Screening of recurrent hot spot SLC12A3 mutations may provide an early diagnosis of GS. Besides gender effect, the nature of homozygous and deep intronic mutations may influence the phenotype. Patients with GS may be at increased risk for the development of chronic kidney disease and type 2 diabetes.

OBJECTIVEDisturbances in podocytes are typically associated with marked proteinuria, a hallmark of diabetic nephropathy. This study was conducted to investigate modulation of Notch-1 signaling in high glucose (HG)-stressed human podocytes and in a diabetic animal model.RESEARCH DESIGN AND METHODSExpression of the Notch signaling components was examined in HG-treated podocytes, human embryonic kidney cells (HEK293), and kidneys from diabetic animals by RT-qPCR, Western blot analysis, and immunohistochemical staining. The association between the Notch signaling, VEGF expression, and podocyte integrity was evaluated.RESULTSNotch-1 signaling was significantly activated in HG-cultured human podocytes and HEK293 cells and kidneys from diabetic animals. HG also augmented VEGF expression, decreasing nephrin expression and podocyte number—a critical event for the development of proteinuria in diabetic nephropathy. After use of pharmacological modulators or specific shRNA knockdown strategies, inhibition of Notch-1 signaling significantly abrogated VEGF activation and nephrin repression in HG-stressed cells and ameliorated proteinuria in the diabetic kidney.CONCLUSIONSOur findings suggest that upregulation of Notch-1 signaling in HG-treated renal podocytes induces VEGF expression and subsequent nephrin repression and apoptosis. Modulation of Notch-1 signaling may hold promise as a novel therapeutic strategy for the treatment of diabetic nephropathy.

Notch receptors are evolutionarily conserved fromDrosophila to human and play important roles in cell fate decisions. After ligand binding, Notch receptors are cleaved to release their intracellular domains. The intracellular domains, the activated form of Notch receptors, are then translocated into the nucleus where they interact with other transcriptional machinery to regulate the expression of cellular genes. To dissect the molecular mechanisms of Notch signaling, the cellular targets that interact with Notch1 receptor intracellular domain (N1IC) were screened. In this study, we found that endogenous transcription factor Ying Yang 1 (YY1) was associated with exogenous N1IC in human K562 erythroleukemic cells. The ankyrin (ANK) domain of N1IC and zinc finger domains of YY1 were essential for the association of N1IC and YY1 according to the pulldown assay of glutathione S-transferase fusion proteins. Furthermore, both YY1 and N1IC were present in a large complex of the nucleus to suppress the luciferase reporter activity transactivated by Notch signaling. The transcription factor YY1 indirectly regulated the transcriptional activity of the wild-type CBF1-response elements via the direct interaction of N1IC and CBF1. We also demonstrated the association between endogenous N1IC and intrinsic YY1 in human acute T-cell lymphoblastic leukemia cell lines. Taken together, these results indicate that transcription factor YY1 may modulate Notch signaling via association with the high molecular weight Notch complex.

Although DNA hypermethylation within promoter CpG islands is highly correlated with tumorigenesis, it has not been established whether DNA hypermethylation within a specific tumor suppressor gene (TSG) is sufficient to fully transform a somatic stem cell. In this study, we addressed this question using a novel targeted DNA methylation technique to methylate the promoters of HIC1 and RassF1A, two well-established TSGs, along with a two-component reporter system to visualize successful targeting of human bone marrow-derived mesenchymal stem cells (MSC) as a model cell system. MSCs harboring targeted promoter methylations of HIC1/RassF1A displayed several features of cancer stem/initiating cells including loss of anchorage dependence, increased colony formation capability, drug resistance, and pluripotency. Notably, inoculation of immunodeficient mice with low numbers of targeted MSC resulted in tumor formation, and subsequent serial xenotransplantation and immunohistochemistry confirmed the presence of stem cell markers and MSC lineage in tumor xenografts. Consistent with the expected mechanism of TSG hypermethylation, treatment of the targeted MSC with a DNA methyltransferase inhibitor reversed their tumorigenic phenotype. To our knowledge, this is the first direct demonstration that aberrant TSG hypermethylation is sufficient to transform a somatic stem cell into a fully malignant cell with cancer stem/initiating properties. Cancer Res; 71(13); 4653-63. Ó2011 AACR.

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A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/ oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc T58M/T58M mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4 D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/ Osr1-Ncc, were crossed with Ncc T58M/T58M mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II.

SummaryBackground and objectives Gitelman's syndrome (GS) is an autosomal recessive renal tubular disorder caused by mutations in the SLC12A3 gene encoding the thiazide-sensitive Na ϩ -Cl Ϫ cotransporter (NCC). Despite meticulous sequencing of genomic DNA, approximately one-third of GS patients are negative or heterozygotes for the known mutations.Design, Setting, Participants, & Measurements Because blood leukocytes express NCC mRNA, we evaluate whether deep intronic mutations contribute to GS patients with uniallelic or undetectable SLC12A3 mutations. Twenty-nine patients with GS (men/women ϭ 16/13), including eight negative and 21 uniallelic SLC12A3 mutations from 19 unrelated families, and normal controls were enrolled in an academic medical center. Analysis of cDNA from blood leukocytes, sequencing of the corresponding introns of genomic DNA for abnormal transcript, and analysis of NCC protein expression from renal biopsy were performed. Results We identified nine Taiwan aboriginal patients carrying c.1670 -191C3 T mutations in intron 13and 10 nonaboriginal patients carrying c.2548ϩ253C3 T mutations in intron 21 from 14 families (14/ 19). These two mutations undetected in 100 healthy subjects created pseudoexons containing new premature termination codons. Haplotype analysis with markers flanking SLC12A3 revealed that both mutations did not have founder effects. Apical NCC expression in the DCT of renal tissue was markedly diminished in two patients carrying deep intronic mutations.Conclusions Deep intronic mutations in SLC12A3 causing defective NCC expression can be identified with the RNA-based approach in patients with GS. c.1670 -191C3 T and c.2548ϩ253C3 T are hot spot mutations that can be screened in GS patients with uniallelic or negative SLC12A3 mutations.

Curcumin (CUR) has been shown to possess a preventive effect against various cancers and interfere with multiple-cell signaling pathways. We evaluated the protective effects of CUR in regression of UVB-induced skin tumor formation in SKH-1 hairless mice and its underlying early molecular biomarkers associated with carcinogenesis. Mice irradiated with UVB at 180 mJ/cm2 twice per week elicited 100% tumor incidence at 20 weeks. Topical application of CUR prior to UVB irradiation caused delay in tumor appearance, multiplicity, and size. Topical application of CUR prior to and immediately after a single UVB irradiation (180 mJ/cm2) resulted in a significant decrease in UVB-induced thymine dimer-positive cells, expression of proliferative cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, and apoptotic sunburn cells together with an increase in p53 and p21/Cip1-positive cell population in epidermis. Simultaneously, CUR also significantly inhibited NF-κB, cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and nitric oxide (NO) levels. The results suggest that the protective effect of CUR against photocarcinogenesis is accompanied by downregulation of cell proliferative controls, involving thymine dimer, PCNA, apoptosis, transcription factors NF-κB, and of inflammatory responses involving COX-2, PGE2, and NO, while upregulation of p53 and p21/Cip1 to prevent DNA damage and facilitate DNA repair.