Following ischemic injury, surviving kidney proximal tubule cells dedifferentiate and proliferate to regenerate the tubules. After reaching critical cell densities, growth arrest and differentiation follow. The signaling mechanisms that govern this process are important and critically determine structural and functional recovery from ischemic injury. Our research suggests that the phosphatidylinositol 3-kinase (PI3K) signaling pathway may play an important role in proximal tubule cell proliferation and regeneration. We observed that the suppression of PI3K signaling associated with growth arrest of cultured proximal tubule cells is accompanied by an increase in the cellular content of the tumor suppressor protein PTEN. This suggests that PTEN might regulate proximal tubule proliferation through the lipid phosphatase actions of PTEN that would decrease the levels of D-3 phosphorylated inositol phospholipids made by PI3K. Our preliminary studies suggest that the increase of PTEN associated with growth inhibition is caused by at least two feedback mechanisms of autoregulatory signaling: one is driven by Akt, a downstream member of the PI3K pathway and the other by intercellular contact as cell numbers increase. We plan to use cell culture and in vivo models to unravel molecular mechanisms of proximal tubule cell growth regulation. Our research will use 5 approaches to study how PTEN regulates growth, and how PTEN is regulated. (1) We will employ PTEN vectors and siRNA methods to increase or decrease PTEN protein and activity. (2) We will use a constitutively expressed Akt construct that permits rapid and selective induction of Akt kinase activity to study how Akt activation leads to PTEN upregulation. (3) We will disrupt and restore cell junctions in cultured proximal tubule cells to study how cell contact leads to regulation of PTEN. Regulation of PTEN will be studied at the levels of transcription, mRNA stability and protein turnover. (4) We will use genomics and proteomics approaches to identify novel proteins involved in PTEN regulation. (5) We will study regulation of PI3K signaling by PTEN in vivo in rats and mice with ischemic acute renal failure using biochemical methods, immuno histochemistry and in situ hybridization. We will employ PTEN antisense oligonucleotides to suppress kidney PTEN in vivo to study how this intervention affects the course of proximal tubule regeneration and recovery.
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