Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch.

The serine-threonine kinase Akt plays a fundamental role in cell survival, metabolism, proliferation, and migration. To keep these essential processes under control, Akt activity and stability must be tightly regulated; otherwise, life-threatening conditions might prevail. Although it is well understood that phosphorylation regulates Akt activity, much remains to be known about how its stability is maintained. Here, we characterize BAG5, a chaperone regulator, as a novel Akt-interactor and substrate that attenuates Akt stability together with Hsp70. BAG5 switches monoubiquitination to polyubiquitination of Akt and increases its degradation caused by Hsp90 inhibition and Hsp70 overexpression. Akt interacts with BAG5 at the linker region that joins the first and second BAG domains and phosphorylates the first BAG domain. The Akt-BAG5 complex is formed in serum-starved conditions and dissociates in response to HGF, coincident with BAG5 phosphorylation. BAG5 knockdown attenuated Akt degradation and facilitated its activation, whereas the opposite effect was caused by BAG5 overexpression. Altogether, our results indicate that Akt stability and signaling are dynamically regulated by BAG5, depending on growth factor availability.

AT1R antagonism improves metabolic and vascular changes of obesity-induced gestational diabetes mellitus.

Obesity can overload glucose homeostasis and physiological insulin resistance during gestation which increases the risk of complications like diabetes mellitus or preeclampsia. Angiotensin II /AT1 receptors are involved in the pathogenesis of vascular effects of obesity/insulin resistance but its role during gestation is not as clear. We sought to determine angiotensin II- AT1R participation on a diet-induced gestational diabetes mellitus (GDM) experimental model. Female Wistar rats were fed with a standard or hypercaloric diet for 7 weeks. Half of the animals were mated and became pregnant from week 4-7. Animals were treated with saline, irbesartan (30 mg/kg) or metformin (320 mg/kg) for the last two weeks of the protocol. Weight gain, systolic blood pressure (BP), oral glucose tolerance test and vascular contractility were measured at the last day of the protocol (day 19-20 of pregnancy). Hypercaloric diet increased blood glucose, impaired glucose tolerance test, and increased BP in pregnant rats, fulfilling criteria for GDM. Both drugs decreased impaired GTT and relative hyperglycemia. Metformin had no effect on BP but prevented weight increase. In isolated aortas, irbesartan and metformin decreased vasoconstriction only of non-pregnant hypercaloric diet fed animals. Results support angiotensin II/ AT1R involvement in BP and glucose homeostasis disturbances observed in present GDM model. Also, provide evidence that a hypercaloric diet can mask pregnancy´s physiological hypoglycemia and hypotension without surpassing non-pregnant values. Then, we conclude overweight during pregnancy causes subtle but significant vascular and metabolic damage that might be dismissed in clinical practice.

Time course of angiotensin II dependent vascular and metabolic effects of preeclampsia.

Pregnancy is characterized by a blunted pressor response to angiotensin II that is progressively lost during preeclampsia complicated pregnancies. Renin angiotensin system (RAS) plays a pivotal role in cardiovascular and renal function but its role in normal and pathological pregnancy is far from being clarified. It is not as clear if hypertension and particularly pregnancy-induced hypertension as the initial event, can trigger some of the metabolic syndrome components, and if these changes are angiotensin II mediated. The aim of this study was to determine the time course of angiotensin II contribution to the vascular and eventual metabolic changes of preeclampsia. An experimental model was developed by reducing feto-placental circulation through a subrenal aorta coarctation before pregnancy in rats. Control and pregnant (preeclamptic) animals were treated with captopril (5mg/kgpo) or saline solution for 21, 14 or 7days before delivery, and their body weight, plasma glucose andblood pressure were registered. Phenylephrine (Phe) induced contraction was evaluated using isolated aorta rings. Preeclampsia increased blood pressure (2nd and 3rd wk) but also weight (3rd wk) and glucose values (2nd and 3rd week). Captopril (for 21 or 14days) treatment prevented increases in blood pressure and plasma glucose but not in body weight. Also, captopril treatment significantly increased aorta contractility. These results provide evidence that cardiovascular and metabolic disturbances of preeclampsia appear simultaneously and are angiotensin II dependent.

Evidence of changes in alpha-1/AT1 receptor function generated by diet-induced obesity.

To study whether hypercaloric diet-induced obesity deteriorates vascular contractility of rat aorta through functional changes in α1 adrenergic and/or AT1 Angiotensin II receptors. Angiotensin II- or phenylephrine-induced contraction was tested on isolated aorta rings with and without endothelium from female Wistar rats fed for 7 weeks with hypercaloric diet or standard diet. Vascular expression of Angiotensin II Receptor type 1 (AT1R), Angiotensin II Receptor type 2 (AT2R), Cyclooxygenase-1 (COX-1), Cyclooxygenase-2 (COX-2), inducible Nitric Oxide Synthase (iNOS) and endothelial Nitric Oxide Synthase (eNOS), as well as blood pressure, glucose, insulin and angiotensin II blood levels were measured. Diet-induced obesity did not significantly change agonist-induced contractions (Emax and pD2 hypercaloric diet vs standard diet n.s.d.) of both intact (e+) or endothelium free (e-) vessels but significantly decrease both phenylephrine and angiotensin II contraction (Emax p < 0.01 hypercaloric diet vs standard diet) in the presence of both prazosin and losartan but only in endothelium-intact vessels. Diet-induced obesity did not change angiotensin II AT1, AT2 receptor proteins expression but reduced COX-1 and NOS2 ( p < 0.05 vs standard diet). Seven-week hypercaloric diet-induced obesity produces alterations in vascular adrenergic and angiotensin II receptor dynamics that suggest an endothelium-dependent adrenergic/angiotensin II crosstalk. These changes reflect early-stage vascular responses to obesity.

Gßγ Pathways in Cell Polarity and Migration Linked to Oncogenic GPCR Signaling: Potential Relevance in Tumor Microenvironment.

Cancer cells and stroma cells in tumors secrete chemotactic agonists that exacerbate invasive behavior, promote tumor-induced angiogenesis, and recruit protumoral bone marrow-derived cells. In response to shallow gradients of chemotactic stimuli recognized by G protein-coupled receptors (GPCRs), Gßγ-dependent signaling cascades contribute to specifying the spatiotemporal assembly of cytoskeletal structures that can dynamically alter cell morphology. This sophisticated process is intrinsically linked to the activation of Rho GTPases and their cytoskeletal-remodeling effectors. Thus, Rho guanine nucleotide exchange factors, the activators of these molecular switches, and their upstream signaling partners are considered participants of tumor progression. Specifically, phosphoinositide-3 kinases (class I PI3Ks, ß and γ) and P-Rex1, a Rac-specific guanine nucleotide exchange factor, are fundamental Gßγ effectors in the pathways controlling directionally persistent motility. In addition, GPCR-dependent chemotactic responses often involve endosomal trafficking of signaling proteins; coincidently, endosomes serve as signaling platforms for Gßγ In preclinical murine models of cancer, inhibition of Gßγ attenuates tumor growth, whereas in cancer patients, aberrant overexpression of chemotactic Gßγ effectors and recently identified mutations in Gß correlate with poor clinical outcome. Here we discuss emerging paradigms of Gßγ signaling in cancer, which are essential for chemotactic cell migration and represent novel opportunities to develop pathway-specific pharmacologic treatments.

Experimental gestational diabetes mellitus induces blunted vasoconstriction and functional changes in the rat aorta.

Diabetic conditions increase vascular reactivity to angiotensin II in several studies but there are scarce reports on cardiovascular effects of hypercaloric diet (HD) induced gestational diabetes mellitus (GDM), so the objective of this work was to determine the effects of HD induced GDM on vascular responses. Angiotensin II as well as phenylephrine induced vascular contraction was tested in isolated aorta rings with and without endothelium from rats fed for 7 weeks (4 before and 3 weeks during pregnancy) with standard (SD) or hypercaloric (HD) diet. Also, protein expression of AT1R, AT2R, COX-1, COX-2, NOS-1, and NOS-3 and plasma glucose, insulin, and angiotensin II levels were measured. GDM impaired vasoconstrictor response (P < 0.05 versus SD) in intact (e+) but not in endothelium-free (e-) vessels. Losartan reduced GDM but not SD e- vasoconstriction (P < 0.01 versus SD). AT1R, AT2R, and COX-1 and COX-2 protein expression were significantly increased in GDM vessels (P < 0.05 versus SD). Results suggest an increased participation of endothelium vasodilator mediators, probably prostaglandins, as well as of AT2 vasodilator receptors as a compensatory mechanism for vasoconstrictor changes generated by experimental GDM. Considering the short term of rat pregnancy findings can reflect early stage GDM adaptations.

mTORC1- and mTORC2-interacting proteins keep their multifunctional partners focused.

The mammalian target of rapamycin, best known as mTOR, is a phylogenetically conserved serine/threonine kinase that controls life-defining cellular processes such as growth, metabolism, survival, and migration under the influence of multiple interacting proteins. Historically, the cellular activities blocked by rapamycin in mammalian cells were considered the only events controlled by mTOR. However, this paradigm changed with the discovery of two signaling complexes differentially sensitive to rapamycin, whose catalytic component is mTOR. The one sensitive to rapamycin, known as mTORC1, promotes protein synthesis in response to growth factors and nutrients via the phosphorylation of p70S6K and 4EBP1; while the other, known as mTORC2, promotes cell migration and survival via the activation of Rho GTPases and the phosphorylation of AKT, respectively. Although mTORC2 kinase activity is not inhibited by rapamycin, hours of incubation with this antibiotic can impede the assembly of this signaling complex. The direct mechanism by which mTORC2 leads to cell migration depends on its interaction with P-Rex1, a Rac-specific guanine nucleotide exchange factor, while additional indirect pathways involve the intervention of PKC or AKT, multifunctional ubiquitous serine/threonine kinases that activate effectors of cell migration upon being phosphorylated by mTORC2 in response to chemotactic signals. These mTORC2 effectors are altered in metastatic cancer. Numerous clinical trials are testing mTOR inhibitors as potential antineoplasic drugs. Here, we briefly review the actions of mTOR with emphasis on the controlling role of mTORC1 and mTORC2-interacting proteins and highlight the mechanisms linked to cell migration.

Regulation of mTORC1 complex assembly and signaling by GRp58/ERp57.

The mammalian target of rapamycin (mTOR) regulates cell growth and survival via two different multiprotein complexes, mTORC1 and mTORC2. The assembly of these serine-threonine kinase multiprotein complexes occurs via poorly understood molecular mechanisms. Here, we demonstrate that GRp58/ERp57 regulates the existence and activity of mTORC1. Endogenous mTOR interacts with GRp58/ERp57 in different mammalian cells. In vitro, recombinant GRp58/ERp57 preferentially interacts with mTORC1. GRp58/ERp57 knockdown reduces mTORC1 levels and phosphorylation of 4E-BP1 and p70(S6K) in response to insulin. In contrast, GRp58/ERp57 overexpression increases mTORC1 levels and activity. A redox-sensitive mechanism that depends on GRp58/ERp57 expression activates mTORC1. Although GRp58/ERp57 is known as an endoplasmic reticulum (ER) resident, we demonstrate its presence at the cytosol, together with mTOR, Raptor, and Rictor as well as a pool of these proteins associated to the ER. In addition, the presence of GRp58/ERp57 at the ER decreases in response to insulin or leucine. Interestingly, a fraction of p70(S6K), but not 4E-BP1, is associated to the ER and phosphorylated in response to serum, insulin, or leucine. Altogether, our results suggest that GRp58/ERp57 is involved in the assembly of mTORC1 and positively regulates mTORC1 signaling at the cytosol and the cytosolic side of the ER.

Increased alpha-1 adrenoceptor expression in pregnant rats with subrenal aortic coarctation.

UNLABELLED: The progression of pregnancy is associated with attenuation in vasopressor response to adrenergic agonists. In pregnancy-induced hypertension this attenuation is reverted. It is not known if this reversion involves alpha-1 adrenoceptor expression. OBJECTIVE: In this work we propose that in pregnant rats with subrenal aortic coarctation there are changes in the expression of alpha-1 adrenergic receptors in the thoracic and abdominal aorta during pregnancy. METHODS: We used non-pregnant, normal pregnant and pregnant with subrenal aortic coarctation female Wistar rats. Pregnancy-induced hypertension indicators, systolic blood pressure, 24 hours proteinuria, pup weight and maternal weight were measured. Dose response curves to phenylephrine were carried out to determine vascular reactivity along pregnancy. Alpha 1-adrenoceptors were detected from thoracic and abdominal aorta using immunoblot. RESULTS: Results show significant increases in arterial pressure and proteinuria in pregnant rats with SRAC at the end of the third week. Pregnancy reduces alpha-(1-A, -B) and (-D) adrenoceptor expression and this event is reverted by subrenal aortic coarctation. This phenomenon is more apparent in the abdominal segment of the aorta. CONCLUSIONS: These findings suggest that subrenal aortic coarctation is a good animal model of pregnancy-induced hypertension and that alpha1-adrenoceptors participate in its physiopathology increasing their expression in a segment-dependent manner.

Effect of pregnancy on the roles of nitric oxide and prostaglandins in 5-hydroxytryptamine-induced contractions in rat isolated thoracic and abdominal aorta.

1. Vascular resistance and sensitivity to circulating pressor and vasoconstrictor substances are blunted during pregnancy. This has been attributed mainly to an increased production of endothelium-derived mediators. The aim of the present study was to determine whether pregnancy changes the relative participation of nitric oxide (NO) and prostaglandins (PG) in the modulation of the contractile response to 5-hydroxytryptamine (5-HT) in two anatomically distint segments of the rat aorta. 2. Full concentration-response curves to 5-HT were obtained in isolated rings from the thoracic and abdominal portion of the aorta from pregnant and non-pregnant rats in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (L-NAME; 10 micromol/L) or the PG synthesis inhibitor indomethacin (10 micromol/L). Cyclo-oxygenase (COX)-1, COX-2 and endothelial (e) NOS protein expression were determined in the same tissues by immunoblot. 3. The effects of pregnancy were accentuated in the abdominal compared with the thoracic aorta. In addition, the relative participation of the NO and PG pathways seems to be changed during pregnancy. Although NO seems to be the mediator mainly responsible for the effect of pregnancy in the thoracic aorta, our results suggest a complex interaction between NO and PG in the abdominal aorta. Indomethacin significantly reduced the contractile response of both segments of the aorta, whereas expression of COX-1, COX-2 and eNOS were increased only in the abdominal segment of pregnant animals. 4. These results show that the effect of pregnancy is not homogeneous along the aorta. There seems to be a mutual interaction between PG and NO in the abdominal, but not in the thoracic, aorta from pregnant rats: the role of NO becomes evident in the absence of vasodilatory PG, whereas the participation of the latter increases in the absence of NO working as a compensatory mechanism.