine or phenylalanine mutations did not alter ß1 structure or interactions between α and ß1 subunit transmembrane/cytoplasmic domains; however, they did decrease talin and kindlin binding. Thus, these studies highlight the fact that the functional roles of the NPXY motifs are organ dependent. Moreover, the ß1 cytoplasmic tail, in the context of the adjacent transmembrane domain in bicelles, is significantly different from the more ordered, membrane-associated ß3 integrin tail. Finally, tyrosine mutations of ß1 NPXY motifs induce phenotypes by disrupting their interactions with critical integrin binding proteins like talins and kindlins.

CD98 increases renal epithelial cell proliferation by activating MAPKs.

CD98 heavy chain (CD98hc) is a multifunctional transmembrane spanning scaffolding protein whose extracellular domain binds with light chain amino acid transporters (Lats) to form the heterodimeric amino acid transporters (HATs). It also interacts with ß1 and ß3 integrins by its transmembrane and cytoplasmic domains. This interaction is proposed to be the mechanism whereby CD98 mediates cell survival and growth via currently undefined signaling pathways. In this study, we determined whether the critical function of CD98-dependent amino acid transport also plays a role in cell proliferation and defined the signaling pathways that mediate CD98-dependent proliferation of murine renal inner medullary collecting duct (IMCD) cells. We demonstrate that downregulating CD98hc expression resulted in IMCD cell death. Utilizing overexpression studies of CD98hc mutants that either lacked a cytoplasmic tail or were unable to bind to Lats we showed that CD98 increases serum-dependent cell proliferation by a mechanism that requires the CD98hc cytoplasmic tail. We further demonstrated that CD98-dependent amino acid transport increased renal tubular epithelial cell proliferation by a mechanism that does not require the CD98hc cytoplasmic tail. Both these mechanisms of increased renal tubular epithelial cell proliferation are mediated by Erk and p38 MAPK signaling. Although increased amino transport markedly activated mTor signaling, this pathway did not alter cell proliferation. Thus, these studies demonstrate that in IMCD cells, the cytoplasmic and extracellular domains of CD98hc regulate cell proliferation by distinct mechanisms that are mediated by common MAPK signaling pathways.

Integrin-linked kinase regulates p38 MAPK-dependent cell cycle arrest in ureteric bud development.

The integrin-linked kinase (ILK), pinch and parvin ternary complex connects the cytoplasmic tails of beta1 integrins to the actin cytoskeleton. We recently showed that constitutive expression of ILK and alpha parvin in both the ureteric bud and the metanephric mesenchyme of the kidney is required for kidney development. In this study, we define the selective role of ILK in the ureteric bud of the mouse kidney in renal development by deleting it in the ureteric cell lineage before the onset of branching morphogenesis (E10.5). Although deleting ILK resulted in only a moderate decrease in branching, the mice died at 8 weeks of age from obstruction due to the unprecedented finding of intraluminal collecting duct cellular proliferation. ILK deletion in the ureteric bud resulted in the inability of collecting duct cells to undergo contact inhibition and to activate p38 mitogen-activated protein kinase (MAPK) in vivo and in vitro. p38 MAPK activation was not dependent on the kinase activity of ILK. Thus, we conclude that ILK plays a crucial role in activating p38 MAPK, which regulates cell cycle arrest of epithelial cells in renal tubulogenesis.

TGF-beta receptor deletion in the renal collecting system exacerbates fibrosis.

TGF-beta plays a key role in upregulating matrix production in injury-induced renal fibrosis, but how TGF-beta signaling in distinct compartments of the kidney, such as specific segments of the nephron, affects the response to injury is unknown. In this study, we determined the role of TGF-beta signaling both in development of the renal collecting system and in response to injury by selectively deleting the TGF-beta type II receptor in mice at the initiation of ureteric bud development. These mice developed normally but demonstrated a paradoxic increase in fibrosis associated with enhanced levels of active TGF-beta after unilateral ureteral obstruction. Consistent with this observation, TGF-beta type II receptor deletion in cultured collecting duct cells resulted in excessive integrin alphavbeta6-dependent TGF-beta activation that increased collagen synthesis in co-cultured renal interstitial fibroblasts. These results suggest that inhibiting TGF-beta receptor-mediated function in collecting ducts may exacerbate renal fibrosis by enhancing paracrine TGF-beta signaling between epithelial and interstitial cells.

Integrin alpha1beta1 regulates epidermal growth factor receptor activation by controlling peroxisome proliferator-activated receptor gamma-dependent caveolin-1 expression.

Integrin alpha1beta1 negatively regulates the generation of profibrotic reactive oxygen species (ROS) by inhibiting epidermal growth factor receptor (EGFR) activation; however, the mechanism by which it does this is unknown. In this study, we show that caveolin-1 (Cav-1), a scaffolding protein that binds integrins and controls growth factor receptor signaling, participates in integrin alpha1beta1-mediated EGFR activation. Integrin alpha1-null mesangial cells (MCs) have reduced Cav-1 levels, and reexpression of the integrin alpha1 subunit increases Cav-1 levels, decreases EGFR activation, and reduces ROS production. Downregulation of Cav-1 in wild-type MCs increases EGFR phosphorylation and ROS synthesis, while overexpression of Cav-1 in the integrin alpha1-null MCs decreases EGFR-mediated ROS production. We further show that integrin alpha1-null MCs have increased levels of activated extracellular signal-regulated kinase (ERK), which leads to reduced activation of peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor that positively regulates Cav-1 expression. Moreover, activation of PPARgamma or inhibition of ERK increases Cav-1 levels in the integrin alpha1-null MCs. Finally, we show that glomeruli of integrin alpha1-null mice have reduced levels of Cav-1 and activated PPARgamma but increased levels of phosphorylated EGFR both at baseline and following injury. Thus, integrin alpha1beta1 negatively regulates EGFR activation by positively controlling Cav-1 levels, and the ERK/PPARgamma axis plays a key role in regulating integrin alpha1beta1-dependent Cav-1 expression and consequent EGFR-mediated ROS production.

Modification of collagen IV by glucose or methylglyoxal alters distinct mesangial cell functions.

Diabetic nephropathy (DN) affects both glomerular cells and the extracellular matrix (ECM), yet the pathogenic mechanisms involving cell-matrix interactions are poorly understood. Glycation alters integrin-dependent cell-ECM interactions, and perturbation of these interactions results in severe renal pathology in diabetic animals. Here, we investigated how chemical modifications of the ECM by hyperglycemia and carbonyl stress, two major features of the diabetic milieu, affect mesangial cell functions. Incubation of collagen IV with pathophysiological levels of either the carbonyl compound methylglyoxal (MGO) or glucose resulted in modification of arginine or lysine residues, respectively. Mouse mesangial cells plated on MGO-modified collagen IV showed decreased adhesion and migration. Cells plated on glucose-modified collagen IV showed reduced proliferation and migration and increased collagen IV production. Inhibiting glucose-mediated oxidative modification of collagen IV lysine residues rescued the alterations in cell growth, migration, and collagen synthesis. We propose that diabetic ECM affects mesangial cell functions via two distinct mechanisms: modification of arginine residues by MGO inhibits cell adhesion, whereas oxidative modification of lysine residues by glucose inhibits cell proliferation and increases collagen IV production. These mechanisms may contribute to mesangial cell hypertrophy and matrix expansion in DN.

Functional analysis of the cytoplasmic domain of the integrin {alpha}1 subunit in endothelial cells.

Integrin alpha1beta1, the major collagen type IV receptor, is expressed by endothelial cells and plays a role in both physiologic and pathologic angiogenesis. Because the molecular mechanisms whereby this collagen IV receptor mediates endothelial cell functions are poorly understood, truncation and point mutants of the integrin alpha1 subunit cytoplasmic tail (amino acids 1137-1151) were generated and expressed into alpha1-null endothelial cells. We show that alpha1-null endothelial cells expressing the alpha1 subunit, which lacks the entire cytoplasmic tail (mutant alpha1-1136) or expresses all the amino acids up to the highly conserved GFFKR motif (mutant alpha1-1143), have a similar phenotype to parental alpha1-null cells. Pro(1144) and Leu(1145) were shown to be necessary for alpha1beta1-mediated endothelial cell proliferation; Lys(1146) for adhesion, migration, and tubulogenesis and Lys(1147) for tubulogenesis. Integrin alpha1beta1-dependent endothelial cell proliferation is primarily mediated by ERK activation, whereas migration and tubulogenesis require both p38 MAPK and PI3K/Akt activation. Thus, distinct amino acids distal to the GFFKR motif of the alpha1 integrin cytoplasmic tail mediate activation of selective downstream signaling pathways and specific endothelial cell functions.

Apoptosis of the thick ascending limb results in acute kidney injury.

Ischemia- or toxin-induced acute kidney injury is generally thought to affect the cells of the proximal tubule, but it has been difficult to define the involvement of other tubular segments because of the widespread damage caused by ischemia/reperfusion or toxin-induced injury in experimental models. For evaluation of whether thick ascending limb (TAL)-specific epithelial injury results in acute kidney injury, a novel transgenic mouse model that expresses the herpes simplex virus 1 thymidine kinase gene under the direction of the TAL-specific Tamm-Horsfall protein promoter was generated. After administration of gancyclovir, these mice demonstrated apoptosis only in TAL cells, with little evidence of neutrophil infiltration. Compared with control mice, blood urea nitrogen and creatinine levels were at least five-fold higher in the transgenic mice, which also developed oliguria and impaired urinary concentrating ability. These findings suggest that acute injury targeted only to the TAL is sufficient to cause severe acute kidney injury in mice with features similar to those observed in humans.

H-Ras, R-Ras, and TC21 differentially regulate ureteric bud cell branching morphogenesis.

The collecting system of the kidney, derived from the ureteric bud (UB), undergoes repetitive bifid branching events during early development followed by a phase of tubular growth and elongation. Although members of the Ras GTPase family control cell growth, differentiation, proliferation, and migration, their role in development of the collecting system of the kidney is unexplored. In this study, we demonstrate that members of the R-Ras family of proteins, R-Ras and TC21, are expressed in the murine collecting system at E13.5, whereas H-Ras is only detected at day E17.5. Using murine UB cells expressing activated H-Ras, R-Ras, and TC21, we demonstrate that R-Ras-expressing cells show increased branching morphogenesis and cell growth, TC21-expressing cells branch excessively but lose their ability to migrate, whereas H-Ras-expressing cells migrated the most and formed long unbranched tubules. These differences in branching morphogenesis are mediated by differential regulation/activation of the Rho family of GTPases and mitogen-activated protein kinases. Because most branching of the UB occurs early in development, it is conceivable that R-Ras and TC-21 play a role in facilitating branching and growth in early UB development, whereas H-Ras might favor cell migration and elongation of tubules, events that occur later in development.

CD98 modulates integrin beta1 function in polarized epithelial cells.

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 beta1 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 beta1 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 transmembrane domain of CD98 abrogated this effect. CD98 and mutants that interacted with beta1 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 beta1, 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 beta1 integrins and can alter diverse cellular functions such as amino acid transport as well as cell adhesion, migration and branching morphogenesis.