Hypoxic stress‐induced changes in adrenergic function: role of HIF1α

Sustaining epinephrine‐elicited behavioral and physiological responses during stress requires replenishment of epinephrine stores. Egr‐1 and Sp1 contribute by stimulating the gene encoding the epinephrine‐synthesizing enzyme, phenylethanolamine N‐methyltransferase (PNMT), as shown for immobilization stress in rats in adrenal medulla and for hypoxic stress in adrenal medulla‐derived PC12 cells. Hypoxia (5% O₂) also activates hypoxia inducible factor (HIF) 1α, increasing mRNA, nuclear protein and nuclear protein/hypoxia response element binding complex formation. Hypoxia and HIF1α over‐expression also elevate PNMT promoter‐driven luciferase activity in PC12 cells. Hypoxia may be limiting as HIF1α over‐expression increases luciferase expression to no greater extent than oxygen reduction alone. HIF1α inducers CoCl₂ or deferoxamine elevate luciferase as well. PC12 cells harboring a HIF1α expression construct show markedly higher levels of Egr‐1 and Sp1 mRNA and nuclear protein and PNMT mRNA and cytoplasmic protein. Inactivation of Egr‐1 and Sp1 binding sites in the proximal ?893 bp of PNMT promoter precludes HIF1α stimulation while a potential hypoxia response element (?282 bp) in the promoter shows weak HIF1α affinity at best. These findings are the first to suggest that hypoxia activates the proximal rat PNMT promoter primarily via HIF1α induction of Egr‐1 and Sp1 rather than by co‐activation by Egr‐1, Sp1 and HIF1α. In addition, the rise in HIF1α protein leading to Egr‐1 and Sp1 stimulation of PNMT appears to include HIF1α gene activation rather than simply prevention of HIF1α proteolytic degradation.     

Combined Use of the Canine Adenovirus-2 and DREADD-Technology to Activate Specific Neural Pathways In Vivo

We here describe a technique to transiently activate specific neural pathways in vivo. It comprises the combined use of a CRE-recombinase expressing canine adenovirus-2 (CAV-2) and an adeno-associated virus (AAV-hSyn-DIO-hM₃D(G_q)-mCherry) that contains the floxed inverted sequence of the designer receptor exclusively activated by designer drugs (DREADD) hM₃D(G_q)-mCherry. CAV-2 retrogradely infects projection neurons, which allowed us to specifically express hM₃D(G_q)-mCherry in neurons that project from the ventral tegmental area (VTA) to the nucleus accumbens (Acb), the majority of which were dopaminergic. Activation of hM₃D(G_q)-mCherry by intraperitoneal (i.p.) injections of clozapine-N-oxide (CNO) leads to increases in neuronal activity, which enabled us to specifically activate VTA to Acb projection neurons. The VTA to Acb pathway is part of the mesolimbic dopamine system and has been implicated in behavioral activation and the exertion of effort. Injections of all doses of CNO led to increases in progressive ratio (PR) performance. The effect of the lowest dose of CNO was suppressed by administration of a DRD1-antagonist, suggesting that CNO-induced increases in PR-performance are at least in part mediated by DRD1-signaling. We hereby validate the combined use of CAV-2 and DREADD-technology to activate specific neural pathways and determine consequent changes in behaviorally relevant paradigms.     

Age‐dependent dystonia in striatal Gγ7 deficient mice is reversed by the dopamine D2 receptor agonist pramipexole

Gγ7 is enriched in striatum and forms a heterotrimeric complex with Gα_olf/Gβ, which is coupled to D1 receptor (D1R). Here, we attempted to characterize the pathophysiological, neurochemical, and pharmacological features of mice deficient of Gγ7 gene. Gγ7 knockout mice exhibited age‐dependent deficiency in rotarod behavior and increased dystonia‐like clasping reflex without loss of striatal neurons. The neurochemical basis for the motor manifestations using immunoblot analysis revealed increased levels of D1R, ChAT and NMDA receptor subunits (NR1 and NR2B) concurrent with decreased levels of D2R and Gα_olf, possibly because of the secondary changes of decreased Gα_olf/Gγ7‐mediated D1R transmission. These behavioral and neurochemical changes are closely related to those observed in Huntington's disease (HD) human subjects and HD model mice. Taking advantage of the finding of D2R down‐regulation in Gγ7 knockout mice and the dopamine‐mediated synergistic relationship in the control of locomotion between D2R‐striatopallidal and D1R‐stritonigral neurons, we hypothesized that D2‐agonist pramipexole would reverse behavioral dyskinesia caused by defective D1R/Gα_olf signaling. Indeed, the rotarod deficiency and clasping reflex were reversed by pramipexole treatment under chronic administration. These findings suggest that Gγ7 knockout mice could be a new type of movement disorders, including HD and useful for the evaluation of therapeutic candidates.     

Activation of ras-dependent signaling pathways by G(14) -coupled receptors requires the adaptor protein TPR1

Many G_q‐coupled receptors mediate mitogenic signals by stimulating extracellular signal‐regulated protein kinases (ERKs) that are typically regulated by the small GTPase Ras. Recent studies have revealed that members of the Gα_q family may possess the ability to activate Ras/ERK by interacting with the adaptor protein tetratricopeptide repeat 1 (TPR1). Within the Gα_q family, the highly promiscuous Gα₁₄ can relay signals from numerous receptors. Here, we examined if Gα₁₄ interacts with TPR1 to stimulate Ras signaling pathways. Expression of the constitutively active Gα₁₄QL mutant in HEK293 cells led to the formation of GTP‐bound Ras as well as increased phosphorylations of downstream signaling molecules including ERK and IκB kinase. Stimulation of endogenous G₁₄‐coupled somatostatin type 2 and α₂‐adrenergic receptors produced similar responses in human hepatocellular HepG2 carcinoma cells. Co‐immunoprecipitation assays using HEK293 cells demonstrated a stronger association of TPR1 for Gα₁₄QL than Gα₁₄, suggesting that TPR1 preferentially binds to the GTP‐bound form of Gα₁₄. Activated Gα₁₄ also interacted with the Ras guanine nucleotide exchange factors SOS1 and SOS2. Expression of a dominant negative mutant of TPR1 or siRNA‐mediated knockdown of TPR1 effectively abolished the ability of Gα₁₄ to induce Ras signaling in native HepG2 or transfected HEK293 cells. Although expression of the dominant negative mutant of TPR1 suppressed Gα₁₄QL‐induced phosphorylations of ERK and IκB kinase, it did not affect Gα₁₄QL‐induced stimulation of phospholipase Cβ or c‐Jun N‐terminal kinase. Our results suggest that TPR1 is required for Gα₁₄ to stimulate Ras‐dependent signaling pathways, but not for the propagation of signals along Ras‐independent pathways. J. Cell. Biochem. 113: 3486–3497, 2012. © 2012 Wiley Periodicals, Inc.     

Valsartan independent of AT1 receptor inhibits tissue factor,TLR‐2 and ‐4 expression by regulation of Egr‐1 through activation of AMPK in diabetic conditions

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)‐1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT₁R) can reduce tissue factor (TF) and toll‐like receptor (TLR)‐2 and ‐4 by regulating Egr‐1 in THP‐1 cells and aorta in streptozotocin‐induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr‐1, TF, TLR‐2 and ‐4 which were significantly reduced by valsartan. HG increased Egr‐1 expression by activation of PKC and ERK1/2 in THP‐1 cells. Valsartan increased AMPK phosphorylation in a concentration and time‐dependent manner via activation of LKB1. Valsartan inhibited Egr‐1 without activation of PKC or ERK1/2. The reduced expression of Egr‐1 by valsartan was reversed by either silencing Egr‐1, or compound C, or DN‐AMPK‐transfected cells. Valsartan inhibited binding of NF‐κB and Egr‐1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF‐α, IL‐6 and IL‐1β) production and NF‐κB activity in HG‐activated THP‐1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT₁R in THP‐1 cells or CHO cells, which were devoid of AT₁R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr‐1, TLR‐2, ‐4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin‐induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr‐1 regulation.     

AGAP1/AP‐3‐dependent endocytic recycling of M5 muscarinic receptors promotes dopamine release

Of the five mammalian muscarinic acetylcholine (ACh) receptors, M₅ is the only subtype expressed in midbrain dopaminergic neurons, where it functions to potentiate dopamine release. We have identified a direct physical interaction between M₅ and the AP‐3 adaptor complex regulator AGAP1. This interaction was specific with regard to muscarinic receptor (MR) and AGAP subtypes, and mediated the binding of AP‐3 to M₅. Interaction with AGAP1 and activity of AP‐3 were required for the endocytic recycling of M₅ in neurons, the lack of which resulted in the downregulation of cell surface receptor density after sustained receptor stimulation. The elimination of AP‐3 or abrogation of AGAP1–M₅ interaction in vivo decreased the magnitude of presynaptic M₅‐mediated dopamine release potentiation in the striatum. Our study argues for the presence of a previously unknown receptor‐recycling pathway that may underlie mechanisms of G‐protein‐coupled receptor (GPCR) homeostasis. These results also suggest a novel therapeutic target for the treatment of dopaminergic dysfunction.     

Signaling mechanisms and physiological functions of G‐protein Gα13 in blood vessel formation,bone homeostasis,and cancer

Heterotrimeric G‐proteins are cellular signal transducers. They mainly relay signals from G‐protein‐coupled receptors (GPCRs). GPCRs function as guanine nucleotide‐exchange factors to active these G‐proteins. Based on the sequence and functional similarities, these G‐proteins are grouped into four subfamilies: G_s, G_i, G_q, and G_12/13. The G_12/13 subfamily consists of two members: G₁₂ and G₁₃. G_12/13‐mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. Here we summarize the signaling mechanisms and physiological functions of Gα₁₃ in blood vessel formation and bone homeostasis. We further discuss the expanding roles of Gα₁₃ in cancers, serving as oncogenes as well as tumor suppressors.