Centrosomal BRCA2 is a target protein of membrane type-1 matrix metalloproteinase (MT1-MMP)

BRCA2 localizes to centrosomes between G1 and prophase and is removed from the centrosomes during mitosis, but the underlying mechanism is not clear. Here we show that BRCA2 is cleaved into two fragments by membrane type-1 matrix metalloproteinase (MT1-MMP), and that knockdown of MT1-MMP prevents the removal of BRCA2 from centrosomes during metaphase. Mass spectrometry mapping revealed that the MT1-MMP cleavage site of human BRCA2 is between Asn-2135 and Leu-2136 (²¹³²LSNN/LNVEGG²¹⁴¹), and the point mutation L2136D abrogated MT1-MMP cleavage. Our data demonstrate that MT1-MMP proteolysis of BRCA2 regulates the abundance of BRCA2 on centrosomes.     

NDRG2 is a new HIF-1 target gene necessary for hypoxia-induced apoptosis in A549 cells.

The NDRG2 gene belongs to a family of N-Myc downstream-regulated genes (NDRGs) and is expressed in many normal tissues. NDRG2 gene expression has been shown to be regulated in the stress response of certain cells. However, its function is not yet fully understood. Many studies have demonstrated that hypoxia, one of the stress responses, induced apoptosis in several cell types. In the current study, we investigated NDRG2 involvement in hypoxia response and found that NDRG2 expression was markedly up-regulated in several tumor cell lines exposed to hypoxic conditions or similar stresses at the mRNA and protein level. We also observed that the expression of NDRG2 was regulated by Hypoxia-inducible factor 1 (HIF-1) in tumor cells under hypoxia. Three hypoxia-responsive elements (HREs) in the NDRG2 promoter were identified. HRE1 could directly bind Hif-1 in vivo. Importantly, we found that silencing or enforcing the expression of NDRG2 could strongly inhibit or increase apoptosis. In addition, our data also showed that Ndrg2 was able to be translocated from the cytoplasm to the nucleus, and the segment from 101 to 178 amino acids of Ndrg2 is responsible for its translocation. Taken together, this study suggests that NDRG2 is a Hif-1 target gene and closely related with hypoxia-induced apoptosis in A549 cells.     

The human sex-determining gene SRY is a direct target of WT1

The product of the Wilms' tumor gene, WT1, is essential for male sex determination and differentiation in mammals. In addition to causing Wilms' tumor, mutations in WT1 often cause two distinct but overlapping urogenital defects in men, Denys-Drash syndrome and Frasier syndrome. In this study we investigated the regulation of the sex determination gene SRY by WT1. Our results showed that WT1 up-regulates the SRY gene through the proximal early growth response gene-1-like DNA-binding sequences in the core promoter. Mutant WT1 proteins in Denys-Drash syndrome patients were unable to activate this promoter. These mutants did not act in a dominant negative manner, as expected over the wild-type WT1 in this promoter. We also found that WT1 could transactivate the endogenous SRY gene. These observations, together with the overlapping expression patterns of WT1 and SRY in human gonads, led us to propose that WT1 regulates SRY in the initial sex determination process in humans and activates a cascade of genes ultimately leading to the complete organogenesis of the testis.     

Cdx4 is a Cdx2 target gene

The products of the Cdx genes, Cdx1, Cdx2 and Cdx4, play multiple roles in early vertebrate development, and have been proposed to serve to relay signaling information from Wnt, RA and FGF pathways to orchestrate events related to anterior-posterior vertebral patterning and axial elongation. In addition, Cdx1 and Cdx2 have been reported to both autoregulate and to be subject to cross regulation by other family members. We have now found that Cdx4 expression is significantly down regulated in Cdx2(-/-) mutants suggesting previously unrecognized cross-regulatory interactions. Moreover, we have previously shown that Cdx4 is a direct target of the canonical Wnt signaling pathway, and that Cdx1 physically interacts with LEF/TCF members in an autoregulatory loop. We therefore investigated the means by which Cdx2 impacted on Cdx4 expression and assessed potential interaction between Cdx2 and canonical Wnt signaling on the Cdx4 promoter. We found that the Cdx4 promoter was regulated by Cdx2 in transient transfection assays. Electrophoretic mobility shift assays showed that Cdx2 bound to predicted Cdx response elements in the Cdx4 promoter which, when mutated, significantly reduced activity. Consistent with these data, chromatin immunoprecipitation assays from embryos demonstrated occupancy of the Cdx4 promoter by Cdx2 in vivo. However, we failed to observe an interaction between Cdx2 and components of the canonical Wnt signaling pathway. These findings suggest that, while both canonical Wnt and Cdx2 can regulate the activity of the Cdx4 promoter, they appear to operate through distinct mechanisms.     

HMGA1 is a new target of miR-195 involving isoprenaline-induced cardiomyocyte hypertrophy

Emerging data have shown that microRNAs (miRNAs) have important functions in the processes of cardiac hypertrophy and heart failure that occur during the postnatal period. Cardiac overexpression of miR-195 results in pathological cardiac growth and heart failure in transgenic mice. In the present study, we analyzed the roles of miR-195 in cardiomyocyte hypertrophy and found that miR-195 was greatly upregulated during isoprenaline-induced cardiomyocyte hypertrophy. By using mRNA microarray and molecular approach, we identified a novel putative target of miR-195 called high-mobility group A1 (HMGA1). Total mRNA microarray showed that HMGA1 was downregulated in primary cardiomyocytes that overexpressed miR-195. Using luciferase activity assay, we demonstrated that miR-195 interacts with the 3′-untranslated region of HMGA1 mRNA. Moreover, we showed that miR-195 in primary cardiomyocytes downregulates the expression of HMGA1 at the protein level. Taken together, our data demonstrated that miR-195 can negatively regulate a new target, HMGA1, which is involved in cardiomyocyte hypertrophy.     

Nutrient stress is a target for new antibiotics

Novel approaches are required to address the looming threat of pan-resistant Gram-negative pathogens and forestall the rise of untreatable infections. Unconventional targets that are uniquely important during infection and tractable to high-throughput drug discovery methods hold high potential for innovation in antibiotic discovery programs. In this context, inhibitors of bacterial nutrient stress are particularly exciting candidates for future antibiotic development. Amino acid, nucleotide, and vitamin biosynthesis pathways are critical for bacterial growth in nutrient-limiting conditions in the laboratory and the host. Although historically dismissed as dispensable for pathogens, a wealth of transposon mutagenesis and single-mutant studies have emerged which demonstrate that several such pathways are critical for infection. Indeed, high-throughput screens of diverse synthetic compounds and natural products have uncovered inhibitors of nutrient biosynthesis. Herein, we review bacterial nutrient biosynthesis and its role during host infection. Further, we explore screening platforms developed to search for inhibitors of these targets and highlight successes among these. Finally, we feature important and sometimes surprising connections between bacterial nutrient biosynthesis, antibiotic activity, and antibiotic resistance.     

ASK-1 (apoptosis signal-regulating kinase 1) is a direct E2F target gene

S1P (sphingosine 1-phosphate) receptor expression and the effects of S1P on migration were studied in one papillary (NPA), two follicular (ML-1, WRO) and two anaplastic (FRO, ARO) thyroid cancer cell lines, as well as in human thyroid cells in primary culture. Additionally, the effects of S1P on proliferation, adhesion and calcium signalling were addressed in ML-1 and FRO cells. All cell types expressed multiple S1P receptors. S1P evoked intracellular calcium signalling in primary cultures, ML-1 cells and FRO cells. Neither proliferation nor migration was affected in primary cultures, whereas S1P partly inhibited proliferation in ML-1 and FRO cells. Low nanomolar concentrations of S1P inhibited migration in FRO, WRO and ARO cells, but stimulated ML-1 cell migration. Consistently, S1P1 and S1P3, which mediate migratory responses, were strongly expressed in ML-1 cells, and S1P2, which inhibits migration, was the dominating receptor in the other cell lines. The migratory effect in ML-1 cells was mediated by G(i) and phosphatidylinositol 3-kinase. Both S1P and the S1P1-specific agonist SEW-2871 induced Akt phosphorylation at Ser473. However, SEW-2871 failed to stimulate migration, whereas the S1P1/S1P3 antagonist VPC 23019 inhibited S1P-induced migration. The results suggest that aberrant S1P receptor expression may enhance thyroid cancer cell migration and thus contribute to the metastatic behaviour of some thyroid tumours.     

Stanniocalcin-2 is a HIF-1 target gene that promotes cell proliferation in hypoxia

Stanniocalcin-2 (STC2), the paralog of STC1, has been suggested as a novel target of oxidative stress response to protect cells from apoptosis. The expression of STC2 has been reported to be highly correlated with human cancer development. In this study, we reported that STC2 is a HIF-1 target gene and is involved in the regulation of cell proliferation. STC2 was shown to be up-regulated in different breast and ovarian cancer cells, following exposure to hypoxia. Using ovarian cancer cells (SKOV3), the underlying mechanism of HIF-1 mediated STC2 gene transactivation was characterized. Hypoxia-induced STC2 expression was found to be HIF-1α dependent and required the recruitment of p300 and HDAC7. Using STC2 promoter deletion constructs and site-directed mutagenesis, two authentic consensus HIF-1 binding sites were identified. Under hypoxic condition, the silencing of STC2 reduced while the overexpression of STC2 increased the levels of phosphorylated retinoblastoma and cyclin D in both SKOV3 and MCF7 cells. The change in cell cycle proteins correlated with the data of the serial cell counts. The results indicated that cell proliferation was reduced in STC2-silenced cells but was increased in STC2-overexpressing hypoxic cells. Solid tumor progression is usually associated with hypoxia. The identification and functional analysis of STC2 up-regulation by hypoxia, a feature of the tumor microenvironment, sheds light on a possible role for STC2 in tumors.