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Hyperglycemia induces a wide array of signaling pathways in the kidney that lead to hypertrophy and matrix expansion, eventually culminating in progressive kidney failure. High glucoseinduced reduction of the tumor suppressor protein phosphatase and tensin homolog deleted in chromosome 10 (PTEN) contributes to renal cell hypertrophy and matrix expansion. We identified microRNA-21 (miR-21) as the molecular link between high glucose and PTEN suppression. Renal cortices from OVE26 type 1 diabetic mice showed significantly elevated levels of miR-21 associated with reduced PTEN and increased fibronectin content. In renal mesangial cells, high glucose increased the expression of miR-21, which targeted the 3-UTR of PTEN mRNA to inhibit PTEN protein expression. Overexpression of miR-21 mimicked the action of high glucose, which included a reduction in PTEN expression and a concomitant increase in Akt phosphorylation. In contrast, expression of miR-21 Sponge, to inhibit endogenous miR-21, prevented down-regulation of PTEN and phosphorylation of Akt induced by high glucose. Interestingly, high glucose-stimulated miR-21 inactivated PRAS40, a negative regulator of TORC1. Finally, miR-21 enhanced high glucose-induced TORC1 activity, resulting in renal cell hypertrophy and fibronectin expression. Thus, our results identify a previously unrecognized function of miR-21 that is the reciprocal regulation of PTEN levels and Akt/TORC1 activity that mediate critical pathologic features of diabetic kidney disease.

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Sustained activation of Akt kinase acts as a focal regulator to increase cell growth and survival, which cause tumorigenesis including breast cancer. Statins, potent inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, display anticancer activity. The molecular mechanisms by which statins block cancer cell growth are poorly understood. We demonstrate that in the tumors derived from MDA-MB-231 human breast cancer cell xenografts, simvastatin significantly inhibited phosphorylation of Akt with concomitant attenuation of expression of the anti-apoptotic protein BclXL. In many cancer cells, BclXL is a target of NFκB. Simvastatin inhibited the DNA binding and transcriptional activities of NF κ B resulting in marked reduction in transcription of BclXL. Signals transmitted by anti-neoplastic mechanism implanted in the cancer cells serve to obstruct the initial outgrowth of tumors. One such mechanism represents the action of the tumor suppressor protein PTEN, which negatively regulates Akt kinase activity. We provide the first evidence for significantly increased levels of PTEN in the tumors of simvastatin-administered mice. Importantly, simvastatin markedly prevented binding of NFκB to the two canonical recognition elements, NFRE-1 and NFRE-2 present in the PTEN promoter. Contrary to the transcriptional suppression of BclXL, simvastatin significantly increased the transcription of PTEN. Furthermore, expression of NFκ B p65 subunit inhibited transcription of PTEN, resulting in reduced protein expression, which leads to enhanced phosphorylation of Akt. Taken together, our data present a novel bifaceted mechanism where simvastatin acts on a nodal transcription factor NFκ B, which attenuates the expression of anti-apoptotic BclXL and simultaneously derepresses the expression of anti-proliferative/proapoptotic tumor suppressor PTEN to prevent breast cancer cell growth.

Diabetic nephropathy manifests aberrant activation of TORC1, which senses key signals to modulate protein synthesis and renal hypertrophy. PRAS40 has recently been identified as a raptor-interacting protein and is a component and a constitutive inhibitor of TORC1. The mechanism by which high glucose stimulates TORC1 activity is not known. PRAS40 was identified in the mesangial cells in renal glomeruli and in tubulointerstitium of rat kidney. Streptozotocin-induced diabetic renal hypertrophy was associated with phosphorylation of PRAS40 in the cortex and glomeruli. In vitro, high glucose concentration increased PRAS40 phosphorylation in a PI 3 kinase-and Akt-dependent manner, resulting in dissociation of raptor-PRAS40 complex in mesangial cells. High glucose augmented the inactivating and activating phosphorylation of 4EBP-1 and S6 kinase, respectively with concomitant induction of protein synthesis and hypertrophy. Expression of TORC1-nonphosphorylatable mutant of 4EBP-1 and dominant negative S6 kinase significantly inhibited high glucose-induced protein synthesis and hypertrophy. PRAS40 knockdown mimicked the effect of high glucose on phosphorylation of 4EBP-1 and S6 kinase, protein synthesis and hypertrophy. To elucidate the role of PRAS40 phosphorylation, we used phosphorylation-deficient mutant of PRAS40, which in contrast to PRAS40 knockdown inhibited phosphorylation of 4EBP-1 and S6 kinase, leading to reduced mesangial cell hypertrophy. Thus our data identify high glucose-induced phosphorylation and inactivation of PRAS40 as a central node for mesangial cell hypertrophy in diabetic nephropathy.

Lovastatin promotes osteoblast differentiation by increasing bone morphogenetic protein-2 (BMP-2) expression. We demonstrate that lovastatin stimulates tyrosine phosphorylation of the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K), leading to an increase in its kinase activity in osteoblast cells. Inhibition of PI3K ameliorated expression of the osteogenic markers alkaline phosphatase, type I collagen, osteopontin, and BMP-2. Expression of dominant-negative PI3K and PTEN, an inhibitor of PI3K signaling, significantly attenuated lovastatin-induced transcription of BMP-2. Akt kinase was also activated in a PI3K-dependent manner. However, our data suggest involvement of an additional signaling pathway. Lovastatin-induced Erk1/2 activity contributed to BMP-2 transcription. Inhibition of PI3K abrogated Erk1/2 activity in response to lovastatin, indicating the presence of a signal relay between them. We provide, as a mechanism of this cross-talk, the first evidence that lovastatin stimulates rapid activation of Ras, which associates with and activates PI3K in the plasma membrane, which in turn regulates Akt and Erk1/2 to induce BMP-2 expression for osteoblast differentiation.Statins block cholesterol biosynthesis by competitively inhibiting the rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase, which converts 3-hydroxy-3-methylglutaryl-CoA to mevalonate (1). Statins have recently been shown to reduce osteoclast activity and to stimulate osteoblast differentiation in vitro and bone formation in vivo (2-4). The role of statins in increasing bone mineral density in experimental animals and their role in protecting against fractures in cross-sectional or retrospective case control studies have led to testing this group of drugs for osteoporosis management (3, 5-9).The lipophilic statins, viz. lovastatin, fluvastatin, simvastatin, and mevastatin, specifically activate the bone morphogenetic protein-2 (BMP-2) 3 gene promoter (3). The more water-soluble pravastatin does not, however, induce BMP-2 promoter activity or BMP-2 mRNA and protein levels (10). Pravastatin does not stimulate new bone formation in neonatal murine calvaria (3). Transient exposure of bone cultures to lipophilic statins is sufficient to initiate the cascade resulting in bone formation, most probably because of the local production of BMP-2. Simvastatin-induced differentiation of MC3T3-E1 cells is accompanied by an increase in mRNA expression of BMP-2, vascular endothelial growth factor, alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (11). Although the expression of Cbfa-1/Runx2 was found to be unchanged by simvastatin treatment in the previous study (11), an earlier report demonstrated that lovastatin increases Cbfa-1/Runx2 expression while stimulating osteogenic differentiation of bone marrow mesenchymal cells (12). These studies clearly demonstrate a role for statins in osteoblast differentiation.Here we report that lovastatin stimulates osteoblast differentiation by activating phosphatidylinositol...

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Substantial data from clinical trials and epidemiological studies show promising results for use of statins in many cancers, including mammary carcinoma. Breast tumor primarily metastasizes to bone to form osteolytic lesions, causing severe pain and pathological fracture. Here, we report that simvastatin acts as an inhibitor of osteolysis in a mouse model of breast cancer skeletal metastasis of human mammary cancer cell MDA-MB-231, which expresses the mutant p53R280K. Simvastatin and lovastatin attenuated migration and invasion of MDA-MB-231 and BT-20 breast tumor cells in culture. Acquisition of phenotype to express the cancer stem cell marker, CD44, leads to invasive potential of the tumor cells. Interestingly, statins significantly decreased the expression of CD44 protein via a transcriptional mechanism. shRNA-mediated down-regulation of CD44 markedly reduced the migration and invasion of breast cancer cells in culture. We identified that in the MDA-MB-231 cells, simvastatin elevated the levels of mutated p53R280K, which was remarkably active as a transcription factor. shRNAderived inhibition of mutant p53R280K augmented the expression of CD44, leading to increased migration and invasion. Finally, we demonstrate an inverse correlation between expression of p53 and CD44 in the tumors of mice that received simvastatin. Our results reveal a unique function of statins, which foster enhanced expression of mutant p53R280K to prevent breast cancer cell metastasis to bone.Statins are potent inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate pathway for the biosynthesis of cholesterol (1). These compounds have been used for decades as safe and effective drugs in the control of hypercholesterolemia. The mevalonate pathway also produces a number of important end products, which include isoprenoid precursors, ubiquinone, dolichol, and isopentenyladenine (2). Statins also show anti-carcinogenic effects in rodent models of lung, prostate, melanoma, colon, glioma, and mammary tumorigenesis, and beneficial effects of statins have been seen in different cancers, including breast cancer (2, 3). For example, a significant 20% reduction in overall cancer risk was observed in patients with statin use (4). A recent study conducted in women who used statins showed significantly reduced risk of breast cancer as compared with nonusers (5). Moreover, an independent study of women who used statins for more than 4 years reported a significantly lower risk of breast cancer in this group (6).Several distinct mechanisms have been proposed whereby statins block tumor cell proliferation and induce apoptosis. For example, inhibition of geranylgeranyl pyrophosphate and farnesyl pyrophosphate production by statins prevents the post-translational modification of Rho and Ras GTPases necessary for their membrane localization (2). Rho proteins regulate the proliferative and invasive potential of various tumor cells, including breast cancer cells (2). Thus, suppression of geranylge...