BackgroundKlotho was originally characterized as an anti-aging gene that predisposed Klotho-deficient mice to a premature aging-like syndrome. Recently, KLOTHO was reported to function as a secreted Wnt antagonist and as a tumor suppressor. Epigenetic gene silencing of secreted Wnt antagonists is considered a common event in a wide range of human malignancies. Abnormal activation of the canonical Wnt pathway due to epigenetic deregulation of Wnt antagonists is thought to play a crucial role in cervical tumorigenesis. In this study, we examined epigenetic silencing of KLOTHO in human cervical carcinoma.ResultsLoss of KLOTHO mRNA was observed in several cervical cancer cell lines and in invasive carcinoma samples, but not during the early, preinvasive phase of primary cervical tumorigenesis. KLOTHO mRNA was restored after treatment with either the DNA demethylating agent 2'-deoxy-5-azacytidine or histone deacetylase inhibitor trichostatin A. Methylation-specific PCR and bisulfite genomic sequencing analysis of the promoter region of KLOTHO revealed CpG hypermethylation in non-KLOTHO-expressing cervical cancer cell lines and in 41% (9/22) of invasive carcinoma cases. Histone deacetylation was also found to be the major epigenetic silencing mechanism for KLOTHO in the SiHa cell line. Ectopic expression of the secreted form of KLOTHO restored anti-Wnt signaling and anti-clonogenic activity in the CaSki cell line including decreased active β-catenin levels, suppression of T-cell factor/β-catenin target genes, such as c-MYC and CCND1, and inhibition of colony growth.ConclusionsEpigenetic silencing of KLOTHO may occur during the late phase of cervical tumorigenesis, and consequent functional loss of KLOTHO as the secreted Wnt antagonist may contribute to aberrant activation of the canonical Wnt pathway in cervical carcinoma.

Complement-C1q TNF-related protein 1 (CTRP1), a member of the CTRP superfamily, is expressed at high levels in adipose tissues of obese Zucker diabetic fatty (fa/fa) rats, and CTRP1 expression is induced by proinflammatory cytokines, including TNF-alpha and IL-1beta. In the present study, we investigated stimulation of aldosterone production by CTRP1, since it was observed that CTRP1 was specifically expressed in the zona glomerulosa of the adrenal cortex, where aldosterone is produced. Increased aldosterone production by CTRP1 in cells of the human adrenal cortical cell line H295R was dose-dependent. Expression levels of aldosterone synthase CYP11B2 were examined to investigate the molecular mechanisms by which CTRP1 enhances the production of aldosterone. The expression of CYP11B2 was greatly increased by treatment with CTRP1, as was the expression of the transcription factors NGFIB and NURR1, which play critical roles in stimulation of CYP11B2 gene expression. It was also revealed that angiotensin II-induced aldosterone production is, at least in part, mediated by the stimulation of CTRP1 secretion, not by the increase of CTRP1 mRNA transcription. In addition, the levels of CTRP1 were significantly up-regulated in hypertensive patients' serum. As CTRP1 was highly expressed in obese subjects as well as up-regulated in hypertensive patients, CTRP1 may be a newly identified molecular link between obesity and hypertension.

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Low serum levels of adiponectin are a high risk factor for various types of cancer. Although adiponectin inhibits proliferation and metastasis of breast cancer cells, the underlying molecular mechanisms remain obscure. In this study, we show that adiponectin-activated AMPK reduces the invasiveness of MDA-MB-231 cells by stimulating dephosphorylation of AKT by increasing protein phosphatase 2A (PP2A) activity. Among the various regulatory B56 subunits, B56; was directly phosphorylated by AMPK at Ser 298 and Ser 336 , leading to an increase of PP2A activity through dephosphorylation of PP2Ac at Tyr 307 . We also show that both the blood levels of adiponectin and the tissue levels of PP2A activity were decreased in breast cancer patients and that the direct administration of adiponectin into tumor tissues stimulates PP2A activity. Taken together, these findings show that adiponectin, derived from adipocytes, negatively regulates the invasiveness of breast cancer cells by activating the tumor suppressor PP2A. [Cancer Res 2009;69(9):4018-26]

SummaryArabidopsis thaliana homeobox 12 (ATHB12), a homeodomain-leucine zipper class I (HDZip I) gene, is highly expressed in leaves and stems, and induced by abiotic stresses, but its role in development remains obscure.To understand its function during plant development, we studied the effects of loss and gain of function. Expression of ATHB12 fused to the EAR-motif repression domain (SRDX) -P 35S ::ATHB12SRDX (A12SRDX) and P ATHB12 ::ATHB12SRDX -slowed both leaf and root growth, while the growth of ATHB12-overexpressing seedlings (A12OX) was accelerated.Microscopic examination revealed changes in the size and number of leaf cells. Ploidy was reduced in A12SRDX plants, accompanied by decreased cell expansion and increased cell numbers. By contrast, cell size was increased in A12OX plants, along with increased ploidy and elevated expression of cell cycle switch 52s (CCS52s), which are positive regulators of endoreduplication, indicating that ATHB12 promotes leaf cell expansion and endoreduplication. Overexpression of ATHB12 led to decreased phosphorylation of Arabidopsis thaliana ribosomal protein S6 (AtRPS6), a regulator of cell growth. In addition, induction of ATHB12 in the presence of cycloheximide increased the expression of several genes related to cell expansion, such as EXPANSIN A10 (EXPA10) and DWARF4 (DWF4).Our findings strongly suggest that ATHB12 acts as a positive regulator of endoreduplication and cell growth during leaf development.

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 49,6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/ or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology.

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Downregulation of the N-myc downstream-regulated gene 2 (NDRG2) gene is involved in the progression of aggressive forms of cancer, along with the poor prognosis of cancer patients. In the current study, we examined the effect of NDRG2 expression on the metastatic potential of HT1080 human fibrosarcoma and B16F10 murine melanoma cells in both in vitro and in vivo systems. In gelatin zymography, NDRG2 expression remarkably suppressed the matrix metalloproteinase (MMP)-9 activity and slightly inhibited MMP-2 activity of both cell lines. Tumor migration and invasion in vitro were significantly reduced by NDRG2 expression, and NDRG2 inhibited tumor cell proliferation in an anchorage-independent semisolid agar assay. Specifically, we found that NDRG2 affects invasion through suppression of nuclear factor kappa B (NF-kappaB) activity. In animal experiments, subcutaneously injected B16F10-NDRG2 cells showed delayed tumor growth compared with B16F10-mock cells. Furthermore, severe metastasis from primary tumor mass into the draining lymph nodes was observed after injection of B16F10-mock cells, but not with B16F10-NDRG2 cells. Pulmonary metastasis after intravenous injection of B16F10 cells was also reduced by NDRG2 expression. Intra- and peritumoral angiogenesis that is critical for the tumor growth and metastasis was clearly found in tumors after injection with B16F10-mock cells, whereas it was impaired in tumors after injection with B16F10-NDRG2 cells. Collectively, our data show that NDRG2 expression significantly suppresses tumor invasion by inhibiting MMP activities, which are regulated through the NF-kappaB signaling. Moreover, results from animal experiments provide evidence for the regulatory role of the NDRG2 gene in metastatic tumors.