cis-Acting elements within CFTR 5'-flanking DNA are not sufficient to decrease gene expression in response to phorbol ester

The cystic fibrosis transmembrane conductance regulator gene (CFTR) is regulated in a tissue-specific and developmental fashion. Although it has been known for some time that phorbol esters decrease CFTR expression in cell lines that have high CFTR mRNA levels, the cis-acting elements that control this down-regulation remain ill-defined. The role of cis-acting elements within the CFTR minimal promoter in modulating responses to phorbol 12-myristate 13-acetate (PMA) and forskolin was assessed using luciferase reporter gene (luc)-containing plasmids transfected into Calu-3 and HT-29 cells. PMA treatment had no effect on luciferase activity in Calu-3 cells transiently transfected with plasmids containing luc driven by up to 2.3 kb of CFTR 5'-flanking DNA. PMA increased luciferase activity in transfected HT-29 cells. A more extensive region of DNA was evaluated using a yeast artificial chromosome (YAC) containing luc driven by approximately 335 of CFTR 5'-flanking DNA (y5'luc) stably introduced into HT-29 cells. Clonal cell lines containing y5'luc were created and assessed for luciferase activity at baseline and in response to forskolin and PMA. There was a wide range of baseline luciferase activities among the clones (42-1038 units/microg protein) that was not entirely due to the number of luc copies present within the cells. Treatment with both PMA and forskolin led to increased luciferase activity in six randomly selected clonal cell lines. As expected, endogenous CFTR expression increased in response to forskolin and decreased in response to PMA. These studies demonstrate that luc-containing YAC vectors can be used to study CFTR expression in human cells. In addition, these data suggest that important regulatory elements responsible for decreased CFTR expression in response to PMA are not located upstream of CFTR in the approximately 335 kb 5'-flanking sequence included in this YAC construct.     

Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPKα2-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction.     

Phosphate and R2D2 restrict the substrate specificity of Dicer-2, an ATP-driven ribonuclease

Drosophila Dicer-2 generates small interfering RNAs (siRNAs) from long double-stranded RNA (dsRNA), whereas Dicer-1 produces microRNAs (miRNAs) from pre-miRNA. What makes the two Dicers specific for their biological substrates? We find that purified Dicer-2 can efficiently cleave pre-miRNA, but that inorganic phosphate and the Dicer-2 partner protein R2D2 inhibit pre-miRNA cleavage. Dicer-2 contains C-terminal RNase III domains that mediate RNA cleavage and an N-terminal helicase motif, whose function is unclear. We show that Dicer-2 is a dsRNA-stimulated ATPase that hydrolyzes ATP to ADP; ATP hydrolysis is required for Dicer-2 to process long dsRNA, but not pre-miRNA. Wild-type Dicer-2, but not a mutant defective in ATP hydrolysis, can generate siRNAs faster than it can dissociate from a long dsRNA substrate. We propose that the Dicer-2 helicase domain uses ATP to generate many siRNAs from a single molecule of dsRNA before dissociating from its substrate.     

Phenotype sequencing: identifying the genes that cause a phenotype directly from pooled sequencing of independent mutants

Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50-100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a "Phenotype Sequencing" approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110-$340.     

Enhanced antiviral activity against foot-and-mouth disease virus by a combination of type I and II porcine interferons

Previously, we showed that type I interferon (alpha/beta interferon [IFN-alpha/beta]) can inhibit foot-and-mouth disease virus (FMDV) replication in cell culture, and swine inoculated with 10(9) PFU of human adenovirus type 5 expressing porcine IFN-alpha (Ad5-pIFN-alpha) were protected when challenged 1 day later. In this study, we found that type II pIFN (pIFN-gamma) also has antiviral activity against FMDV in cell culture and that, in combination with pIFN-alpha, it has a synergistic antiviral effect. We also observed that while each IFN alone induced a number of IFN-stimulated genes (ISGs), the combination resulted in a synergistic induction of some ISGs. To extend these studies to susceptible animals, we inoculated groups of swine with a control Ad5, 10(8) PFU of Ad5-pIFN-alpha, low- or high-dose Ad5-pIFN-gamma, or a combination of Ad5-pIFN-alpha and low- or high-dose Ad5-pIFN-gamma and challenged all groups with FMDV 1 day later. The control group and the groups inoculated with either Ad5-pIFN-alpha or a low dose of Ad5-pIFN-gamma developed clinical disease and viremia. However, the group that received the combination of both Ad5-IFNs with the low dose of Ad5-pIFN-gamma was completely protected from challenge and had no viremia. Similarly the groups inoculated with the combination of Ad5s with the higher dose of Ad5-pIFN-gamma or with only high-dose Ad5-pIFN-gamma were protected. The protected animals did not develop antibodies against viral nonstructural (NS) proteins, while all infected animals were NS protein seropositive. No antiviral activity or significant levels of IFNs were detected in the protected groups, but there was an induction of some ISGs. The results indicate that the combination of type I and II IFNs act synergistically to inhibit FMDV replication in vitro and in vivo.     

Regulation of the Pro-apoptotic scaffolding protein POSH by Akt

POSH (Plenty of SH3 domains) binds to activated Rac and promotes apoptosis by acting as a scaffold to assemble a signal transduction pathway leading from Rac to JNK activation. Overexpression of POSH induces apoptosis in a variety of cell types, but apoptosis can be prevented by co-expressing the pro-survival protein kinase Akt. We report here that POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro, and we identify a major site of Akt phosphorylation as serine 304 of POSH, which lies within the Rac-binding domain. We further show that phosphorylation of POSH results in a decreased ability to bind activated Rac, as does phosphomimetic S304D and S304E mutation of POSH. S304D mutant POSH also shows a strongly reduced ability to induce apoptosis. These findings identify a novel mechanism by which Akt promotes cell survival.     

The role of LEF1 in endometrial gland formation and carcinogenesis

Endometrial carcinoma is the most common gynecologic cancer, yet the mechanisms underlying this disease process are poorly understood. We hypothesized that Lef1 is required for endometrial gland formation within the uterus and is overexpressed in endometrial cancer. Using Lef1 knockout (KO) mice, we compared uterine gland development to wild-type (WT) controls, with respect to both morphology and expression of the Lef1 targets, cyclin D1 and MMP7. We characterized the dynamics of Lef1 protein expression during gland development and the mouse estrus cycle, by immunostaining and Western blot. Finally, we investigated the roles of cyclin D1 and MMP7 in gland and cancer formation in the mouse, and assessed the relevance of Lef1 to human cancer by comparing expression levels in cancerous and normal endometrial tissues. Lef1 upregulation in mouse endometrium correlates with the proliferative stages of the estrus cycle and gland development during the neonatal period. WT mice endometrial glands began to develop by day 5 and were easily identified by day 9, whereas Lef1 KO mice endometrial glands had not developed by day 9 although the endometrial lining was intact. We found that during gland development cyclin D1 is elevated and localized to the gland buds, and that this requires the presence of Lef1. We also noted that Lef1 protein was expressed at higher levels in endometrial cancers within mice and humans when compared to normal endometrium. Our loss-of-function data indicate that Lef1 is required for the formation of endometrial glands in the mouse uterus. Lef1 protein elevation corresponds to gland formation during development, and varies cyclically with the mouse estrus cycle, in parallel with gland regeneration. Finally, Lef1 is overexpressed in human and mouse endometrial tumors, consistent with it playing a role in gland proliferation.