Role of Short Chain Fatty Acid Receptors in Intestinal Physiology and Pathophysiology.

Nutrient sensing is a mechanism for organisms to sense their environment. In larger animals, including humans, the intestinal tract is a major site of nutrient sensing for the body, not surprisingly, as this is the central location where nutrients are absorbed. In the gut, bacterial fermentation results in generation of short chain fatty acids (SCFAs), a class of nutrients, which are sensed by specific membrane bound receptors, FFA2, FFA3, GPR109a, and Olfr78. These receptors are expressed uniquely throughout the gut and signal through distinct mechanisms. To date, the emerging data suggests a role of these receptors in normal and pathological conditions. The overall function of these receptors is to regulate aspects of intestinal motility, hormone secretion, maintenance of the epithelial barrier, and immune cell function. Besides in intestinal health, a prominent role of these receptors has emerged in modulation of inflammatory and immune responses during pathological conditions. Moreover, these receptors are being revealed to interact with the gut microbiota. This review article updates the current body of knowledge on SCFA sensing receptors in the gut and their roles in intestinal health and disease as well as in whole body energy homeostasis. © 2017 American Physiological Society. Compr Physiol 8:1091-1115, 2018.

MicroRNA-21 Contributes to Reduced Microvascular Function in Binge Drinking Young Adults.

BACKGROUND: Binge drinking is associated with increased risk for cardiovascular (CV) disease. MicroRNA-21 (miR21) is up-regulated in the setting of excessive alcohol consumption and CV disease. Therefore, the goal of this study was to examine the vasodilatory responses to flow and acetylcholine (ACh) in the absence and presence of an anti-miR21 inhibitor in the microcirculation of young adult repeated binge drinkers (BDs). METHODS: Gluteal subcutaneous adipose tissue biopsies were obtained from young adults (18 to 30 years, n = 35 vessels from BDs and n = 28 vessels from abstainers). Resistance arteries (RAs) were isolated, incubated with anti-miR21 or a negative control (NC) to miR21 (12 hours; 50 nM), and lumen diameters measured with video microscopy. miR21 of adipose tissues was determined by quantitative polymerase chain reaction. RESULTS: Flow-induced dilation and ACh-induced dilation (AChID) were reduced in BDs as compared to abstainers. The miR21 inhibitor but not the NC abrogated these effects in BDs, but did not affect vasodilation in abstainers. Nitric oxide synthase inhibition with L-NAME reduced vasodilation in abstainers but not in BDs. In BDs, vasodilation was reduced by L-NAME in the presence of anti-miR21 but not the NC. Scavenging the reactive oxygen species, hydrogen peroxide with polyethylene glycol catalase reduced dilation in BDs but did not affect the restored dilation by the miR21 inhibitor. Maximum dilation to papaverine (endothelium independent) was similar between groups and unaffected by pharmacological inhibition. Finally, vascular endogenous miR21 was increased in BDs compared to abstainers. CONCLUSIONS: Endogenous miR21 is increased in RAs of young BDs, leading to reduced flow and AChID in the microcirculation.

Heterogeneous nuclear ribonucleoprotein A1 is a novel cellular target of atrial natriuretic peptide signaling in renal epithelial cells.

Two classes of guanylyl cyclases (GC) form intracellular cGMP. One is a receptor for atrial natriuretic peptide (ANP) and the other for nitric oxide (NO). The ANP receptor guanylyl cyclase (GC-A) is a membrane-bound, single subunit protein. Nitric oxide activated or soluble guanylyl cyclases (NOGC) are heme-containing heterodimers. These have been shown to be important in cGMP mediated regulation of arterial vascular resistance and renal sodium transport. Recent studies have shown that cGMP produced by both GCs is compartmentalized in the heart and vascular smooth muscle cells. To date, however, how intracellular cGMP generated by ANP and NO is compartmentalized and how it triggers specific downstream targets in kidney cells has not been investigated. Our studies show that intracellular cGMP formed by NO is targeted to cytosolic and cytoskeletal compartments whereas cGMP formed by ANP is restricted to nuclear and membrane compartments. We used two dimensional difference in gel electrophoresis and MALDI-TOF/TOF to identify distinct sub-cellular targets that are specific to ANP and NO signaling in HK-2 cells. A nucleocytoplasmic shuttling protein, heterogeneous nuclear ribonucleo protein A1 (hnRNP A1) is preferentially phosphorylated by ANP/cGMP/cGK signaling. ANP stimulation of HK-2 cells leads to increased cGK activity in the nucleus and translocation of cGK and hnRNP A1 to the nucleus. Phosphodiestaerase-5 (PDE-5 inhibitor) sildenafil augmented ANP-mediated effects on hnRNPA1 phosphorylation, translocation to nucleus and nuclear cGK activity. Our results suggest that cGMP generated by ANP and SNAP is differentially compartmentalized, localized but not global changes in cGMP, perhaps at different sub-cellular fractions of the cell, may more closely correlate with their effects by preferential phosphorylation of cellular targets.

Implication of microRNAs in atrial natriuretic peptide and nitric oxide signaling in vascular smooth muscle cells.

MicroRNAs (miRs) are endogenous small RNA molecules that suppress gene expression by binding to complementary sequences in the 3' untranslated regions of their target genes. miRs have been implicated in many diseases, including heart failure, ischemic heart disease, hypertension, cardiac hypertrophy, and cancers. Nitric oxide (NO) and atrial natriuretic peptide (ANP) are potent vasorelaxants whose actions are mediated through receptor guanylyl cyclases and cGMP-dependent protein kinase. The present study examines miRs in signaling by ANP and NO in vascular smooth muscle cells. miR microarray analysis was performed on human vascular smooth muscle cells (HVSMC) treated with ANP (10 nM, 4 h) and S-nitroso-N-acetylpenicillamine (SNAP) (100 µM, 4 h), a NO donor. Twenty-two shared miRs were upregulated, and 21 shared miRs were downregulated, by both ANP and SNAP (P < 0.05). Expression levels of four miRs (miRs-21, -26b, -98, and -1826), which had the greatest change in expression, as determined by microarray analysis, were confirmed by quantitative RT-PCR. Rp-8-Br-PET-cGMPS, a cGMP-dependent protein kinase-specific inhibitor, blocked the regulation of these miRs by ANP and SNAP. 8-bromo-cGMP mimicked the effect of ANP and SNAP on their expression. miR-21 was shown to inhibit HVSMC contraction in collagen gel lattice contraction assays. We also identified by computational algorithms and confirmed by Western blot analysis new intracellular targets of miR-21, i.e., cofilin-2 and myosin phosphatase and Rho interacting protein. Transfection with pre-miR-21 contracted cells and ANP and SNAP blocked miR-21-induced HVSMC contraction. Transfection with anti-miR-21 inhibitor reduced contractility of HVSMC (P < 0.05). The present results implicate miRs in NO and ANP signaling in general and miR-21 in particular in cGMP signaling and vascular smooth muscle cell relaxation.

Evidence for cross-talk between atrial natriuretic peptide and nitric oxide receptors.

Guanylyl cyclases (GCs), a ubiquitous family of enzymes that metabolize GTP to cyclic GMP (cGMP), are traditionally divided into membrane-bound forms (GC-A-G) that are activated by peptides and cytosolic forms that are activated by nitric oxide (NO) and carbon monoxide. However, recent data has shown that NO activated GC's (NOGC) also may be associated with membranes. In the present study, interactions of guanylyl cyclase A (GC-A), a caveolae-associated, membrane-bound, homodimer activated by atrial natriuretic peptide (ANP), with NOGC, a heme-containing heterodimer (alpha/beta) beta1 isoform of the beta subunit of NOGC (NOGCbeta1) was specifically focused. NOGCbeta1 co-localized with GC-A and caveolin on the membrane in human kidney (HK-2) cells. Interaction of GC-A with NOGCbeta1 was found using immunoprecipitations. In a second set of experiments, the possibility that NOGCbeta1 regulates signaling by GC-A in HK-2 cells was explored. ANP-stimulated membrane guanylyl cyclase activity (0.05 +/- 0.006 pmol/mg protein/5 min; P < 0.01) and intra cellular GMP (18.1 +/- 3.4 vs. 1.2 +/- 0.5 pmol/mg protein; P < 0.01) were reduced in cells in which NOGCbeta1 abundance was reduced using specific siRNA to NOGCbeta1. On the other hand, ANP-stimulated cGMP formation was increased in cells transiently transfected with NOGCbeta1 (530.2 +/- 141.4 vs. 26.1 +/- 13.6 pmol/mg protein; P < 0.01). siRNA to NOGCbeta1 attenuated inhibition of basolateral Na/K ATPase activity by ANP (192 +/- 22 vs. 92 +/- 9 nmol phosphate/mg protein/min; P < 0.05). In summary, the results show that NOGCbeta1 and GC-A interact and that NOGCbeta1 regulates ANP signaling in HK-2 cells. The results raise the novel possibility of cross-talk between NOGC and GC-A signaling pathways in membrane caveolae.

GAGE, an antiapoptotic protein binds and modulates the expression of nucleophosmin/B23 and interferon regulatory factor 1.

The GAGE family of highly related tumor antigens is expressed in a variety of tumors. This albeit silent gene expression resulted in resistance of cells to various apoptotic agents such as Fas, interferon-gamma, Taxol, or gamma-radiation. We now report that GAGE overexpression in either HeLa (expressing endogenous GAGE) or HEK293 (devoid of GAGE expression) rendered those cells unsusceptible to cell death induced by IFN-gamma. We investigated the underlying mechanism of GAGE-induced cell survival upon treatment with IFN-gamma in this report. We showed that GAGE overexpression resulted in down-regulation of a key player of IFN-gamma-signaling pathway, interferon regulatory factor 1 (IRF1), and its target genes caspase-1 and caspase-7. An interaction between GAGE and IRF1 is detected in cells. Furthermore, GAGE interacted with a multifunctional protein nucleophosmin (NPM)/B23 and increased its abundance by stabilizing the protein. Increased level of NPM/B23 in conjunction with decreased level of IRF1 could aid GAGE-induced resistance to IFN-gamma. Our results suggest that GAGE could rescue cell death induced by IFN-gamma by altering the level of key players in cell death pathways. As GAGE is silent in most healthy tissues, targeting GAGE could result in therapeutic interventions in cancer therapy.

Nrf2 is an inhibitor of the Fas pathway as identified by Achilles' Heel Method, a new function-based approach to gene identification in human cells.

Here we describe the Achilles' Heel Method (AHM), a new function-based approach for identification of inhibitors of signaling pathways, optimized for human cells. The principle of AHM is the identification of 'sensitizing' cDNAs based on their decreased abundance following selection. As a proof of principle, we have employed AHM for the identification of Fas/CD95/APO-1 pathway inhibitors. HeLa cells were transfected with an antisense cDNA expression library in an episomal vector followed by selection with a suboptimal dose of the apoptotic inducer. Antisense inactivation of Fas inhibitors rendered the cells more sensitive to apoptosis resulting in their preferential death and consequent loss of their sensitizing episomes that were identified by subtraction. We show that the resulting products were enriched for sensitizing cDNAs as seven out of eight candidates tested were confirmed as inhibitors of Fas-induced killing either by transfection or by pharmacological inhibition. Furthermore, we demonstrate by multiple approaches that one candidate, NF-E2 related factor 2 (Nrf2), is an inhibitor of Fas-induced apoptosis. Inactivation of Nrf2 by antisense or by a membrane permeable dominant-negative polypeptide sensitized cells while overexpression of Nrf2 protected cells from Fas-induced apoptosis. In addition, dicumarol, an inhibitor of the phase II detoxifying enzyme NQO1, a downstream target of Nrf2, sensitized cells. Nrf2 induces the production of Glutathione (GSH) and we demonstrated that N-acetyl L-cysteine (NAC), a precursor to GSH, protected cells from Fas-mediated killing. Taken together, AHM is a powerful approach for the identification of inhibitors of a signaling pathway with a low rate of false positives that opens new avenues for function profiling of human genes and discovery of new drug targets.

Cell death inhibiting RNA (CDIR) derived from a 3'-untranslated region binds AUF1 and heat shock protein 27.

Regulators of programmed cell death were previously identified using a technical knockout genetic screen. Among the elements that inhibited interferon-gamma-induced apoptosis of HeLa cells was a 441-nucleotide fragment derived from the 3'-untranslated region (UTR) of KIAA0425, a gene of unknown function. This fragment was termed cell death inhibiting RNA (CDIR). Deletion and mutation analyses of CDIR were employed to identify the features required for its anti-apoptotic activity. Single nucleotide alterations within either copy of the duplicated U-rich motif found in the CDIR sequence abolished the anti-apoptotic activity of CDIR and altered its in vitro association with a protein complex. Further analysis of the CDIR-binding complex indicated that it contained heat shock protein 27 (Hsp27) and the regulator of mRNA turnover AUF1 (heterogeneous nuclear ribonucleoprotein D). In addition, recombinant AUF1 bound directly to CDIR. Furthermore, expression of another AUF1-binding RNA element, derived from the 3'-UTR of c-myc, inhibited apoptosis. We also demonstrate that the level and the stability of p21(waf1/Cip1/sdi1) mRNA, a target of AUF1 with anti-apoptotic activity, were increased in CDIR-transfected cells. The level of mRNA and protein of Bcl-2, another anti-apoptotic gene, containing an AUF1 binding site in its 3'-UTR was also increased in CDIR-transfected cells. Our data suggest that AUF1 regulates apoptosis by altering mRNA turnover. We propose that CDIR inhibits apoptosis by acting as a competitive inhibitor of AUF1, preventing AUF1 from binding to its targets.