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Introduction While PTEN displays phosphatase activity for both protein and lipid substrates (1), accumulating evidence reveals that its lipid phosphatase activity, which dephosphorylates the 3'-position phosphate from the inositol ring [...], Inactivation of phosphatase and tensin homolog (PTEN) is a critical step during tumorigenesis, and PTEN inactivation by genetic and epigenetic means has been well studied. There is also evidence suggesting that PTEN negative regulators (PTEN-NRs) have a role in PTEN inactivation during tumorigenesis, but their identity has remained elusive. Here we have identified shank-interacting protein-like 1 (SIPL1) as a PTEN-NR in human tumor cell lines and human primary cervical cancer cells. Ectopic SIPL1 expression protected human U87 glioma cells from PTEN-mediated growth inhibition and promoted the formation of HeLa cell-derived xenograft tumors in immunocompromised mice. Conversely, siRNA-mediated knockdown of SIPL1 expression inhibited the growth of both HeLa cells and DU145 human prostate carcinoma cells in vitro and in vivo in a xenograft tumor model. These inhibitions were reversed by concomitant knockdown of PTEN, demonstrating that SIPL1 affects tumorigenesis via inhibition of PTEN function. Mechanistically, SIPL1 was found to interact with PTEN through its ubiquitin-like domain (UBL), inhibiting the phosphatidylinositol 3,4,5-trisphosphate (PIP3) phosphatase activity of PTEN. Furthermore, SIPL1 expression correlated with loss of PTEN function in PTEN-positive human primary cervical cancer tissue. Taken together, these observations indicate that SIPL1 is a PTEN-NR and that it facilitates tumorigenesis, at least in part, through its PTEN inhibitory function.

Glomerular sclerosis of diverse etiologies is characterized by mesangial matrix accumulation, with transforming growth factor-J3 (TGF[beta]) an important pathogenic factor. The GTPase RhoA mediates TGF[beta]-induced matrix accumulation in some settings. Here we study the role of the membrane microdomain caveolae in TGF[beta]-induced RhoA activation and fibronectin up-regulation in mesangial cells (MC). In primary rat MC, TGF[beta]1 time dependently increased RhoA and downstream Rho kinase activation. Rho pathway inhibition blocked TGF[beta]1-induced upregulation of fibronectin transcript and protein. TGF[beta]1-induced RhoA activation was prevented by disrupting caveolae with cholesterol depletion and rescued by cholesterol repletion. Compared with wild types, RhoA/ Rho kinase activation was absent in MC lacking caveolae. Re-expression of caveolin-1 (and caveolae) restored these responses. Phosphorylation of caveolin-I on Y14, effected by Src kinases, has been implicated in signaling responses. Overexpression of nonphosphorylatable caveolin-1 Y14A prevented TGF[beta]1-induced RhoA activation. TGF[beta]1 also activated Src, and its inhibition blocked RhoA activation. Furthermore, TGF[beta]1 led to association of RhoA and caveolin-1. This was prevented by Src or TGF[beta] receptor I inhibition, and by caveolin-1 Y14A overexpression. Last, fibronectin upregulation by TGF[beta]1 was blocked by Src inhibition, not seen in caveolin-1 knockout MC, and restored by caveolin-1 reexpression in the latter. TGF[beta]1-induced collagen I accumulation also required caveolae. TGF[beta]1-mediated Smad2/3 activation, however, did not require caveolae. We conclude that RhoA/Rho kinase mediates TGF[beta]-induced fibronectin upregulation. This requires caveolae and caveolin-1 interaction with RhoA. Interference with caveolin/caveolae or RhoA signaling thus represents a potential target for the treatment of fibrotic renal disease. extracellular matrix; glomerular sclerosis; caveolin-1

Background. Fischer 344 (F344) rats display focal and diffuse glomerulosclerosis with aging postulated to result from loss of normal mesangial cell intrinsic function, e.g., vasoactive hormone signaling, or preservation of normal responsiveness to extrinsic growth factors. Methods. ln 3-, 17-, and 24-month-old F344 male rats, glomerular structure; measured by PC-based morphometry, and function were compared. Immunoperoxidase staining of glomerular proliferating cell nuclear antigen (PCNA) detected cellular proliferation. Primary cultured mesangial cells from the 3 age groups were studied in parallel. Calcium ([Ca.sup.2+]) signaling, measured by Fura-2 fluorescence, contraction to vasopressin (AVP) 1 [mu]M, measured by videomicroscopy, and proliferative response to platelet-derived growth factor-[beta][beta] (PDGF) were compared. Results. Proteinuria was 13 [+ or -] 4, 38 [+ or -] 17, and 110 [+ or -]35 mg/24 hours at 3, 17, and 24 months, respectively (n = 5, mean [+ or -] SE, p < .01, 3 vs 24 months), with no change in 24-hour creatinine clearances. Glomerular volumes (n = 200/ group) for 3, 17, and 24 months, respectively, were .30 [+ or -] .01, .60 [+ or -] .02, .74 [+ or -] .02 x [10.sup.6] [mu][m.sup.3] (p < .001, 3 months vs 17 months, and 17 vs 24 months). Glomerular basement membrane (GBM) widths and fractional mesangial volumes increased significantly with aging. Glomerular cell PCNA staining remained positive at 24 months. Cultured mesangial cell [Ca.sup.2+] signaling and contraction to AVP were unchanged with aging. Proliferation to PDGF, which was partially inhibited with verapamil, was similar at 3 and 24 months. Conclusions. In the Fischer 344 rat, mesangial cell [Ca.sup.2+] signaling, contraction, and proliferation responsiveness are unchanged with aging. Continued growth is associated with the glomerulosclerosis of aging.

Angiotensin II activates the GFAT promoter in mesangial cells. Am J Physiol Renal Physiol 281: F151-F162, 2001.--Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme for glucose entry into the hexosamine pathway, is transcriptionally regulated. Immunohistochemical studies of human kidney biopsies demonstrate increased GFAT expression in diabetic glomeruli, but the mechanism responsible for this overexpression is unknown. Given the role of ANG II in diabetic kidney disease, we chose to study the effect of ANG II on GFAT promoter activity in mesangial cells (MC). Exposure of MC to ANG II ([10.sup.-7] M) increased GFAT promoter activity (2.5-fold), mRNA (3-fold), and protein (1.6-fold). ANG II-mediated GFAT promoter activation was inhibited by the ANG II type I receptor antagonist candesartan ([10.sup.-8] M) but was unaffected by the ANG II type II receptor antagonist PD-123319 ([10.sup.-8] M). The intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid ([10.sup.-6] M), protein kinase C (PKC) inhibitors bisindoylmaleimide-4 ([10.sup.-6] M) and calphostin C ([10.sup.-7] M), protein tyrosine kinase (PTK) inhibitor genistein ([10.sup.-4] M), Src family kinase inhibitor PP2 (2.5 x [10.sup.-7] M), p42/44 mitogen-activated protein kinase (MAPK) inhibitor PD-98059 ([10.sup.-5] M), and the epidermal growth factor (EGF) inhibitor AG-1478 all attenuated GFAT promoter activation by ANG II. We conclude that the GFAT promoter is activated by ANG II via the [AT.sub.1] receptor. Promoter activation is calcium dependent and PKC dependent but also involves PTK signaling pathways including Src, the EGF receptor, and p42/44 MAPK. angiotensin II; signaling; glomerulus; mesangial cells; glutamine:fructose-6-phosphate amidotransferase