Aveline Hewetson

Minocycline reduces ethanol drinking.

Alcoholism is a disease characterized by continued alcohol consumption despite recurring negative consequences. Thus, medications that reduce the drive to consume alcohol can be beneficial in treating alcoholism. The neurobiological systems that regulate alcohol consumption are complex and not fully understood. Currently, medications are available to treat alcoholism that act either by causing accumulation of a toxic metabolite of ethanol, or by targeting specific transmitter receptors. The purpose of our study was to investigate a new potential therapeutic pathway, neuroimmune interactions, for effects on ethanol consumption. We hypothesized that neuroimmune activity of brain glia may have a role in drinking. We utilized minocycline, a second generation tetracycline antibiotic that has immune modulatory actions, to test our hypothesis because it is known to suppress microglia, and to a lesser extent astroglia, activity following many types of insults to the brain. Treatment with 50mg/kg minocycline significantly reduced ethanol intake in male and female C57Bl/6J mice using a free choice voluntary drinking model. Saline injections did not alter ethanol intake. Minocycline had little effect on water intake or body weight change. The underlying mechanism whereby minocycline reduced ethanol intake requires further study. The results suggest that drugs that alter neuroimmune pathways may represent a new approach to developing additional therapies to treat alcoholism.

Prolactin and growth hormone signaling.

Prolactin (PRL) and growth hormone (GH) act by way of their receptors as either hormones (systemically) or cytokines (locally). The Jak2/Stat5 pathway is the principal route by which PRL/GH activate target genes. The availability of knockout mice for each member of this signaling cascade has provided opportunities to understand their unique interactions. Jak2 is important in alternative signal transduction schema such as the MAP kinase and PI3K/Akt pathways. The putative Jak2/RUSH pathway is based on the fact that RUSH mediates the ability of PRL to augment progesterone-dependent gene transcription. New evidence shows that suppressors, regulators, and degraders control Jak2/Stat5. This review focuses on the most recent advances in the field of PRL/GH signal transduction.

An Sp1-NF-Y/progesterone receptor DNA binding-dependent mechanism regulates progesterone-induced transcriptional activation of the rabbit RUSH/SMARCA3 gene

Steroids regulate alternative splicing of rabbit RUSH/SMARCA3, an SWI/SNF-related transcription factor. Transactivation was evaluated in 2057 bp of genomic sequence. Truncation analysis identified a minimal 252-bp region with strong basal promoter activity in transient transfection assays. The size of the 5′-untranslated region (233 bp) and the transcription start site were determined by primer extension analysis. The transcription start site mapped to a consensus initiator (Inr) element in a TATA-less region with a downstream promoter element (+29). These elements were authenticated by mutation/deletion analysis. The Inr/downstream promoter element combination is conserved in the putative core promoter (-35/+35) of the human ortholog, suggesting that transcription initiation is similarly conserved. Two Sp1 sites that are also conserved in the putative promoter of human SMARCA3 and a RUSH binding site (-616/-611) that is unique to the rabbit promoter repress basal transcription. These sites were variously authenticated by gel shift and chromatin immunoprecipitation assays. Analysis of the proximal promoter showed the -162/+90 region was required for progesterone responsiveness in transient transfection assays. Subsequent mutation/deletion analysis revealed a progesterone receptor half-site mediated induction by progesterone. An overlapping Y-box (in the reverse ATTGG orientation) repressed basal transcription and progesterone-induced transcriptional activation in the presence of the Sp1 sites. The specificity of progesterone receptor and transcription factor NF-Y binding were authenticated by gel shift assays. Chromatin immunoprecipitation assays confirmed the Y-box effects were mediated in a DNA binding-dependent fashion. This represents a unique regulatory scenario in which ligand-dependent transactivation by the progesterone receptor is subject to Sp1/NF-Y repression.

Altered sodium pump alpha and gamma subunit gene expression in nephron segments from hypertensive rats.

OBJECTIVE To determine the qualitative and quantitative expression of alpha and gamma sodium pump subunits in whole kidney and nephron segment RNA from Sprague Dawley rats, spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. DESIGN A novel reverse transcription polymerase chain reaction technique was devised which provides accurate and precise measurement of the number of molecules of specific transcript abundance, a measurement of gene expression. This allows the quantitative comparison of multiple samples across multiple subjects and, since the estimates are accurate rather than relative, can also be used to make quantitative comparisons across expressed genes, such as isoforms and subunits of the heterotrimeric renal sodium pump. METHODS We examined which catalytic isoforms were expressed and then quantified transcript abundance in whole kidney and convoluted and straight segments of the proximal tubule. RESULTS Alpha 1 and gamma transcripts, but not alpha 2, alpha 3 or alpha 4 isoforms, were consistently observed in nephron segments. Levels of alpha 1 were lower in kidney RNA from 15-16-week-old SHR than in WKY rats of the same age (P = 0.001), but were not different between SHR and WKY in 4-5-week-old animals. No significant difference was observed in gamma subunit abundance in kidney RNA from 4-5-week-old animals; however, at 15-16 weeks, the expression in SHR was one-third that in WKY rats (P = 0.003). In proximal convoluted tubules from 4-5-week-old animals, the level of alpha 1 RNA expression was lower (P = 0.03) in SHR than in WKY rats. In addition, levels of alpha 1 in proximal straight tubule from the 4-5-week-old SHR were also lower than in WKY rats (P = 0.02). This difference was even greater in 15-16-week-old animals: in SHR, alpha 1 expression was less than 20% of the level of expression in WKY rats (P = 0.0003). Expression of the gamma subunit exhibited a similar pattern of downregulation in SHR. In RNA from proximal convoluted tubules and proximal straight tubules from both 4-5- and 15-16-week-old animals, expression of the gamma subunit was demonstrated to be significantly lower in SHR than in WKY rats. CONCLUSION The results indicate a coordinate reduction in the abundance of sodium pump alpha and gamma subunits in the proximal tubules of SHR, which occurs early during the development of hypertension.

Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency.

Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.

Alcohol-induced One-carbon Metabolism Impairment Promotes Dysfunction of DNA Base Excision Repair in Adult Brain

Background: DNA repair dysfunction leads to genomic instability and neuron death. Results: Long term alcohol exposure results in reduced DNA repair, increased DNA damage, and neuron death in adult brain. Conclusion: Long term alcohol exposure in adult brain promotes genomic instability mediated by impairment in one-carbon metabolism. Significance: This is the first demonstration of alcohol-induced genomic instability in brain. The brain is one of the major targets of chronic alcohol abuse. Yet the fundamental mechanisms underlying alcohol-mediated brain damage remain unclear. The products of alcohol metabolism cause DNA damage, which in conditions of DNA repair dysfunction leads to genomic instability and neural death. We propose that one-carbon metabolism (OCM) impairment associated with long term chronic ethanol intake is a key factor in ethanol-induced neurotoxicity, because OCM provides cells with DNA precursors for DNA repair and methyl groups for DNA methylation, both critical for genomic stability. Using histological (immunohistochemistry and stereological counting) and biochemical assays, we show that 3-week chronic exposure of adult mice to 5% ethanol (Lieber-Decarli diet) results in increased DNA damage, reduced DNA repair, and neuronal death in the brain. These were concomitant with compromised OCM, as evidenced by elevated homocysteine, a marker of OCM dysfunction. We conclude that OCM dysfunction plays a causal role in alcohol-induced genomic instability in the brain because OCM status determines the alcohol effect on DNA damage/repair and genomic stability. Short ethanol exposure, which did not disturb OCM, also did not affect the response to DNA damage, whereas additional OCM disturbance induced by deficiency in a key OCM enzyme, methylenetetrahydrofolate reductase (MTHFR) in Mthfr+/− mice, exaggerated the ethanol effect on DNA repair. Thus, the impact of long term ethanol exposure on DNA repair and genomic stability in the brain results from OCM dysfunction, and MTHFR mutations such as Mthfr 677C→T, common in human population, may exaggerate the adverse effects of ethanol on the brain.

Prolactin Signals Through RUSH/SMARCA3 in the Absence of a Physical Association with Stat5a

Abstract Jak/Stat-mediated prolactin signal transduction culminates in the sequence-selective binding of Stat5a. However, in the absence of Stat-binding sites, a RUSH-binding element mediates the prolactin signal in the rabbit uteroglobin promoter. Speculation about the existence of a Jak/RUSH pathway prompted this series of experiments to examine potential interactions between RUSH and Stat5a. Profiles of Jak/Stat pathway-specific genes by RT-PCR showed that mRNA for Jak2 and Stat5a is expressed in the endometrium of estrous, progesterone-treated, and 5-day pseudopregnant rabbits. Interspecies microarrays showed that transcripts for Stat5a were present at equal concentrations in the endometrium regardless of hormone treatment. The absence of a physical interaction between RUSH and individual Stat proteins bound to enhancer sites was demonstrated with transcription factor interaction arrays. These studies confirm that transmission of the prolactin signal through RUSH occurs in the absence of a physical association with Stat5a. Although a strong physical interaction between RUSH and Egr-1 was identified with the same arrays, no Egr-1 consensus sites were found in the region of the uteroglobin promoter (−175/−80) that contains the authentic RUSH site. Because the major transducer molecules (Jak2, Stat5a) are activated by tyrosine phosphorylation, Western analysis of immunoprecipitated samples, and gel shift assays were used to show that tyrosine phosphorylation is required for RUSH-DNA binding. The precise role for Jak2 in this process remains undefined. By comparison, serine-threonine-specific protein phosphorylation had no effect on RUSH-DNA binding.

Prolactin-induced Jak2 phosphorylation of RUSH: a key element in Jak/RUSH signaling

Jak2/Stat-mediated prolactin signaling culminates in Stat5a-DNA-binding. However, not all Jak2-dependent genes have Stat5 sites. Western analysis with inhibitors showed Jak2 is a proximal intermediate in prolactin-induced RUSH phosphorylation. Transfection assays with HRE-H9 cells showed the RUSH-binding site mediated the ability of prolactin to augment progesterone-dependent transcription of the RUSH gene. Jak2 inhibitors or targeted RUSH-site mutation blocked the prolactin effect. RUSH co-immunoprecipitated with phospho-Jak2 from nuclear extracts. Jak2 inhibitors abolished the nuclear pool of phospho-RUSH not the nuclear content of RUSH in HRE-H9 cells. Nucleolar-affiliated partners, e.g. nucleolin, were identified by microLC/MS/MS analysis of nuclear proteins that co-immunoprecipitated with RUSH/GST-RING. RUSH did not exclusively co-localize with fibrillarin to the nucleolus. MG-132 (proteasomal inhibitor) failed to block Tyrene CR4-mediated decrease in phospho-RUSH, and did not promote RUSH accumulation in the nucleolus. These studies authenticate prolactin-dependent Jak2 phosphorylation of RUSH, and provide functional implications on the RUSH network of nuclear interactions.

After chromatin is SWItched-on can it be RUSHed?

Repressive chromatin must be remodeled to allow for transcriptional activation of genes in eukaryotic cells. Factors that alter chromatin structure to permit access of transcriptional activators, RNA polymerase II and the polymerase-associated general transcription factors to nucleosomal promoter sequences are as highly conserved as the basic mechanism of transcription. One group of promoter restructuring factors that perturbs chromatin in an ATP-dependent manner includes NURF, CHRAC, ACF, the SWI/SNF complex, and SWI/SNF-related proteins. Each member of this group contains a subunit homologous to the DNA-dependent ATPase; however, their individual mechanisms of action are unique. The small amount of SWI/SNF complex (100-200 copies/cell), its affiliation with a select number of inducible genes, and its interaction with the glucocorticoid and estrogen receptors, suggests the SWI/SNF complex might be preferentially targeted to active promoters. The SWI/SNF-related family of RUSH proteins which includes RUSH-1alpha and beta, hHLTF, HIP116, Zbu1, P113, and the transcription factor RUSH-1alpha isolog has been implicated as a highly conserved DNA binding site-specific ATPase.

Progesterone regulation of RUSH/SMARCA3/HLTF includes DNA looping

RUSH/SMARCA3 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A, member 3) is capable of sequence-selective DNA binding and ATP-dependent DNA unwinding. In rabbit uterine epithelial cells, RUSH-1alpha (113 kDa) is the progesterone-dependent splice variant and RUSH-1beta (95 kDa) is the oestrogen-dependent splice variant. Rabbit RUSH/SMARCA3 mRNA is primarily regulated at the proximal promoter (-162/+90) via a PRE (progesterone-response element) half-site/overlapping Y-box domain (-38/-26) and two Sp (specificity protein) 3 sites centred at -128 and -58. We investigated hormone regulation by exploring binding of transcription factors to a putative RUSH/SMARCA3 site (-616/-611) and the distal Sp3 (-131/-126) site. In response to progesterone, RUSH-1alpha binds the RUSH site and the Sp3 site becomes a functional binding site for Egr-1 (early growth-response gene product 1)/Sp (specificity protein)1/3/MAZ (Myc-associated zinc-finger protein)/MZF1 (myeloid zinc finger 1)/c-Rel. TransSignal TF-TF Interaction Arrays, supershift assays and ChIP (chromatin immunoprecipitation) analyses confirmed strong physical interactions between RUSH and Egr-1/c-Rel. Higher-order long-range interactions between RUSH and the Egr-1/c-Rel derivative of the anisotropic flexibility of the intervening DNA sequence were shown with 3C (chromosome conformation capture) assays. Transient transfection assays with mutant constructs showed the co-operative interaction between RUSH and Egr-1 mediates repression by c-Rel. Thus DNA-bound RUSH/SMARCA3 communicates with its own proximal promoter by looping the intervening DNA. Moreover, progesterone-dependent DNA looping is an adjunct to progesterone induction of the RUSH/SMARCA3 gene because the availability of RUSH isoforms and relevant binding partners is progesterone-regulated.