TP63 Is Significantly Upregulated in Diabetic Kidney.

The role of tumor protein 63 (TP63) in regulating insulin receptor substrate 1 (IRS-1) and other downstream signal proteins in diabetes has not been characterized. RNAs extracted from kidneys of diabetic mice (db/db) were sequenced to identify genes that are involved in kidney complications. RNA sequence analysis showed more than 4- to 6-fold increases in TP63 expression in the diabetic mice's kidneys, compared to wild-type mice at age 10 and 12 months old. In addition, the kidneys from diabetic mice showed significant increases in TP63 mRNA and protein expression compared to WT mice. Mouse proximal tubular cells exposed to high glucose (HG) for 48 h showed significant decreases in IRS-1 expression and increases in TP63, compared to cells grown in normal glucose (NG). When TP63 was downregulated by siRNA, significant increases in IRS-1 and activation of AMP-activated protein kinase (AMPK (p-AMPK-Th172)) occurred under NG and HG conditions. Moreover, activation of AMPK by pretreating the cells with AICAR resulted in significant downregulation of TP63 and increased IRS-1 expression. Ad-cDNA-mediated over-expression of tuberin resulted in significantly decreased TP63 levels and upregulation of IRS-1 expression. Furthermore, TP63 knockdown resulted in increased glucose uptake, whereas IRS-1 knockdown resulted in a decrease in the glucose uptake. Altogether, animal and cell culture data showed a potential role of TP63 as a new candidate gene involved in regulating IRS-1 that may be used as a new therapeutic target to prevent kidney complications in diabetes.

mTOR Activation Initiates Renal Cell Carcinoma Development by Coordinating ERK and p38MAPK.

Renal cell carcinoma (RCC) mainly originates from renal proximal tubules. Intriguingly, disruption of genes frequently mutated in human RCC samples thus far has only generated RCC originated from other renal tubule parts in mouse models. This hampers our understanding of the pathogenesis of RCC. Here we show that mTOR signaling, often activated in RCC samples, initiates RCC development from renal proximal tubules. Ablation of Tsc1, encoding an mTOR suppressor, in proximal tubule cells led to multiple precancerous renal cysts. mTOR activation increased MEK1 expression and ERK activation, and Mek1 ablation or inhibition diminished cyst formation in Tsc1-deficient mice. mTOR activation also increased MKK6 expression and p38MAPK activation, and ablation of the p38α-encoding gene further enhanced cyst formation and led to RCC with clear cell RCC features. Mechanistically, Tsc1 deletion induced p53 and p16 expression in a p38MAPK-dependent manner, and deleting Tsc1 and Trp53 or Cdkn2a (encoding p16) enhanced renal cell carcinogenesis. Thus, mTOR activation in combination with inactivation of the p38MAPK-p53/p16 pathway drives RCC development from renal proximal tubules. Moreover, this study uncovers previously unidentified mechanisms by which mTOR controls cell proliferation and suggests the MEK-ERK axis to be a potential target for treatment of RCC. SIGNIFICANCE: Mouse modeling studies show that mTOR activation in combination with inactivation of the p38MAPK-p53/p16 axis initiates renal cell carcinoma that mimics human disease, identifying potential therapeutic targets for RCC treatment.

Galectin-1 is a new fibrosis protein in type 1 and type 2 diabetes.

Chronic exposure of tubular renal cells to high glucose contributes to tubulointerstitial changes in diabetic nephropathy. In the present study, we identified a new fibrosis gene called galectin-1 (Gal-1), which is highly expressed in tubular cells of kidneys of type 1 and type 2 diabetic mouse models. Gal-1 protein and mRNA expression showed significant increase in kidney cortex of heterozygous Akita+/- and db/db mice compared with wild-type mice. Mouse proximal tubular cells exposed to high glucose showed significant increase in phosphorylation of Akt and Gal-1. We cloned Gal-1 promoter and identified the transcription factor AP4 as binding to the Gal-1 promoter to up-regulate its function. Transfection of cells with plasmid carrying mutations in the binding sites of AP4 to Gal-1 promoter resulted in decreased protein function of Gal-1. In addition, inhibition of Gal-1 by OTX-008 showed significant decrease in p-Akt/AP4 and protein-promoter activity of Gal-1 and fibronectin. Moreover, down-regulation of AP4 by small interfering RNA resulted in a significant decrease in protein expression and promoter activity of Gal-1. We found that kidney of Gal-1-/- mice express very low levels of fibronectin protein. In summary, Gal-1 is highly expressed in kidneys of type 1 and 2 diabetic mice, and AP4 is a major transcription factor that activates Gal-1 under hyperglycemia. Inhibition of Gal-1 by OTX-008 blocks activation of Akt and prevents accumulation of Gal-1, suggesting a novel role of Gal-1 inhibitor as a possible therapeutic target to treat renal fibrosis in diabetes.-Al-Obaidi, N., Mohan, S., Liang, S., Zhao, Z., Nayak, B. K., Li, B., Sriramarao, P., Habib, S. L. Galectin-1 is a new fibrosis protein in type 1 and type 2 diabetes.

A new drug combination significantly reduces kidney tumor progression in kidney mouse model.

Tuberous sclerosis complex (TSC) disease is associated with tumors in many organs, particularly angiomyolipoma (AML) in the kidneys. Loss or inactivation of TSC1/2 results in high levels of HIF-α activity and VEGF expression. mTOR inhibitor (rapamycin) and the AMPK activator 5-aminoimidazole-4-carboxamide (AICA)-riboside (AICAR) are currently used separately to treat cancer patients. Here, we investigated the effect of a novel combination of rapamycin and AICAR on tumor progression. Our data show that treatment of AML human cells with drug combinations resulted in 5-7-fold increase in cell apoptosis compared to each drug alone. In addition, drug combinations resulted in 4-5-fold decrease in cell proliferation compared to each drug alone. We found that drug combinations abolished Akt and HIF activity in AML cells. The drug combinations resulted in decrease in cell invasion and cell immigration by 70% and 84%, respectively in AML cells. The combined drugs also significantly decreased the VEGF expression compare to each drug alone in AML cells. Drug combinations effectively abolished binding of HIF-2α to the putative Akt site in the nuclear extracts isolated from AML cells. Treatment TSC mice with drug combinations resulted in 75% decrease in tumor number and 88% decrease in tumor volume compared to control TSC mice. This is first evidence that drug combinations are effective in reducing size and number of kidney tumors without any toxic effect on kidney. These data will provide evidence for initiating a new clinical trial for treatment of TSC patients.

NOX4 functions as a mitochondrial energetic sensor coupling cancer metabolic reprogramming to drug resistance.

The molecular mechanisms that couple glycolysis to cancer drug resistance remain unclear. Here we identify an ATP-binding motif within the NADPH oxidase isoform, NOX4, and show that ATP directly binds and negatively regulates NOX4 activity. We find that NOX4 localizes to the inner mitochondria membrane and that subcellular redistribution of ATP levels from the mitochondria act as an allosteric switch to activate NOX4. We provide evidence that NOX4-derived reactive oxygen species (ROS) inhibits P300/CBP-associated factor (PCAF)-dependent acetylation and lysosomal degradation of the pyruvate kinase-M2 isoform (PKM2). Finally, we show that NOX4 silencing, through PKM2, sensitizes cultured and ex vivo freshly isolated human-renal carcinoma cells to drug-induced cell death in xenograft models and ex vivo cultures. These findings highlight yet unidentified insights into the molecular events driving cancer evasive resistance and suggest modulation of ATP levels together with cytotoxic drugs could overcome drug-resistance in glycolytic cancers.

HIF-1 Mediates Renal Fibrosis in OVE26 Type 1 Diabetic Mice.

Hypoxia-inducible factor (HIF)-1 mediates hypoxia- and chronic kidney disease-induced fibrotic events. Here, we assessed whether HIF-1 blockade attenuates the manifestations of diabetic nephropathy in a type 1 diabetic animal model, OVE26. YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole], an HIF-1 inhibitor, reduced whole kidney glomerular hypertrophy, mesangial matrix expansion, extracellular matrix accumulation, and urinary albumin excretion as well as NOX4 protein expression and NADPH-dependent reactive oxygen species production, while blood glucose levels remained unchanged. The role of NOX oxidases in HIF-1-mediated extracellular matrix accumulation was explored in vitro using glomerular mesangial cells. Through a series of genetic silencing and adenoviral overexpression studies, we have defined GLUT1 as a critical downstream target of HIF-1α mediating high glucose-induced matrix expression through the NADPH oxidase isoform, NOX4. Together, our data suggest that pharmacological inhibition of HIF-1 may improve clinical manifestations of diabetic nephropathy.

Estrogen receptor-alpha binds p53 tumor suppressor protein directly and represses its function.

Estrogen receptor-alpha (ERalpha) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ERalpha binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERalpha and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERalpha by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ERalpha and p53. Ionizing radiation together with ERalpha knock down results in additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the pro-proliferative role of ERalpha.

Stabilization of p53 and transactivation of its target genes in response to replication blockade.

Although it is clear that p53 plays a pivotal role in G1/G2 checkpoints to conserve genomic integrity, its role in S phase checkpoint is less well understood. Recently, it has been reported that p53 is transcriptionally impaired even though it is stabilized during replication blockade. However, the mechanisms underlying this phenomenon are not known. In the present study, it has been shown that p53 accumulates and transactivates its target genes such as p21, gadd45 and bax in response to replication blockade in normal and cancer cells. Lack of transcriptional activation under similar conditions in cells lacking p53 shows that p53-target gene activation during replication blockade is indeed p53-dependent. Further, transactivation of p21 in response to replication blockade by hydroxyurea and aphidicolin is similar to that in response to ionizing radiation except that the latter is more immediate compared to the response to replication blockade. These findings suggest that impairment of transcriptionally active p53 in response to replication blockade is not a general phenomenon.