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Kidney function decreases with age and may soon limit millions of lives as the proportion of the population over 70 years of age increases. Glycogen synthase kinase 3p (GSK3[beta]) is involved with metabolism and may have a role in kidney senescence, positioning it as a target for complications from chronic kidney disease. However, different studies suggest GSK3 has contrasting effects. In this issue of the JCI, Fang et al. explored the function of GSK3[beta] and the interplay with lithium using human tissue and mouse models. Notably, GSK3[beta] was overexpressed and activated in aging mice, and depleting GSK3[beta] reduced senescence and glomerular aging. In this Commentary, we explore the similarities and differences between Fang et al. and previous findings by Hurcombe et al. These findings should prompt further study of lithium and other GSK3[beta] inhibitors as a means of extending glomerular function in individuals with chronic kidney disease., GSK3 affects diverse processes The population of the world is aging and it is estimated that, over the next half a century, as much as 30% of the population may [...]

The integrin alpha3beta1 mediates cellular adhesion to the matrix ligand laminin-5. A second integrin ligand, the urokinase receptor (uPAR), associates with alpha3beta1 via a surface loop within the alpha3 beta-propeller (residues 242-246) but outside the laminin binding region, suggesting that uPAR-integrin interactions could signal differently from matrix engagement. To explore this, alpha3-/- epithelial cells were reconstituted with wild-type (wt) alpha3 or alpha3 with Ala mutations within the uPAR-interacting loop (H245A or R244A). Wt or mutant-bearing cells showed comparable expression and adhesion to laminin-5. Cells expressing wt alpha3 and uPAR dissociated in culture, with increased Src activity, up-regulation of SLUG, and down-regulation of E-cadherin and gamma-catenin. Src kinase inhibition or expression of Src 1-251 restored the epithelial phenotype. The H245A and R244A mutants were unaffected by coexpression of uPAR. We conclude that alpha3beta1 regulates both cell-cell contact and matrix adhesion, but through distinct protein interaction sites within its beta-propeller. These studies reveal an integrin- and Src-dependent pathway for SLUG expression and mesenchymal transition.

Embryonic retinal neurons lose the ability to extend neurites on laminin-1 (LN-1) with increasing developmental age yet still do so on other laminin isoforms. However, after treatment of LN-1 with antibodies to "short-arm" regions or removal of the short arms proteolytically, LN-1 supports attachment and extension of neurites even by late embryonic retinal neurons. We have mapped a domain for antibody-mediated "activation" of LN-1 to the N-terminal end of the alpha1 chain. Furthermore, we show that the primary receptors used in the retinal neuron response to "activated" LN-1 are integrins alpha3 beta1 and alpha6 beta1; these are the same receptors used by these neurons for outgrowth on other LN isoforms. Interestingly, alpha3 beta1 is preferentially involved in neurite outgrowth, whereas alpha6beta1 preferentially mediates attachment and spreading. However, in cultures from alpha3 integrin-deficient mice, alpha6 beta1 mediates retinal ganglion cell neurite outgrowth and compensates for the absence of alpha3 beta1. Finally, we show that key features of the retinal neuron response to LN-1 also characterize neurons of the hippocampus, thalamus, and cerebral cortex; these include poor response to untreated LN-1, extensive neurite outgrowth on antibody-activated LN-1 or on fragment E8, and dependence of this response on integrin alpha6 beta1 and at least one other long arm-binding beta1 integrin. These data suggest that regulation of LN-1 function via the process of activation could have important consequences for axonal regeneration. Curiously, the data also imply that the mechanism of laminin activation involves enhanced function at sites that cannot be considered cryptic.

The discovery of microRNAs (miRNAs) as novel regulators of gene expression has led to a marked change in how gene regulation is viewed, with important implications for development and disease. MiRNAs are endogenous, small, noncoding RNAs that largely repress their target mRNAs post-transcriptionally. The regulation of gene expression by miRNAs represents an evolutionarily conserved mechanism that is broadly applicable to most biological processes. Recent studies have begun to define the role of miRNAs in different cell lineages during kidney development, and to implicate specific miRNAs in developmental and pathophysiological processes in the kidney. This review will focus on novel insights into the role(s) of miRNAs in kidney development, and discuss the implications for pediatric renal disease. Keywords microRNAs * Kidney development * Dicer, Introduction The initial discovery that a small regulatory RNA could specifically silence the function of a gene in the nematode, Caenorhabditis elegans [1], has subsequently led to revolutionary changes in [...]

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that is caused by mutations at two loci, polycystin 1 (PKD1) and polycystin 2 (PKD2). It is characterized by the formation of multiple cysts in the kidneys that can lead to chronic renal failure. Previous studies have suggested a role for hyperactivation of mammalian target of rapamycin (mTOR) in cystogenesis, but the etiology of mTOR hyperactivation has not been fully elucidated. In this report we have shown that mTOR is hyperactivated in Pkd1-null mouse cells due to failure of the HGF receptor c-Met to be properly ubiquitinated and subsequently degraded after stimulation by HGF. In Pkd1-null cells, Casitas B-lineage lymphoma (c-Cbl), an E3-ubiquitin ligase for c-Met, was sequestered in the Golgi apparatus with [α.sub.3][β.sub.1] integrin, resulting in the inability to ubiquitinate c-Met. Treatment of mouse Pkd1-null cystic kidneys in organ culture with a c-Met pharmacological inhibitor resulted in inhibition of mTOR activity and blocked cystogenesis in this mouse model of ADPKD. We therefore suggest that blockade of c-Met is a potential novel therapeutic approach to the treatment of ADPKD., Introduction Polycystic kidney disease (PKD) is one of the most common inherited disorders that result in severe and debilitating disease. There are two predisposing loci, PKD1 and PKD2, residing on [...]

Cortical GABAergic interneurons, most of which originate in the ganglionic eminences, take distinct tangential migratory trajectories into the developing cerebral cortex. However, the ligand-receptor systems that modulate the tangential migration of distinct groups of interneurons into the emerging cerebral wall remain unclear. Here, we show that netrin-1, a diffusible guidance cue expressed along the migratory routes traversed by GABAergic interneurons, interacts with [alpha]3[beta]1 integrin to promote interneuronal migration. In vivo analysis of interneuron-specific [alpha]3[beta]1 integrin, netrin-1-deficient mice ([alpha][3.sup.lox/-]DIx5/6-CIE, netrin-[1.sup.-/-]) reveals specific deficits in the patterns of interneuronal migration along the top of the developing cortical plate, resulting in aberrant interneuronal positioning throughout the cerebral cortex and hippocampus of conditional [alpha][3.sup.lox/-]DIx5/6-CIE, netrin-[1.sup.-/-] mice. These results indicate that specific guidance mechanisms, such as netrin-1-[alpha]3[beta]1 integrin interactions, modulate distinct routes of interneuronal migration and the consequent positioning of groups of cortical interneurons in the developing cerebral cortex.

Pulmonary fibrosis, in particular idiopathic pulmonary fibrosis (IPF), results from aberrant wound healing and scarification. One population of fibroblasts involved in the fibrotic process is thought to originate from lung epithelial cells via epithelial-mesenchymal transition (EMT). Indeed, alveolar epithelial cells (AECs) undergo EMT in vivo during experimental fibrosis and ex vivo in response to TGF-[beta]1. As the ECM critically regulates AEC responses to TGF-[beta]1, we explored the role of the prominent epithelial integrin [alpha]3[beta]1 in experimental fibrosis by generating mice with lung epithelial cell--specific loss of [alpha]3 integrin expression. These mice had a normal acute response to bleomycin injury, but they exhibited markedly decreased accumulation of lung myofibroblasts and type I collagen and did not progress to fibrosis. Signaling through [beta]-catenin has been implicated in EMT; we found that in primary AECs, [alpha]3 integrin was required for [beta]-catenin phosphorylation at tyrosine residue 654 (Y654), formation of the pY654-[beta]-catenin/pSmad2 complex, and initiation of EMT, both in vitro and in vivo during the fibrotic phase following bleomycin injury. Finally, analysis of lung tissue from IPF patients revealed the presence of pY654-[beta]-catenin/pSmad2 complexes and showed accumulation of pY654-[beta]-catenin in myofibroblasts. These findings demonstrate epithelial integrin--dependent profibrotic crosstalk between [beta]-catenin and Smad signaling and support the hypothesis that EMT is an important contributor to pathologic fibrosis., Introduction Progressive fibrosis of the lung, especially that of idiopathic pulmonary fibrosis (IPF), is thought to be a consequence of aberrant wound healing resulting in progressive scarification (1), (2). Fibroblast [...]

Differential expression of collagen- and laminin-binding integrins mediates ureteric bud and inner medullary collecting duct cell tubulogenesis. Am J Physiol Renal Physiol 287:F602-F611, 2004. First published June 8, 2004; 10.1152/ajprenal.00015.2004.--Inner medullary collecting ducts (IMCD) are terminally differentiated structures derived from the ureteric bud (UB). UB development is mediated by changes in the temporal and spatial expression of integrins and their respective ligands. We demonstrate both in vivo and in vitro that the UB expresses predominantly laminin receptors ([[alpha].sub.3][[beta].sub.1]-, [[alpha].sub63][[beta].sub.1]-, and [[alpha].sub.6][[beta].sub.4]-integrins), whereas the IMCD expresses both collagen ([[alpha].sub.1][[beta].sub.1]-and [[alpha].sub.2][[beta].sub.1]-integrins) and laminin receptors. Cells derived from the IMCD, but not the UB. undergo tabulogenesis in collagen-I (CI) gels in an [[alpha].sub.1][[beta].sub.1]- and [[alpha].sub.2][[beta].sub.1]-dependent manner. UB cells transfected with the [[alpha].sub.2]-integrin subunit undergo tubulogenesis in CI, suggesting that collagen receptors are required for branching morphogenesis in CI. In contrast, both UB and IMCD cells undergo tubulogenesis in CI/ Matrigel gels. UB cells primarily utilize [[alpha].sub.3][[beta].sub.1]- and [[alpha].sub.6]-integrins, whereas IMCD cells mainly employ [[alpha].sub.1][[beta].sub.1] for this process. These results demonstrate a switch in integrin expression from primarily laminin receptors in the early UB to both collagen and laminin receptors in the mature IMCD, which has functional consequences for branching morphogenesis in three-dimensional cell culture models. This suggests that temporal and spatial changes in integrin expression could help organize the pattern of branching morphogenesis of the developing collecting system in vivo. kidney development; branching morphogenesis; three-dimensional culture; basement membranes

The Wilms' tumor suppressor gene, Wt1, encodes a transcription factor critical for development of the urogenital system. To identify lineages within the developing urogenital system that have a cell-autonomous requirement for Wt1, chimeric mice were generated from, Wt1-null ES cells. Males with large contributions of Wt1-/- cells showed hypoplastic and dysgenic testes, with seminiferous tubules lacking spermatogonia. Wt1-null cells contributed poorly to both somatic and germ cell lineages within the developing gonad, suggesting an unexpected role for Wt1 in germ cell development in addition to a role in the development of the somatic lineages of the gonad. Wt1 expression was detected in embryonic germ cells beginning at embryonic day 11.5 after migrating primordial germ cells (PGCs) have entered the gonad. Germ cells isolated flora Wt1-null embryos showed impaired growth in culture, further demonstrating a role for Wt1 in germ cell proliferation or survival. Therefore, Wt1 plays important, and in some cases previously unrecognized, roles in multiple lineages during urogenital development. Keywords: Wt1; Germ cells; Gonadal development

The [beta]1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for [alpha]3[beta]1 integrin in association with the tetraspanin CD151 as a component of a cell-cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin-tetraspanin complex affects epithelial cell-cell adhesion at the level of gene expression both by regulating expression of PTP[mu] and by organizing a multimolecular complex containing PKC[beta]II, RACK1, PTP[mu], [beta]-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.

Antibody inhibition and [alpha]6[beta]1 ligand binding experiments indicate that the egg integrin [alpha]6[beta]1 functions as a receptor for sperm during gamete fusion; yet, eggs null for the [alpha]6 integrin exhibit normal fertilization. Alternative integrins may be involved in sperm-egg binding and fusion and could compensate for the absence of [alpha]6[beta]1. Various [beta]1 integrins and [alpha]v integrins are present on mouse eggs. Some of these integrins are also reported to be receptors for ADAMs, which are expressed on sperm. Using [alpha]3 integrin null eggs, we found that the [alpha]3[beta]1 integrin was not essential for sperm-egg binding and fusion. Oocyte-specific, [beta]1 integrin conditional knockout mice allowed us to obtain mature eggs lacking all [beta]1 integrins. We found that the [beta]1 integrin null eggs were fully functional in fertilization both in vivo and in vitro. Furthermore, neither anti-mouse [beta]3 integrin function-blocking monoclonal antibody (mAb) nor [alpha]v integrin function-blocking mAb inhibited sperm binding to or fusion with [beta]1 integrin null eggs. Thus, function of [beta]3 or [alpha]v integrins does not seem to be involved in compensating for the absence of [beta]1 integrins. These results indicate that none of the integrins known to be present on mouse eggs or to be ADAM receptors are essential for sperm-egg binding/fusion, and thus, egg integrins may not play the role in gamete fusion previously attributed to them. Keywords: Integrin; Sperm-egg binding; Membrane fusion; Null mutation; ADAMs

Author(s): Ohad S. Birk [1]; Delane E. Casiano [1]; Christopher A. Wassif [2]; Tiziana Cogliati [3]; Liping Zhao [4]; Yangu Zhao [1]; Alexander Grinberg [1]; SingPing Huang [1]; Jordan A. [...]

Mouse embryos deficient in fibronectin (FN-null) die at E8.5 with mesodermal defects. Eight integrin heterodimers (alpha)3(beta)1, (alpha)4(beta)1, (alpha)5(beta)1, (alpha)8(beta)1, (alpha)(nu)(beta)1, (alpha)nu)(beta)3, (alpha(nu)(beta)6, and (alpha)IIb(beta)3 can bind to FN. However, embryos deficient in each of these integrins exhibit less severe defects than do FN-null embryos, raising questions as to which integrin(s) are the key FN receptors for these early FN-dependent processes, (alpha)5(beta)1 is believed to be the key receptor and (alpha)5-null embryos display mesodermal defects similar to, although less severe than, those of FN-null. Here we report that the (alpha)5-null mutation exhibits a more severe phenotype on a 129Sv (129) than on a C57BL/6 (B6) background, as does the FN-null mutation. While (alpha)5-null/B6 embryos develop normal headfolds, (alpha)5-null/129 embryos have headfold defects similar to those of FN-null. The differences between FN-null and (alpha)5-null embryos, however, cannot be attributed to genetic background. FN-null embryos never form somites, whereas in (alpha)5-null/129 embryos the somites do condense but fail to epithelialize. Second, we examined double mutants carrying all possible pairwise combinations of null mutations in (alpha)3, (alpha)4, and (alpha)5 integrin genes. There was no evidence for any synergy between paired mutations, suggesting that these integrin genes do not have overlapping functions during early embryonic development. Finally, we examined double-mutant embryos deficient in both (alpha)5 and (alpha)(nu) integrin genes. These double-mutant embryos have an amniotic defect similar to that of FN-null embryos, but die even earlier with a defect in gastrulation. These studies thus revealed a gradation in the severity of defects in the mutations (alpha)5(super -/-); (alpha)(nu)(super -/-)>FN(super -/-)(129)>FN(super -/-)(B6)>(alpha 5(super -/-) (129)>(alpha)5(super -/-)(B6), and in each step in this series there is a certain degree of phenotypic overlap, suggesting that the defects arising from these mutations may result from disruptions of the same embryonic process. Key Words: integrins; fibronectin; somitogenesis; gastrulation.

Alpha3beta1 integrin-deficient neonatal mice were found to develop micro-blisters at the epidermal-dermal junction. The effects of alpha2beta1 deficiency on other keratinocyte integrins have been studied and it was found that the absence of alpha3beta1 leads to a rise in stress fiber formation in keratinocytes. Higher concentrations of actin-associated proteins were also observed.

A mutant mice lacking the alpha-3 integrin subunit was used to study the alpha-3 beta-1 functions in the epidermis. Results suggest that alpha-3 beta-1 is involved in establishing and/or maintaining basement membrane integrity by regulating membrane matrix organization. An overlap of functions in keratinocytes was also proposed for alpha-3 beta-1 and alpha-6 beta-4 as evidenced by the ability of both chains to support initial cell attachment to laminin-5, except that alpha-3 beta-1 is also essential for cell spreading.

Integrin [Alpha]1 is a receptor for laminin and collagen which is expressed widely and dynamically in embryogenesis and has been implicated in various developmental processes including establishment of the placenta and formation of the central and peripheral nervous system. In the adult it is the sole collagen receptor in smooth muscle and liver and is thought to be important for the stability of these tissues. We have generated a null allele of the [Alpha]1 gene in the germline of mice by homologous recombination in embryonic stem cells. Mice homozygous for the mutation are viable and fertile and have no overt phenotype, demonstrating that the molecule is not required for development. Embryonic fibroblasts derived from mutant animals are unable to spread on or migrate into substrata of collagen IV and are deficient in spreading on and migrating into laminin. Further in vitro analysis of cell spreading and migration suggests that [Alpha]1/[Beta]1 is not required for binding to collagen I and implicates a third receptor, possibly integrin [Alpha]3/[Beta]1, in collagen I binding.