Objective: Obesity is a worldwide epidemic and current treatments have limited success thus, novel therapies are warranted. Our objective was to determine whether the prorenin/renin receptor [(P)RR] is implicated in obesity. Methods: Mice received a normal or high-fat/high-carbohydrate diet with the handle region peptide (HRP), a (P)RR blocker, or saline for 10 weeks. Post-menopausal non-diabetic obese women were enrolled in the Complication Associated with Obesity Study and were classified as insulin-resistant (IRO) or -sensitive (ISO) using a hyperinsulinemic-euglycemic clamp. Results: In mice, obesity increased the (P)RR by twofold in adipose tissue. Likewise, renin increased by at least twofold. The HRP reduced weight gain in obese mice by 20% associated to a 19% decrease in visceral fat. This was accompanied by a 48% decrease in leptin mRNA in fat and 33% decrease in circulating leptin. Inflammatory markers were also decreased by the HRP treatment. HRP normalized triglyceridemia and reduced insulinemia by 34% in obese mice. Interestingly, we observed a 33% increase in (P)RR mRNA in the fat of IRO women compared to ISO. Conclusions: This is the first report of a potential implication in obesity of the (P)RR which may be a novel therapeutic target.

The monoclonal antibody CC3 recognizes a phosphorylated epitope present on an interphase protein of 255 kDa. Previous work has shown that p255 is localized mainly to nuclear speckles and remains associated with the nuclear matrix scaffold following extraction with non-ionic detergents, nucleases and high salt. The association of p255 with splicing complexes is suggested by the finding that mAb CC3 can inhibit in vitro splicing and immunoprecipitate pre-messenger RNA and splicing products. Small nuclear RNA immunoprecipitation assays show that p255 is a component of the U5 small nuclear ribonucleoprotein (snRNP) and the [U4/U6.U5] tri-snRNP complex. In RNase protection assays, mAb CC3 immunoprecipitates fragments containing branch site and 3' splice site sequences. As predicted for a [U4/U6.U5]-associated component, the recovery of the branch site-protected fragment requires binding of U2 snRNP and is inhibited by EDTA. p255 may correspond to the previously identified p220 protein, the mammalian analogue of the yeast PRP8 protein. Our results suggest that changes in the phosphorylation of p255 may be part of control mechanisms that interface splicing activity with nuclear organization.

We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca2+ signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP2). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca2+ mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4 -d]pyrimidine (PP1). Peak Ca2+ responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca2+ responses to 5-HT and Ca2+ influx resulting from emptying of Ca2+ stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP2 in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca2+ signaling by regulating PIP2 levels and Ca2+ influx.

To our knowledge, we are the first to clearly demonstrate that exercise training both before and during gestation can reduce preeclampsia features in a mouse model. Consequently, women at risk for this disease could benefit from exercise training to protect themselves and their future fetuses.

The multisubstrate docking protein, growth-factor-receptor-bound protein 2-associated binder 1 (Gab1), which is phosphorylated on tyrosine residues following activation of receptor tyrosine kinases and cytokine receptors, regulates cell proliferation, survival and epithelial morphogenesis. Gab1 is also tyrosine phosphorylated following activation of G-protein-coupled receptors (GPCRs) where its function is poorly understood. To elucidate the role of Gab1 in GPCR signalling, we investigated the mechanism by which the type A endothelin-1 (ET-1) GPCR induced tyrosine phosphorylation of Gab1. Tyrosine phosphorylation of Gab1 induced by endothelin-1 was inhibited by PP1, a pharmacological inhibitor of Src-family tyrosine kinases. ET-1-induced Gab1 tyrosine phosphorylation was also inhibited by LY294002, which inhibits phosphoinositide 3-kinase (PI 3-kinase) enzymes. Inhibition of Src-family tyrosine kinases or PI 3-kinase also inhibited ET-1-induced activation of the mitogen activated protein kinase family member, extracellular signal-regulated kinase (ERK) 1. Thus we determined whether Gab1 regulated ET-1-induced ERK1 activation. Overexpression of wild-type Gab1 potentiated ET-1-induced activation of ERK1. Structure–function analyses of Gab1 indicated that mutant forms of Gab1 that do not bind the Src homology (SH) 2 domains of the p85 adapter subunit of PI 3-kinase or the SH2-domain-containing protein tyrosine phosphatase 2 (SHP-2) were impaired in their ability to potentiate ET-1-induced ERK1 activation. Taken together, our data indicate that PI 3-kinase and Src-family tyrosine kinases regulate ET-1-induced Gab1 tyrosine phosphorylation, which, in turn, induces ERK1 activation via PI 3-kinase- and SHP-2-dependent pathways.

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The CREB-binding protein (CBP) plays a central role in the regulation of gene expression by several different second messenger pathways including serum growth factors, cAMP and phorbol esters. CBP specifically binds to the phosphorylated forms of CREB and c-Jun and is thought to activate transcription through a Cterminal activation domain. In this report, we demonstrate that the C terminus of CBP is dispensable for its ability to stimulate phospho-CREB activity, and, further, that the deletion of this domain produces highly active, mutant forms of CBP. The novel N-terminal activation identified by this deletional analysis consists of the first 714 amino acids of CBP and is sufficient for high levels of transcriptional activity. This domain is also capable of stimulating the activity of a second cAMPregulated factor, ATF-1. Surprisingly, ATF-1 activity is not significantly stimulated by full-length CBP suggesting that the C-terminal domain of CBP may also serve to regulate ATF-1/CBP activity. Additionally, the demonstration that one of our hyperactive CBP mutants is able to activate a nonphosphorylatable mutant of CREB (M1 CREB) provides the first evidence that CBP may play a role in regulating the basal transcriptional activity of CREB.Hormonally induced changes in the levels of intracellular cyclic AMP (cAMP) regulate the expression of many cellular and viral genes through a common promoter element termed the CRE 1 (1). The activity of the CRE is, in turn, governed by a large family of transcription factors known as CREB/ATF proteins. These proteins share several common structural features including a DNA binding and dimerization motif, known as the bZIP domain, and transcriptional activation domains located in the N terminus of the molecule. Although poorly conserved overall, these activation domains share consensus phosphorylation sites for a variety of protein kinases suggesting that their activity, like that of many bZIP factors, is highly regulated by phosphorylation (2, 3).The transcription factor CREB (cAMP response elementbinding protein) represents the prototypical member of this family and it, along with the related factors ATF-1 and CREM, mediates many aspects of cAMP-regulated gene expression (4).The activation domains of all three of these proteins are highly homologous and can be divided into three basic elements, a kinase-inducible domain (KID), which contains a consensus phosphorylation site for the cAMP-dependent kinase (PKA), and two glutamine-rich domains (Q1 and Q2) flanking the KID. The phosphorylation of the CREB KID at a consensus PKA site (serine 133) is required for full transcriptional activity and mutation of this site effectively blocks stimulated activity (5). Although CREB is capable of interacting directly with several components of the basic transcriptional apparatus including TFIIB and TFIID, these interactions are not regulated by phosphorylation (6, 7), and it has been unclear how phosphorylation of the CREB activation domain results in enhanced transcriptional activity. The r...

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