Scrambled Research Articles

Impaired sodium-evoked paraventricular nucleus neuronal activation and blood pressure regulation in conscious Sprague-Dawley rats lacking central Gαi2 proteins.

AimWe determined the role of brain Gαi2 proteins in mediating the neural and humoral responses of conscious male Sprague–Dawley rats to acute peripheral sodium challenge.MethodsRats pre‐treated (24‐h) intracerebroventricularly with a targeted oligodeoxynucleotide (ODN) (25 μg per 5 μL) to downregulate brain Gαi2 protein expression or a scrambled (SCR) control ODN were challenged with an acute sodium load (intravenous bolus 3 m NaCl; 0.14 mL per 100 g), and cardiovascular parameters were monitored for 120 min. In additional groups, hypothalamic paraventricular nucleus (PVN) Fos immunoreactivity was examined at baseline, 40, and 100 min post‐sodium challenge.ResultsIn response to intravenous hypertonic saline (HS), no difference was observed in peak change in mean arterial pressure between groups. In SCR ODN pre‐treated rats, arterial pressure returned to baseline by 100 min, while it remained elevated in Gαi2 ODN pre‐treated rats (P < 0.05). No difference between groups was observed in sodium‐evoked increases in Fos‐positive magnocellular neurons or vasopressin release. V1a receptor antagonism failed to block the prolonged elevation of arterial pressure in Gαi2 ODN pre‐treated rats. A significantly greater number of Fos‐positive ventrolateral parvocellular, lateral parvocellular, and medial parvocellular neurons were observed in SCR vs. Gαi2 ODN pre‐treated rats at 40 and 100 min post‐HS challenge (P < 0.05). In SCR, but not Gαi2 ODN pre‐treated rats, HS evoked suppression of plasma norepinephrine (P < 0.05).ConclusionThis highlights Gαi2 protein signal transduction as a novel central mechanism acting to differentially influence PVN parvocellular neuronal activation, sympathetic outflow, and arterial pressure in response to acute HS, independently of actions on magnocellular neurons and vasopressin release.

Abstract 035: Differential Sodium-evoked Activation of Pvn Magnocellular vs. Parvocellular Neurons in Rats Lacking Central GαI&lt;SUB.2 Proteins Contributes to Sustained Elevations in Blood Pressure

Aim: To determine the role of brain Gαi 2 proteins in mediating sodium-evoked PVN neuronal activation and blood pressure regulation in conscious rats. Methods: 24-h intracerebroventricular scrambled (SCR) or Gαi 2 oligodeoxynucleotide (ODN; 25μg/5μl)-pretreated conscious Sprague-Dawley rats were monitored for changes in MAP in response to HS (IV 3M NaCl; 0.14 ml/100g). Rats were sacrificed at control (C), 10, 40, or 100-min post-HS for PVN cFos IHC and analysis of plasma AVP and NE. Separate groups received a V 1a receptor antagonist (IV; 10 μg/ml/kg) 5-min prior to HS. Results: No difference was observed in sodium-evoked peak change in MAP and MAP remained elevated at 100-min in Gαi 2 but not SCR ODN rats (MAP 100-min post-HS [mmHg] SCR 134±2 vs Gαi 2 146±3, P&lt;0.05). Significant increases in the number of Fos + PVN magnocellular neurons were observed post-HS in SCR and Gαi 2 ODN groups ([Fos + cells] SCR C 3±1 vs 100-min 31±5; Gαi 2 C 2±0 vs 100-min 26±4, P &lt;0.05). A rapid increase in circulating AVP was observed at 10-min in both SCR (plasma AVP [pg/mL] C 12.2±1.6 vs 10-min 62.8±6.9, P &lt;0.05) and Gαi 2 ODN (plasma AVP [pg/mL] C 12.1±1.5 vs 10-min 67.7±7.7 P &lt;0.05) groups and returned to control levels at 40- and 100-min. SCR ODN rats exhibited significant increases in the number of Fos + PVN parvocellular neurons ([Fos+ cells] C 15±1 vs 100-min 67±4, P &lt;0.05) and a significant suppression in circulating NE post-HS (plasma NE [nmol/L] C 44.1±4.9 vs 10-min 17.4±3.9, P &lt;0.05). Gαi 2 ODN rats exhibited significantly less Fos + parvocellular neurons compared to SCR ODN rats (100-min [Fos+ cells] SCR 67±4 vs Gαi 2 30±2, P &lt;0.05) and failed to suppress circulating NE ( P &gt;0.05). V 1a receptor blockade prevented a HS-evoked increase in MAP in SCR ODN rats while Gαi 2 ODN rats exhibited elevated MAP (MAP 100-min [mmHg] SCR 125±2 vs Gαi 2 135±0, P &lt;0.05). Conclusion: Brain Gαi 2 proteins are required to mediate sodium-evoked parvocellular sympathetic, but not magnocellular vasopressinergic, responses to maintain physiological blood pressure regulation. A significant component of blood pressure control in this setting is regulated by the sympathetic nervous system, as supported by the attenuated activation of PVN parvocellular neurons and a failure to suppress circulating levels of NE in Gαi 2 ODN rats.

Abstract 106: Hypothalamic PVN GαI2 Protein-mediated Renal Nerve Dependent Sympathoinhibition Facilitates Suppression of NCC Activity and a Salt-resistant Phenotype

Aim: We hypothesize hypothalamic paraventricular (PVN) specific Gαi2 proteins, which are up-regulated in the PVN during increased sodium intake, mediate global and renal sympathoinhibition to facilitate suppression of the activity of the sodium chloride cotransporter (NCC) and salt-resistance in the Sprague Dawley rat. Methods: Groups of intact or bilateral renal denervated (RDNX) salt-resistant Sprague-Dawley rats received a bilateral PVN infusion of a scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN-300ng/side/day) and a normal 0.4% (NS) or high 8% NaCl (HS) diet for 7-days. On day-7 24h Na+ balance was assessed - in sub-groups MAP, plasma norepinephrine (NE) content, kidney NE content and NCC activity (peak natriuresis to iv bolus hydrochlorothiazide (HCTZ)) was assessed (N=6/gp/study). Results: In SCR ODN infused rats HS-intake evoked a significant 3-fold site-specific increase in PVN Gαi2 proteins and suppressed plasma NE content (plasma NE [nmol/L] SCR NS 72±4 vs HS 40±5, P&lt;0.05), endogenous kidney NE content (NE [pg/m] SCR NS 586±36 vs HS 496±41, P&lt;0.05) and NCC activity (peak ΔUNaV to HCTZ [μeq/min] SCR NS 10.6±0.8 vs HS 7±1, P&lt;0.05) without impacting sodium homeostasis or MAP. ODN-mediated PVN Gαi2 down-regulation caused renal nerve-dependent hypertension (MAP [mmHg] Gαi2 NS 126±2, Gαi2 HS 147±2, Gαi2 HS + RDNX 131±3 P&lt;0.05) sodium retention (24h Na+ balance [meq] Gαi2 NS 0.6±0.3, Gαi2 HS 2.3±0.4, Gαi2 HS + RDNX 0.8±0.3 P&lt;0.05), global and renal sympathoexcitation (plasma NE [nmol/L] Gαi2 NS 67±7, Gαi2 HS 105±11, Gαi2 HS + RDNX 74±6 P&lt;0.05; kidney NE content [pg/mg] Gαi2 NS 614±48 vs HS 823±59, P&lt;0.05) and a failure to suppress NCC activity (peak ΔUNaV to HCTZ [μeq/min] Gαi2 NS 10.2±2, Gαi2 HS 14.7±2, Gαi2 HS + RDNX 7.6±2, P&lt;0.05). Conclusion: PVN Gαi2 protein-gated pathways represent a sodium sensitive CNS mechanism acting to regulate renal nerve-dependent sodium excretion via, in part, actions upon the activity of the NCC. Based on recent studies by other groups we speculate the regulation of NCC in this setting is mediated by the actions of NE released from efferent renal nerves on renal adrenoceptors to impact the signal transduction network that regulates NCC expression.

ANGUSTIFOLIA mediates one of the multiple SCRAMBLED signaling pathways regulating cell growth pattern in Arabidopsis thaliana

In Arabidopsis thaliana, an atypical leucine-rich repeat receptor-like kinase, SCRAMBLED (SCM), is required for multiple developmental processes including root epidermal cell fate determination, silique dehiscence, inflorescence growth, ovule morphogenesis, and tissue morphology. Previous work suggested that SCM regulates these multiple pathways using distinct mechanisms via interactions with specific downstream factors. ANGUSTIFOLIA (AN) is known to regulate cell and tissue morphogenesis by influencing cortical microtubule arrangement, and recently, the AN protein was reported to interact with the SCM protein. Therefore, we examined whether AN might be responsible for mediating some of the SCM-dependent phenotypes. We discovered that both scm and an mutant lines cause an abnormal spiral or twisting growth of roots, but only the scm mutant affected root epidermal patterning. The siliques of the an and scm mutants also exhibited spiral growth, as previously reported, but only the scm mutant altered silique dehiscence. Interestingly, we discovered that the spiral growth of roots and siliques of the scm mutant is rescued by a truncated SCM protein that lacks its kinase domain, and that a juxtamembrane domain of SCM was sufficient for AN binding in the yeast two-hybrid analysis. These results suggest that the AN protein is one of the critical downstream factors of SCM pathways specifically responsible for mediating its effects on cell/tissue morphogenesis through cortical microtubule arrangement.

Abstract 395: Elevated Von Willebran Factor in an in vitro Model of Fabry Disease

Fabry disease is an X-linked disorder caused by a defect in the gene encoding the lysosomal hydrolase, alpha-galactosidase A (GLA). The loss in GLA function causes an age-dependent accumulation of globotriaosylceramide in the endothelial cells, which results in decreased nitric oxide bioavailability and premature mortality in the affected patients. Based on this, we hypothesized that GLA deficiency promotes endothelial inflammation. We observed a significant age-dependent increase in the plasma von Willebrand Factor (vWF) in Fabry mice compared to age-matched wild type mice (p=0.046 at 2 months and p=0.0004 at 17 months). To evaluate the molecular mechanisms of increased vWF in Fabry disease, small interfering RNA (siRNA) technique was used to knock-down GLA in EA.hy926 cells. Our initial studies with this cell line demonstrated that thrombin (2U/mL) induced a time-dependent vWF secretion into the cell culture medium with an 80-fold increase in the level of vWF after 90 min stimulation (p=0.001 compared to basal level; n=3 replicates per group), measured by a human antibody against vWF and AlphaLISA. Next, the siRNA studies revealed a greater than 95% knockdown of GLA protein (via western blotting) in the GLA-siRNA group compared to the control group with no effect in the scrambled (SCR) sequence-siRNA group (CON vs. GLA vs. SCR arbitrary units: 1.0±0.03 vs. 0.01±0.01 vs. 1.1±0.08, p&lt;0.001; GLA vs. SCR). Importantly, no difference in GAPDH protein was observed between groups. To determine the direct effect of GLA deficiency on vWF, the confluent cells in each group were incubated in serum-free medium overnight. Consistent with the hypothesis, we observed approximately 60% increase in vWF in the cell culture medium in the GLA siRNA group compared with the untreated and scrambled-sequence control groups. (CON vs. GLA vs. SCR arbitrary units: 38.5±4.7 vs. 61.3±1.1 vs. 31.4±3.8, p=0.02; GLA vs. SCR; n=3 replicates per group). In conclusion, the data provide evidence of endothelial activation in the setting of GLA deficiency, demonstrated by the robust elevation of vWF mediated by GLA gene disruption. Future study is required to further understand the mechanism of the elevated vWF and decreased nitric oxide bioavailability in Fabry disease.

Central Gαi2 proteins facilitate PVN parvocellular neuronal activation during chronic elevated sodium intake to maintain sodium homeostasis

AimBrain Gαi2 protein‐mediated signal transduction acts as a mechanism to potentiate central sympathoinhibition to maintain fluid and electrolyte homeostasis in settings of chronic high dietary sodium intake. The current study determined if brain Gαi2 proteins impact the pattern of neuronal activation evoked during high‐salt intake to maintain sodium homeostasis.MethodsMale Sprague‐Dawley rats received an ICV miniosmotic‐pump infusion of a scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN; 25μg/day) and were maintained on a low (LS; 0.03% NaCl), normal (NS; 0.4%), or high salt (HS; 8%) diet for 7 days. 24‐hour metabolic balance measurements were recorded on day 5 and rats were sacrificed on day 7 for FosB/ΔFosB IHC to examine dietary sodium evoked neuronal activation.ResultsFollowing 5‐d of a HS, but not LS or NS, diet ICV Gαi2 ODN infusion reduced 24‐h total sodium excretion compared to SCR ODN‐infused rats (Na+ excretion [meEq/24‐h] SCR HS: 27.0±1.2 vs. Gαi2 HS: 19.6±3.3, P&lt;0.05). Chronic HS, but not LS, diet evoked a significant increase in total PVN FosB in SCR infused rats that was not observed in Gαi2 infused rats (Δ from NS [FosB+ cells] SCR: 11±3 vs. Gαi2: 1±2, P&lt;0.05). Specifically, Gαi2 infusion prevented increases in PVN dorsal, ventrolateral, and lateral parvocellular nuclei that were observed in SCR infused rats (P&lt;0.05). Gαi2 infusion had no effect on sodium‐evoked increases in FosB in PVN magnocellular nuclei, the SON, and MnPO.ConclusionThese data highlight Gαi2 protein signal transduction as a novel CNS mechanism acting to influence neuronal activation, likely at the level of PVN parvocellular neurons, upon chronic HS challenge in a salt‐resistant phenotype.

Inhibiting Oncogenic RAS in Multiple Myeloma By Targeting Scaffold-ERK Interactions

The introduction of novel therapeutic agents over the past decade has significantly improved the outcomes of patients with newly diagnosed multiple myeloma (MM). Despite these advances, virtually all patients relapse and develop drug-resistant disease that carries a dismal prognosis. In addition to chemoresistance, relapse and tumor regrowth are dependent on self-renewal to maintain clonogenic growth potential over time. A better understanding of the factors responsible for tumor self-renewal and regrowth may lead to novel therapies and improved survival rates. RAS mutations are common in MM and have been demonstrated to increase in frequency with relapse and disease progression. These findings implicate RAS in chemoresistance, self-renewal, and tumor regrowth upon relapse. We have begun to directly examine the functional role of mutant RAS signaling in MM by stably transducing KRASmut MM cells with a tetracycline-inducible shRNA and found that KRAS knockdown decreases MM clonogenic growth in vitro. Although inhibiting oncogenic RAS may be beneficial for treating relapsed disease it remains an elusive drug target. Recently, our collaborators have developed a novel small peptide RAS signaling inhibitor that mimics the WW domain of IQGAP1 which competes for ERK binding to scaffolds. The WW peptide specifically targets solid tumors containing RAS (or BRAF) mutations and is well tolerated in mouse models. Thus, by targeting only tumorigenic cells it may demonstrate decreased toxicity compared to existing small molecule inhibitors of MAPK signaling.We analyzed gene expression profiling data from the University of Arkansas (GSE2658) and found that elevated IQGAP1 expression levels (top 12.5%) are associated with increased disease-related mortality in MM (log-rank p = 0.0002, HR 4.3, 95% CI 2.0-9.1). This suggests that IQGAP1 may play an important biological role in MM disease progression and that inhibition of IQGAP1 and RAS signaling with the WW peptide may have efficacy and specificity in MM patients. We have treated RAS mutated MM cell lines with the WW peptide or a scrambled (SCR) control peptide and found that the WW peptide effectively decreases pERK levels after 24 hours at doses as low as 20 nM. The WW peptide also decreases cell growth in RAS mutated but not wild-type MM cells by inducing a cell cycle arrest (by BrdU/7-AAD staining). Importantly, treatment of RAS mutated MM cell lines with the WW peptide significantly decreased colony formation in vitro and led to a reduction in the frequency of highly clonogenic CD138neg MM cells. Finally, an immunodeficient mouse xenograft model was used to determine the effect of the WW peptide on engraftment potential. We treated NCI-H929NRAS(G13D) cells with SCR or WW peptide and injected them into NOD/SCID/IL2γchainKO (NSG) mice. Engraftment as detected by kappa light chain ELISA was significantly reduced in the WW peptide treated group. These data further implicate RAS signaling as well as MAPK signaling in MM self-renewal and identify a potential novel therapy for treatment of relapsed/refractory MM. DisclosuresNo relevant conflicts of interest to declare.

TRIPTYCHON, not CAPRICE, participates in feedback regulation of SCM expression in the Arabidopsis root epidermis

The Arabidopsis root epidermal cells decide their fates (root-hair cell and non-hair cell) according to their position. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase (LRR RLK) mediates the positional information to the epidermal cells enabling them to adopt the proper fate. Via feedback regulation, the SCM protein accumulates preferentially in cells adopting the root-hair cell fate. In this study, we determine that TRY, but not the related factor CPC, is responsible for this preferential SCM accumulation. We observed severe reduction of SCM::GUS expression in the try-82 mutant root, but not in the cpc-1 mutant. Furthermore, the overexpression of TRY by CaMV35S promoter caused an increase in the expression of SCM::GUS in the root epidermis. Intriguingly, the overexpression of CPC by CaMV35S promoter repressed the expression of SCM::GUS. Together, these results suggest that TRY plays a unique role in generating the appropriate spatial expression of SCM.

Abstract 323: Impaired PVN Neuronal Activity In Rats Lacking Central Gαi2 Proteins: Implications For The Pathophysiology Of Salt-Sensitive Hypertension

Aim: We have demonstrated downregulation of central Gαi 2 proteins produces global sympathoexcitation and elevated MAP in response to acute and chronic high-salt challenge. The current study sought to define whether alterations in PVN neuronal activity in rats lacking brain Gαi 2 proteins underlie the sympathoexcitatory and pressor responses observed upon acute and chronic high-salt challenge. Methods: In acute studies, 24-h ICV scrambled (SCR) or Gαi 2 oligodeoxynucleotide (ODN; 25μg/5μl)-pretreated conscious Sprague-Dawley rats were monitored for changes in MAP in response to IV bolus NaCl (3M; 0.14 ml/100g). Rats were sacrificed at control, 10-, 40-, or 100-min post IV NaCl and cFos IHC was performed. In chronic studies, rats receiving an ICV miniosmotic-pump infusion of Gαi 2 or SCR ODN (25μg/day) were maintained on a normal (NS; 0.4% NaCl) or high (HS; 8% NaCl) salt diet for 7 days and sacrificed for FosB IHC (N=6/gp). Results: In response to IV sodium, in SCR rats MAP returned to control levels by 100-min whereas Gαi 2 rats remained significantly elevated (MAP 100-min post NaCl [mmHg] SCR: 134±2 vs. Gαi 2 : 146±3, P&lt;0.05). At 40-min post NaCl, a significant reduction in Fos immunoreactivity was observed in SCR but not Gαi 2 rats ([PVN cFos + cells] SCR control: 168±21 vs . post NaCl: 39±16, p&lt;0.05, Gαi 2 control: 155±18 vs . post NaCl: 116±16). Additionally, Gαi 2 prevented the Na + induced decrease of circulating plasma NE at 100-min (Δplasma NE [%control] SCR: -8.46±1.31 vs. Gαi 2 : +5.13±7.90, P&lt;0.05). In SCR infused rats, a chronic HS diet evoked a significant decrease ([FosB + cells] NS: 224±13 vs. HS: 133±16, P&lt;0.05) while a HS diet in Gαi 2 infused rats induced a significant increase ([FosB + cells] NS: 212±10 vs. HS: 296±15, P&lt;0.05) in PVN FosB immunoreactivity. Conclusion: Gαi 2 proteins represent a novel CNS mechanism influencing PVN neuronal activity in an acute and chronic setting. Na + induced decreases in Fos + PVN cells in SCR rats likely reflect inhibition of neuronal activity to facilitate sympathoinhibition and physiological blood pressure control. In Gαi 2 rats, failure to inhibit PVN neuronal activity and sympathetic outflow represents a mechanism by which impairment of Gαi 2 signaling may contribute to the pathophysiology of salt-sensitive hypertension.

Distinct Signaling Mechanisms in Multiple Developmental Pathways by the SCRAMBLED Receptor of Arabidopsis

SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase in Arabidopsis (Arabidopsis thaliana), is required for positional signaling in the root epidermis and for tissue/organ development in the shoot. To further understand SCM action, we generated a series of kinase domain variants and analyzed their ability to complement scm mutant defects. We found that the SCM kinase domain, but not kinase activity, is required for its role in root epidermal patterning, supporting the view that SCM is an atypical receptor kinase. We also describe a previously uncharacterized role for SCM in fruit dehiscence, because mature siliques from scm mutants fail to open properly. Interestingly, the kinase domain of SCM appears to be dispensable for this developmental process. Furthermore, we found that most of the SCM kinase domain mutations dramatically inhibit inflorescence development. Because this process is not affected in scm null mutants, it is likely that SCM acts redundantly to regulate inflorescence size. The importance of distinct kinase residues for these three developmental processes provides an explanation for the maintenance of the conserved kinase domain in the SCM protein, and it may generally explain its conservation in other atypical kinases. Furthermore, these results indicate that individual leucine-rich repeat receptor-like kinases may participate in multiple pathways using distinct signaling mechanisms to mediate diverse cellular communication events.

Positional Signaling and Expression of ENHANCER OF TRY AND CPC1 Are Tuned to Increase Root Hair Density in Response to Phosphate Deficiency in Arabidopsis thaliana

Phosphate (Pi) deficiency induces a multitude of responses aimed at improving the acquisition of Pi, including an increased density of root hairs. To understand the mechanisms involved in Pi deficiency-induced alterations of the root hair phenotype in Arabidopsis (Arabidopsis thaliana), we analyzed the patterning and length of root epidermal cells under control and Pi-deficient conditions in wild-type plants and in four mutants defective in the expression of master regulators of cell fate, CAPRICE (CPC), ENHANCER OF TRY AND CPC 1 (ETC1), WEREWOLF (WER) and SCRAMBLED (SCM). From this analysis we deduced that the longitudinal cell length of root epidermal cells is dependent on the correct perception of a positional signal (‘cortical bias’) in both control and Pi-deficient plants; mutants defective in the receptor of the signal, SCM, produced short cells characteristic of root hair-forming cells (trichoblasts). Simulating the effect of cortical bias on the time-evolving probability of cell fate supports a scenario in which a compromised positional signal delays the time point at which non-hair cells opt out the default trichoblast pathway, resulting in short, trichoblast-like non-hair cells. Collectively, our data show that Pi-deficient plants increase root hair density by the formation of shorter cells, resulting in a higher frequency of hairs per unit root length, and additional trichoblast cell fate assignment via increased expression of ETC1.

Abstract 26: PVN GaI2 Subunit Proteins - The Key to a Salt-resistant Phenotype?

Aim: Endogenous up-regulation of PVN Gαi 2 proteins facilitates sympathoinhibition and normotension during chronic Na + challenge in Sprague-Dawley rats via a renal-nerve dependent mechanism. Therefore, we examined the role of PVN Gαi 2 proteins in MAP regulation during high salt-intake in intact and renal denervated (RDNX) Dahl salt-resistant (DSR) &amp; salt-sensitive (DSS) rats and naive Brown Norway (BN) and DSSBN8 congenic rats. Methods: Sham (S) or RDNX DSR &amp; DSS rats receiving a continuous i.c.v. infusion of a scrambled (SCR) or Gαi 2 oligodeoxynucleotide (25 μg/day), and naïve BN and DSSBN8 rats, were fed a 21-day normal 0.4% (NS) or high 8% NaCl (HS) diet. MAP was recorded continuously by radiotelemetry and Na + homeostasis was assessed via metabolic balance studies. On day-21 plasma norepinephrine (NE), autonomic function and PVN Gαi 2 protein levels were determined (N=6/gp). Results: HS-intake did not alter MAP, suppressed plasma NE (P&lt;0.05), and evoked a site-specific increase in PVN Gαi 2 protein levels in BN &amp; DSR (4.8 and 4.5-fold respectively, P&lt;0.05), but not DSS rats. In DSR rats Gαi 2 down-regulation evoked rapid renal nerve-dependent hypertension (MAP [mmHg] Day 4 HS S 121±2* vs RDNX 101±2, Day 21 HS S 127±2* vs RDNX 101±1), Na + retention and sympathoexcitation (plasma NE [nmol/L] S 87±7* vs RDNX 37±3, peak ΔMAP to chlorisondamine [mmHg] S -68±4* vs RDNX -37±3). In DSS rats, Gαi 2 down-regulation exacerbated salt-induced hypertension (MAP [mmHg] Day 4 HS S 148±5* vs RDNX 133±4, Day 21 HS S 175±4* vs RDNX 145±3), Na + retention, and sympathoexcitation (plasma NE [nmol/L] S 118±9* vs RDNX 88±6) in a renal nerve dependent manner. DSSBN8 rats exhibited PVN specific Gαi 2 protein up-regulation, attenuated hypertension (MAP [mmHg] Day 21 DSSBN8 HS 142±2 τ , DSS HS 155±4), Na + retention and global sympathoexcitation (plasma NE [nmol/L] DSSBN8 HS 52±4 τ , DSS HS 87±6). *p&lt;0.05 vs RDNX + HS, τ p&lt;0.05 vs DSS. Conclusion: PVN Gαi 2 protein-gated pathways represent a conserved central molecular pathway mediating sympathoinhibitory renal-nerve dependent responses evoked to maintain sodium homeostasis and a salt-resistant phenotype. Owing to SNP’s in the human GNAI2 gene (which encodes Gαi 2 ) correlating with hypertension our findings have translational implications.

Low-density lipoprotein receptor-related protein 1b impairs cellular proliferation in human cell lines

Low-density lipoprotein (LDL) receptor-related protein 1b (LRP1b) is one of three very large receptors of the LDL receptor family, several members of which have been implicated in the pathogenesis of atherosclerosis. LRP1b was discovered during studies of lung cancer cell lines, where alterations of the receptor were frequently found and LRP1b was therefore postulated as a putative tumor suppressor. LRP1b is expressed in human brain, thyroid gland, skeletal muscle, and to a lesser amount in testis. Previously, we demonstrated binding of LRP1b to circulating ligands like very low density lipoprotein, high density lipoprotein, and fibrinogen. We generated an expression vector containing the full length 13.800 bp murine LRP1b cDNA for overexpression (OE) of the receptor in LRP1b-negative lung cancer cell lines (A427, A549) and used siRNA for silencing in LRP1b-positive lung cancer cells (Calu-1). Cellular proliferation was analyzed using a BrdU incorporation ELISA and anchorage-independent growth using a soft agar colony formation assay, respectively. Consistent with the postulated tumor suppressor function, overexpression of mLRP1b in A427 and A549 cells significantly reduced both cellular proliferation and anchorage-independent growth compared to mock-transfected control cells. Conversely, siRNA mediated knockdown of hLRP1b in Calu-1 lung cancer cells led to a significant increase in cellular proliferation compared to scrambled (SCR)-siRNA treated control cells (Fig 1). ![Figure][1] Figure 1 Our findings demonstrate that overexpression of full length LRP1b leads to impaired cellular proliferation, while LRP1b knockdown has the opposite effect. These experiments may help in unraveling the largely unknown physiological role of LRP1b and its potential functions in cancer and atherosclerosis. [1]: pending:yes

Focal adhesion kinase is required for IGF-I-mediated growth of skeletal muscle cells via a TSC2/mTOR/S6K1-associated pathway

Focal adhesion kinase (FAK) is an attachment complex protein associated with the regulation of muscle mass through as-of-yet unclear mechanisms. We tested whether FAK is functionally important for muscle hypertrophy, with the hypothesis that FAK knockdown (FAK-KD) would impede cell growth associated with a trophic stimulus. C₂C₁₂ skeletal muscle cells harboring FAK-targeted (FAK-KD) or scrambled (SCR) shRNA were created using lentiviral transfection techniques. Both FAK-KD and SCR myotubes were incubated for 24 h with IGF-I (10 ng/ml), and additional SCR cells (±IGF-1) were incubated with a FAK kinase inhibitor before assay of cell growth. Muscle protein synthesis (MPS) and putative FAK signaling mechanisms (immunoblotting and coimmunoprecipitation) were assessed. IGF-I-induced increases in myotube width (+41 ± 7% vs. non-IGF-I-treated) and total protein (+44 ± 6%) were, after 24 h, attenuated in FAK-KD cells, whereas MPS was suppressed in FAK-KD vs. SCR after 4 h. These blunted responses were associated with attenuated IGF-I-induced FAK Tyr³⁹⁷ phosphorylation and markedly suppressed phosphorylation of tuberous sclerosis complex 2 (TSC2) and critical downstream mTOR signaling (ribosomal S6 kinase, eIF4F assembly) in FAK shRNA cells (all P < 0.05 vs. IGF-I-treated SCR cells). However, binding of FAK to TSC2 or its phosphatase Shp-2 was not affected by IGF-I or cell phenotype. Finally, FAK-KD-mediated suppression of cell growth was recapitulated by direct inhibition of FAK kinase activity in SCR cells. We conclude that FAK is required for IGF-I-induced muscle hypertrophy, signaling through a TSC2/mTOR/S6K1-dependent pathway via means requiring the kinase activity of FAK but not altered FAK-TSC2 or FAK-Shp-2 binding.

296 GPCR STIMULATED HYPOTHALAMIC PVN GαI2 PROTEIN-GATED PATHWAYS – THE KEY TO RENAL SYMPATHETIC NERVE MEDIATED SODIUM HOMEOSTASIS AND THE LONG-TERM CONTROL OF BLOOD PRESSURE

Aim: We have demonstrated CNS Gαi2 proteins, which are highly expressed in the hypothalamic PVN, mediate the renal sympathoinhibition and natriuresis evoked by α2-adrenoceptor activation and volume expansion in-vivo. Therefore, we examined the role(s) of PVN Gαi2 proteins in fluid & electrolyte homeostasis and MAP regulation following elevated dietary salt-intake. Methods: Intact or bilateral renal denervated (RDNX) Sprague-Dawley rats received a bilateral PVN or posterior hypothalamic (PH) infusion of a scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN-300ng/side/day) and a normal 0.4% (NS) or high 8% NaCl (HS) diet for 7-days. On day-7 MAP, 24 h metabolic balance, plasma norepinephrine, (NE) and PVN Gαi2 protein levels were determined (N = 5/group). Results: HS-intake evoked a significant 3-fold site-specific increase in PVN Gαi2 proteins. ODN-mediated PVN Gαi2 down-regulation, but not PH miss-injection or PVN SCR ODN pre-treatment, caused renal nerve-dependent sodium retention (24 h Na+ balance [meq] PH Gαi2 + HS 0.5 ± 0.2, PVN Gαi2 + HS 2.3 ± 0.4*, PVN Gαi2 RDNX + HS 0.8 ± 0.3τ), global sympathoexcitation (plasma norepinephrine [nmol/L] PH Gαi2 + HS 17 ± 4, PVN Gαi2 + HS 75 ± 9*, PVN Gαi2 RDNX + HS 24 ± 6τ) and hypertension (MAP [mmHg] PH Gαi2 + HS 128 ± 3, PVN Gαi2 + HS 140 ± 2*, PVN Gαi2 RDNX + HS 131 ± 3τ) *p<0.05 vs. PH Gαi2 + HS, τp < 0.05 vs. PVN Gαi2 + HS. Conclusion: Renal nerve dependent dysregulation of sympathetically driven sodium retaining mechanisms occurred following PVN Gαi2-protein down-regulation evoking salt-sensitive hypertension. We conclude PVN Gαi2 protein-gated pathways regulate renal nerve-dependent sodium excretion to facilitate sodium homeostasis and maintenance of a salt-resistant phenotype – R01HL107330.

Functional selectivity of central Gα‐subunit proteins in mediating the cardiovascular and renal excretory responses evoked by central α2‐adrenoceptor activation in vivo

Activation of brain α(2) -adrenoceptors in conscious rodents decreases heart rate (HR) and mean arterial blood pressure (MAP) and increases urine output and urinary sodium excretion. In vitro, α(2) -adrenoceptor stimulation activates Gα(i(1-3)) , Gα(o) and Gα(s) -subunit protein-gated signal transduction pathways. Here we have investigated whether these same Gα-subunit protein-gated pathways mediate the cardiovascular and renal excretory responses to central α(2) -adrenoceptor activation in conscious Sprague-Dawley rats. Rats were pre-treated by intracerebroventricular injection (i.c.v.) with an oligodeoxynucleotide (ODN) targeted to a Gα(i1) , Gα(i2) , Gα(i3) , Gα(o) , Gα(s) or a scrambled (SCR) ODN sequence (25 µg, 24 h). On the day of study, the α(2) -adrenoceptor agonist guanabenz (50 µg) or saline vehicle, was injected i.c.v. into ODN-pre-treated conscious rats. MAP and HR were recorded, and urine was collected for 150 min. In vehicle- and SCR ODN-pre-treated rats, i.c.v. guanabenz decreased MAP and HR, and produced marked diuretic and natriuretic responses. Selective ODN-mediated down-regulation of brain Gα(i2) -subunit proteins abolished the central guanabenz-induced hypotension and natriuresis. In contrast, following selective Gα(s) down-regulation, the characteristic hypotensive response to i.c.v. guanabenz was converted to an immediate increase in MAP. The bradycardic and diuretic responses to i.c.v. guanabenz were not blocked by pre-treatment with any ODN. There was functional selectivity of Gα(i2) and Gα(s) subunit protein-gated signal transduction pathways in mediating the hypotensive and natriuretic, but not bradycardic or diuretic, responses evoked by central α(2) -adrenoceptor activation in vivo.

The efficiency of silencing expression of the gene coding STAT3 transcriptional factor and susceptibility of bladder cancer cells to apoptosis

Aim of the studyAbnormalities in signaling as well as altered gene expression have been identified in numerous diseases, including cancer. The biological functions of signal transducer and activator of transcription 3 (STAT3) are very broad. It is thought that STAT3 can also contribute to oncogenesis. RNA interference (RNAi) is one of the most efficient tools for silencing gene expression within cells. The main goal of the study was to verify the effectiveness of STAT3 gene silencing and its influence on cell proliferation and activation of apoptosis in bladder cancer cells.Material and methodsThe study was conducted on cellular material, which was the stable human bladder cancer cell line T24. The synthesis of shRNA (short hairpin RNA) interfering with the STAT3 gene was based on pSUPER. neo expression vector. The gene expression at the mRNA level was determined by the real-time PCR method. The influence of STAT3 gene silencing on apoptosis induced in cells with modulated STAT3 expression was evaluated using parallel quantification of mono- and oligonucleosomal DNA degradation of genomic DNA.ResultsIn transfected T24 cells, the STAT3 mRNA expression decreased to the level of 68.3% compared to the scrambled (SCR) control. Silencing the STAT3 gene induced changes in the phenotype of T24 cells. Statistically significant differences in cell proliferation (p = 0.0318) and apoptosis induction (p = 0.0376) were observed.ConclusionsApplication of the designed shRNA for the STAT3 gene contributed to a decrease of expression of the examined gene. It also decreased the proliferation and increased the susceptibility to apoptosis in T24 bladder cancer cells.

Neurons develop in situ in foregut endoderm of sea urchin embryos

between these genes, we have found that transcriptional feedbackloops acting within and between adjacent cells are important inestablishing the cell type pattern. Specifically, the WER MYB-typeprotein, the GL3/EGL3 bHLH-type proteins, and the TTG WD-proteinappear to interact in a transcriptional complex to positively regulatethe GL2, CPC, TRY, and ETC1 genes. The GL2 homeodomaintranscription factor is involved in regulating genes that generatethe non-hair cell type. The CPC, TRY, and ETC1 proteins arestructurally-related small MYB transcription factors that appear tomove between cells and inhibit the formation of the WER-GL3/EGL3-TTG complex; this negative regulation represents a type of lateralinhibition mechanism. The position-dependent pattern relies on aleucine-rich-repeat receptor-like kinase (SCRAMBLED (SCM)), thatappears to influence WER gene expression causing an unequaldistribution of the transcriptional regulators in the N and H cellpositions. Current research is focused on using genomics, bioinfor-matics, and math modeling to further dissect the regulatory network.These studies are likely to provide new insights into the basicmechanisms of regulatory gene networks and cell-type specificationduring development.doi:10.1016/j.ydbio.2011.05.056Program/Abstract # 40Neurons develop in situ in foregut endoderm of seaurchin embryosZheng Wei

Sensory neuron specification in the neural crest lineage

loops acting within and between adjacent cells are important in establishing the cell type pattern. Specifically, the WER MYB-type protein, the GL3/EGL3 bHLH-type proteins, and the TTG WD-protein appear to interact in a transcriptional complex to positively regulate the GL2, CPC, TRY, and ETC1 genes. The GL2 homeodomain transcription factor is involved in regulating genes that generate the non-hair cell type. The CPC, TRY, and ETC1 proteins are structurally-related small MYB transcription factors that appear to move between cells and inhibit the formation of the WER-GL3/EGL3TTG complex; this negative regulation represents a type of lateral inhibition mechanism. The position-dependent pattern relies on a leucine-rich-repeat receptor-like kinase (SCRAMBLED (SCM)), that appears to influence WER gene expression causing an unequal distribution of the transcriptional regulators in the N and H cell positions. Current research is focused on using genomics, bioinformatics, and math modeling to further dissect the regulatory network. These studies are likely to provide new insights into the basic mechanisms of regulatory gene networks and cell-type specification during development.

Role of c-Cbl Carboxyl Terminus in Serotonin 5-HT2A Receptor Recycling and Resensitization

The 5-hydroxytryptamine 2A receptor (5-HT(2A)R) undergoes constitutive and agonist-dependent internalization. Despite many advances in our understanding of G protein-coupled receptor trafficking, the exact mechanism of endocytic sorting of G protein-coupled receptors remains obscure. Recently, we have reported a novel finding documenting a global role for the ubiquitin ligase c-Cbl in regulating vesicular sorting of epidermal growth factor receptor (Baldys, A., Göoz, M., Morinelli, T. A., Lee, M. H., Raymond, J. R., Jr., Luttrell, L. M., and Raymond, J. R., Sr. (2009) Biochemistry 48, 1462-1473). Thus, we tested the hypothesis that c-Cbl might play a role in 5-HT(2A)R recycling. In this study, we demonstrated an association of 5-HT(2A)R with c-Cbl. Furthermore, down-regulation of c-Cbl by RNA interference blocked efficient recycling of 5-HT(2A)R to the plasma membrane. Immunofluorescence microscopy revealed that 5-HT(2A) receptors were trapped in early endosome antigen 1- and Rab11-positive sorting endosomes in cells overexpressing c-Cbl mutants lacking carboxyl termini. This inhibitory effect was associated with a relative decrease in association of c-Cbl truncation proteins with the 5-HT(2A)R, compared with that observed for the full-length c-Cbl fusion protein. Consistent with the delayed recycling, 5-HT(2A)R resensitization was greatly attenuated in the presence of c-Cbl mutants lacking carboxyl termini, as detected by changes in the cytosolic calcium. Taken together, these studies have led to the discovery that the C-terminal region of c-Cbl plays a crucial role in the temporal and spatial control of 5-HT(2A)R recycling.