Abstract Development of the metanephric kidney crucially depends on proper interactions between cells and the surrounding extracellular matrix. For example, we showed previously that in the absence of alpha8beta1 integrin, invasion by the ureteric bud into the metanephric mesenchyme is inhibited, resulting in renal agenesis. Here we present genetic evidence that the extracellular matrix protein nephronectin is an essential ligand that engages alpha8beta1 integrin during early kidney development. We show that embryos lacking a functional nephronectin gene frequently display kidney agenesis or hypoplasia, which can be traced to a delay in the invasion of the metanephric mesenchyme by the ureteric bud at an early stage of kidney development. Significantly, we detected no defects in extracellular matrix organization in the nascent kidneys of the nephronectin mutants. Instead, we found that Gdnf expression was dramatically reduced in both nephronectin- and alpha8beta1 integrin-null mutants specifically in the metanephric mesenchyme at the time of ureteric bud invasion. We show that this reduction is sufficient to explain the agenesis and hypoplasia observed in both mutants. Interestingly, the reduction in Gdnf expression is transient, and its resumption presumably enables the nephronectin-deficient ureteric buds to invade the metanephric mesenchyme and begin branching. Our results thus place nephronectin and alpha8beta1 integrin in a pathway that regulates Gdnf expression and is essential for kidney development.

Idiopathic pulmonary fibrosis (IPF), or scarring of the lungs, is an incurable progressive disease that has a 5-year mortality rate of 80%. The cytokine, Transforming Growth Factor Beta (TGF-β) is considered a central mediator in the pathogenesis of this fibrotic disorder. Virtually all cells secrete TGF-β as a latent complex that must be activated for the cytokine to exert its biological functions. We previously discovered that the integrin αvβ6 functions to activate TGF-β. However, the signals and mechanisms that regulate this process in epithelial cells are unclear. We discovered that the phospholipid, Sphingosine 1-Phosphate (S1P), induces αvβ6-mediated TGF-β activation by increasing Rho Kinase (ROCK)-mediated actomyosin contraction. Furthermore, we demonstrate that this process requires intracellular mechanical forces applied to the integrin and the generation of cellular tension. Interestingly, lung epithelial cells appear to exert force on latent TGF-β using sub-cortical actin rather than the actin stress fibers utilized by fibroblasts and other traditionally "contractile" cells. In addition, we show that aerosol delivery of small molecule ROCK inhibitors to wild type mice is an effective method of blocking TGF-β signaling in vivo. Finally, we report six candidate biomarkers of inhibiting αvβ6-mediated TGF-β activation and demonstrate the potential of utilizing alveolar macrophages as a biosensor for monitoring this pathway. These findings enhance our understanding of the mechanisms that regulate αvβ6-mediated TGF-β activation, and they demonstrate the potential of blocking this pathway in vivo and monitoring the effectiveness of blockade, defining a potential strategy for developing improved treatments for pulmonary fibrosis.

Secretion and assembly of the extracellular matrix protein fibrobnectin (FN) regulates a plethora of normal cell and tissue functions, including development, cell growth, differentiation and cell migration, and is dysregulated in diseases such as fibrosis, diabetes, and cancer. My thesis work revealed that mislocalized scaffolding by the plasma membrane Na-H exchanger NHE1 dominantly suppresses FN expression, secretion, and assembly, and inhibits cleavage of latent to active TGF-beta;. By catalyzing an electroneutral exchange of extracellular Na+ and intracellular H+, NHE1 has recognized functions in intracellular pH and osmotic homeostasis. Recent evidence indicates NHE1 also functions as a plasma membrane anchor for the actin cytoskeleton by binding directly to ERM (ezrin, moesin, radixin) proteins and a PI(4,5)P2-binding scaffold, which are independent of ion translocation by NHE1. Endogenous NHE1 localizes to the distal margin of membrane protrusions in lamellipodia, however a mutantNHE1-KRA2, lacking binding sites for PI(4,5)P2 and the ERM proteins ezrin, radixin and moesin, is mislocalized and distributed uniformly along the plasma membrane. Fibroblasts expressing NHE1-KRA2 but not wild-type NHE1 have impaired FN expression, secretion, and assembly, and reduced active but not latent TGF-beta;. We found that FN production is not regulated by changes in intracellular pH, nor is it attenuated in NHE1-deficient cells, indicating FN expression and TGF-beta; activation are independent of NHE1 activity. However, treating NHE1-KRA2 fibroblasts with recombinant TGF-beta; restores FN secretion and assembly. These data suggest that scaffolding by NHE1-KRA2 sequesters and mislocalizes signals necessary for FN synthesis and TGF-beta; activation. Although the precise signals sequestered by NHE1-KRA2 remain unknown, these findings suggest that NHE1-KRA2 could be a valuable tool for obtaining a mechanistic understanding of how FN production and TGF-beta; activation are regulated, and more speculatively for therapeutic control of increased FN production in pathological conditions such as fibrosis and inflammation.

Integrins are heterodimeric membrane proteins that mediate cell adhesion, migration and proliferation in a number of physiologic processes, including angiogenesis. The integrin α9β1 binds to a number of ligands, including the angiogenic growth factor, VEGF-A, and mediates cell adhesion and proliferation on VEGF-A. Blockade of α9β1 inhibits angiogenesis in the chick and quail chorioallantoic membranes, but the specific cell type(s) in which α9β1 is expressed and the mechanisms by which integrin α9β1 mediates angiogenesis have not been described. The complete knockout of α9 results in lethal chylothoraces between postnatal day 6 and 10, but has no reported deficits of vasculogenesis or developmental angiogenesis. To study the role of α9β1 during pathologic angiogenesis, we generated a mouse in which α9 can be temporally deleted in all tissues following administration of Doxycyline. Administration of Doxycycline after the development of the lymphatics allowed us to define a role for α9β1 during pathologic angiogenesis in adult mice. Deletion of α9β1 inhibited angiogenesis in vivo induced by VEGF-A, but not by bFGF. We determined that α9β1 is expressed on cells which also express the pericyte markers NG2 and PDGFRβ, and not on endothelial cells expressing PECAM. Pericyte-specific deletion of the integrin using an α-Smooth Muscle Actin promoter was sufficient to inhibit angiogenesis in response to VEGF-A due to a failure of pericyte recruitment before endothelial cell recruitment. Taken together, these results indicate that pathologic angiogenesis in response to VEGF-A requires pericyte recruitment before endothelial cell recruitment, and that this pericyte recruitment requires expression of integrin α9β1 on pericytes.