Mineralocorticoid Receptor Mutations Differentially Affect Individual Gene Expression Profiles in Pseudohypoaldosteronism Type 1

Context: Type 1 pseudohypoaldosteronism (PHA1), a primary form of mineralocorticoid resistance, isdueto inactivating mutations of the NR3C2 gene, coding for the mineralocorticoid receptor (MR). Objective: The objective of the study was to assess whether different NR3C2 mutations have distinct effects on the pattern of MR-dependent transcriptional regulation of aldosterone-regulated genes. Design and Methods: Four MR mutations affecting residues in the ligand binding domain, identified in families with PHA1, were tested. MR proteins generated by site-directed mutagenesis were analyzed for their binding to aldosterone and were transiently transfected into renal cells to explore the functional effects on the transcriptional activity of the receptors by cis-trans-cotrans-activation assays and by measuring the induction of endogenous gene transcription. Results: Binding assays showed very low or absent aldosterone binding for mutants MR(877Pro), MR(848Pro), and MR(947stop) and decreased affinity for aldosterone of MR(843Pro). Compared with wildtype MR, the mutations p.Leu843Pro and p.Leu877Pro displayed half-maximal aldosterone-dependent transactivation of reporter genes driven by mouse mammary tumor virus or glucocorticoid response element-2 dependent promoters, whereas MR(848Pro) and MR(947stop) nearly or completely lost transcriptional activity. Although MR(848Pro) and MR(947stop) were also incapable of inducing aldosterone-dependent gene expression ofendogenoussgk1, GILZ, NDRG2, and SCNN1A, MR(843Pro) retained complete transcriptional activity on sgk1 and GILZ gene expression, and MR(877Pro) negatively affected the expression of sgk1, NDRG2, and SCNN1A. Conclusions: Our data demonstrate that MR mutations differentially affect individual gene expression in a promoter-dependent manner. Investigation of differential gene expression profiles in PHA1 may allow a better understanding of the molecular substrate of phenotypic variability and to elucidate pathogenic mechanisms underlying the disease. (J Clin Endocrinol Metab 96: E519-E527, 2011)

Anabolic steroid associated to physical training induces deleterious cardiac effects

DO CARMO, E. C., T. FERNANDES, D. KOIKE, N. D. DA SILVA JR., K. C. MATTOS, K. T. ROSA, D. BARRETTI, S. F. S. MELO, R. B. WICHI, M. C. C. IRIGOYEN, and E. M. DE OLIVEIRA. Anabolic Steroid Associated to Physical Training Induces Deleterious Cardiac Effects. Med. Sci. Sports Exerc., Vol. 43, No. 10, pp. 1836-1848, 2011. Purpose: Cardiac aldosterone might be involved in the deleterious effects of nandrolone decanoate (ND) on the heart. Therefore, we investigated the involvement of cardiac aldosterone, by the pharmacological block of AT1 or mineralocorticoid receptors, on cardiac hypertrophy and fibrosis. Methods: Male Wistar rats were randomized into eight groups (n = 14 per group): Control (C), nandrolone decanoate (ND), trained (T), trained ND (TND), ND + losartan (ND + L), trained ND + losartan (TND + L), ND + spironolactone (ND + S), and trained ND + spironolactone (TND + S). ND (10 mg.kg(-1).wk(-1)) was administered during 10 wk of swimming training (five times per week). Losartan (20 mg.kg(-1).d(-1)) and spironolactone (10 mg.kg(-1).d(-1)) were administered in drinking water. Results: Cardiac hypertrophy was increased 10% by using ND and 17% by ND plus training (P < 0.05). In both groups, there was an increase in the collagen volumetric fraction (CVF) and cardiac collagen type III expression (P < 0.05). The ND treatment increased left ventricle-angiotensin-converting enzyme I activity, AT1 receptor expression, aldosterone synthase (CYP11B2), and 11-beta hydroxysteroid dehydrogenase 2 (11 beta-HSD2) gene expression and inflammatory markers, TGF beta and osteopontin. Both losartan and spironolactone inhibited the increase of CVF and collagen type III. In addition, both treatments inhibited the increase in left ventricle-angiotensin-converting enzyme I activity, CYP11B2, 11 beta-HSD2, TGF beta, and osteopontin induced by the ND treatment. Conclusions: We believe this is the first study to show the effects of ND on cardiac aldosterone. Our results suggest that these effects may be associated to TGF beta and osteopontin. Thus, we conclude that the cardiac aldosterone has an important role on the deleterious effects on the heart induced by ND.

Vascular proinflammatory responses by aldosterone are mediated via c-Src trafficking to cholesterol-rich microdomains: role of PDGFR

Aims We demonstrated c-Src activation as a novel non-genomic signalling pathway for aldosterone in vascular smooth muscle cells (VSMCs). Here, we investigated molecular mechanisms and biological responses of this phenomenon, focusing on the role of lipid rafts/caveolae and platelet-derived growth factor receptor (PDGFR) in c-Src-regulated proinflammatory responses by aldosterone. Methods and results Studies were performed in cultured VSMCs from Wistar-Kyoto (WKY) rats and caveolin-1 knockout (Cav 1(-/-)) and wild-type mice. Aldosterone stimulation increased c-Src phosphorylation and trafficking to lipid rafts/caveolae. Cholesterol depletion with methyl-beta-cyclodextrin abrogated aldosterone-induced phosphorylation of c-Src and its target, Pyk2. Aldosterone effects were recovered by cholesterol reload. Aldosterone-induced c-Src and cortactin phosphorylation was reduced in caveolin-1-silenced and Cav 1(-/-) VSMCs. PDGFR is phosphorylated by aldosterone within cholesterol-rich fractions of VSMCs. AG1296, a PDGFR inhibitor, prevented c-Src phosphorylation and translocation to cholesterol-rich fractions. Aldosterone induced an increase in adhesion molecule protein content and promoted monocyte adhesion to VSMCs, responses that were inhibited an by cholesterol depletion, caveolin-1 deficiency, AG1296 and PP2, a c-Src inhibitor. Mineralocorticoid receptor (MR) content in flotillin-2-rich fractions and co-immunoprecipitation with c-Src and PDGFR increased upon aldosterone stimulation, indicating MR-lipid raft/signalling association. Conclusion We demonstrate that aldosterone-mediated c-Src trafficking/activation and proinflammatory signalling involve lipid rafts/caveolae via PDGFR.

The prednisolone suppression test in depression: Dose-response and changes with antidepressant treatment

Depressed patients have reduced glucocorticoid receptor (GR) function, as demonstrated by resistance to the suppressive effects of the synthetic glucocorticoid hormone, and GR agonist, dexamethasone. We have developed a suppressive test with prednisolone, a synthetic glucocorticoid that is similar to cortisol in its pharmacodynamics and pharmacokinetics, and binds to both the GR and the mineralocorticoid receptor (MR). We have found that depressed patients suppress normally to prednisolone, unless they are particularly non-responsive to treatment. In the present study, we evaluated 28 inpatients with treatment-resistant depression (TRD), and compared salivary cortisol secretion (at 0900 h, 1200 h and 1700 h) after placebo or after prednisolone (5 mg), before and after an inpatient treatment admission. Half of the patients (n = 14) reached treatment response. When comparing the assessment between admission and discharge, cortisol output after placebo fell (-26% of area under the curve; p = 0.024) while the output after prednisolone did not change. Moreover, there was no change in the response to prednisolone (percentage suppression) between admission at discharge, and this was not influenced by treatment response. Finally, we could confirm and extend our previously published data with prednisolone (5 mg), showing that depressed patients (n = 12) and controls (n = 12) suppressed equally to both 5 and 10 mg doses of prednisolone. This study suggests that the response to prednisolone is similar in depressed patients and controls at different doses of prednisolone, and does not change with symptomatic improvement. This is in contrast with findings, from us and others, using other measures of hypothalamic-pituitary-adrenal axis function, such as basal cortisol levels or the response to dexamethasone. Thus, we propose that the prednisolone suppression test may offer specific biological and clinical information, related to its action at both the GR and the MR. (C) 2010 Elsevier Ltd. All rights reserved.

Genomic and nongenomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments

Leite-Dellova DC, Malnic G, Mello-Aires M. Genomic and non-genomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments. Am J Physiol Renal Physiol 300: F682-F691, 2011. First published December 29, 2010; doi:10.1152/ajprenal.00172.2010.-The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(-)(+)ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 +/- 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10),10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 +/- 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule

Pergher PS, Leite-Dellova D, de Mello-Aires M. Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule. Am J Physiol Renal Physiol 296: F1185-F1193, 2009. First published February 18, 2009; doi:10.1152/ajprenal.90217.2008.-The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO3)(-)) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO3)(-) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO3)(-), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol ( in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO3)(-) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO3)(-) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO3)(-) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO3)(-) in middle proximal tubule.

Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na(+)/H(+) exchanger in proximal S3 segment: role of cytosolic calcium

Leite-Dellova DC, Oliveira-Souza M, Malnic G, Mello-Aires M. Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na(+)/H(+) exchanger in proximal S3 segment: role of cytosolic calcium. Am J Physiol Renal Physiol 295: F1342-F1352, 2008. First published August 20, 2008; doi:10.1152/ajprenal.00048.2008.-The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na(+)/H(+) exchanger and on the [Ca(2+)](i) were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca(2+)](i) and after 6-min pi there was a new increase of [Ca(2+)](i) that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15 -or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca(2+)](i). RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15 -or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca(2+)](i) to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca(2+)](i) and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca(2+)](i) in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca(2+)](i) (at 10(-6) M aldosterone plus RU 486).

History of hypertension and eplerenone in patients with acute myocardial infarction complicated by heart failure

In the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study ( n = 6632), eplerenone- associated reduction in all- cause mortality was significantly greater in those with a history of hypertension ( Hx- HTN). There were 4007 patients with Hx- HTN ( eplerenone: n = 1983) and 2625 patients without Hx- HTN ( eplerenone: n = 1336). Propensity scores for eplerenone use, separately calculated for patients with and without Hx- HTN, were used to assemble matched cohorts of 1838 and 1176 pairs of patients. In patients with Hx- HTN, all- cause mortality occurred in 18% of patients treated with placebo ( rate, 1430/ 10 000 person- years) and 14% of patients treated with eplerenone ( rate, 1058/ 10 000 person- years) during 2350 and 2457 years of follow- up, respectively ( hazard ratio [ HR]: 0.71; 95% CI: 0.59 to 0.85; P < 0.0001). Composite end point of cardiovascular hospitalization or cardiovascular mortality occurred in 33% of placebo-treated patients ( 3029/ 10 000 person- years) and 28% of eplerenone- treated patients (2438/10 000 person- years) with Hx- HTN ( HR: 0.82; 95% CI: 0.72 to 0.94; P = 0.003). In patients without Hx- HTN, eplerenone reduced heart failure hospitalization ( HR: 73; 95% CI: 0.55 to 0.97; P = 0.028) but had no effect on mortality ( HR: 0.91; 95% CI: 0.72 to 1.15; P = 0.435) or on the composite end point ( HR: 0.91; 95% CI: 0.76 to 1.10; P = 0.331). Eplerenone should, therefore, be prescribed to all of the post - acute myocardial infarction patients with reduced left ventricular ejection fraction and heart failure regardless of Hx- HTN.

Activation of the ET-1/ETA Pathway Contributes to Erectile Dysfunction Associated with Mineralocorticoid Hypertension

The cavernosal tissue is highly responsive to endothelin-1 (ET-1), and penile smooth muscle cells not only respond to but also synthesize ET-1. Considering that ET-1 is directly involved in end-organ damage in salt-sensitive forms of hypertension, we hypothesized that activation of the ET-1/ET(A) receptor pathway contributes to erectile dysfunction (ED) associated with mineralocorticoid hypertension. Wistar rats were uninephrectomized and submitted to deoxycorticosterone acetate (DOCA)-salt treatment for 5 weeks. Control (Uni [uninephrectomized control]) animals were uninephrectomized and given tap water. Uni and DOCA-salt rats were simultaneously treated with vehicle or atrasentan (ET(A) receptor antagonist, 5 mg/Kg/day). Cavernosal reactivity to ET-1, phenylephrine (PE), ET(B) receptor agonist (IRL-1620) and electric field stimulation (EFS) were evaluated in vitro. Expression of ROCK alpha, ROCK beta, myosin phosphatase target subunit 1 (MYPT-1), and extracellular signal-regulated kinase 1/2 (ERK 1/2) were evaluated by western blot analysis. ET-1 and ET(A) receptor mRNA expression was evaluated by real-time reverse-transcriptase polymerase chain reaction. Voltage-dependent increase in intracavernosal pressure/mean arterial pressure (ICP/MAP) was used to evaluate erectile function in vivo. ET(A) receptor blockade prevents DOCA-salt-associated ED. Cavernosal strips from DOCA-salt rats displayed augmented preproET-1 expression, increased contractile responses to ET-1 and decreased relaxation to IRL-1620. Contractile responses induced by EFS and PE were enhanced in cavernosal tissues from DOCA-salt hypertensive rats. These functional changes were associated with increased activation of the RhoA/Rho-kinase and ERK 1/2 pathways. Treatment of rats with atrasentan completely prevented changes in cavernosal reactivity in DOCA-salt rats and restored the decreased ICP/MAP, completely preventing ED in DOCA-salt rats. Activation of the ET-1/ET(A) pathway contributes to mineralocorticoid hypertension-associated ED. ET(A) receptor blockade may represent an alternative therapeutic approach for ED associated with salt-sensitive hypertension and in pathological conditions where increased levels of ET-1 are present. Carneiro FS, Nunes KP, Giachini FRC, Lima VV, Carneiro ZN, Nogueira EF, Leite R, Ergul A, Rainey WE, Webb RC, and Tostes RC. Activation of the ET-1/ETA pathway contributes to erectile dysfunction associated with mineralocorticoid hypertension. J Sex Med **;**:**-**.

Dysregulation of renal transient receptor potential melastatin 6/7 but not paracellin-1 in aldosterone-induced hypertension and kidney damage in a model of hereditary hypomagnesemia

Rationale Hyperaldosteronism, important in hypertension, is associated with electrolyte alterations, including hypomagnesemia, through unknown mechanisms. Objective To test whether aldosterone influences renal Mg(2+) transporters, (transient receptor potential melastatin (TRPM) 6, TRPM7, paracellin-1) leading to hypomagnesemia, hypertension and target organ damage and whether in a background of magnesium deficiency, this is exaggerated. Methods and results Aldosterone effects in mice selectively bred for high-normal (MgH) or low (MgL) intracellular Mg(2+) were studied. Male MgH and MgL mice received aldosterone (350 mu g/kg per day, 3 weeks). SBP was elevated in MgL. Aldosterone increased blood pressure and albuminuria and increased urinary Mg(2+) concentration in MgH and MgL, with greater effects in MgL. Activity of renal TRPM6 and TRPM7 was lower in vehicle-treated MgL than MgH. Aldosterone increased activity of TRPM6 in MgH and inhibited activity in MgL. TRPM7 and paracellin-1 were unaffected by aldosterone. Aldosterone-induced albuminuria in MgL was associated with increased renal fibrosis, increased oxidative stress, activation of mitogen-activated protein kinases and nuclear factor-NF-kappa B and podocyte injury. Mg(2+) supplementation (0.75% Mg(2+)) in aldosterone-treated MgL normalized plasma Mg(2+), increased TRPM6 activity and ameliorated hypertension and renal injury. Hence, in a model of inherited hypomagnesemia, TRPM6 and TRPM7, but not paracellin-1, are downregulated. Aldosterone further decreased TRPM6 activity in hypomagnesemic mice, a phenomenon associated with hypertension and kidney damage. Such effects were prevented by Mg(2+) supplementation. Conclusion Amplified target organ damage in aldosterone-induced hypertension in hypomagnesemic conditions is associated with dysfunctional Mg(2+)-sensitive renal TRPM6 channels. Novel mechanisms for renal effects of aldosterone and insights into putative beneficial actions of Mg(2+), particularly in hyperaldosteronism, are identified. J Hypertens 29: 1400-1410 (C) 2011 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

Influência do bloqueador de receptor de angiotensina (Losartana potássica) na função renal e pressão arterial em cães GRMD

A distrofia muscular de Duchenne (DMD) é uma alteração neuromuscular caracterizada por contínua necrose muscular e degeneração, com eventual fibrose e infiltração por tecido adiposo. O aumento progressivo da fibrose intersticial no músculo impede a migração das células miogênicas, necessárias para a formação muscular. O modelo canino constitui-se nas melhores fenocópias da doença em humanos, quando comparados com outros modelos animais com distrofia. O tratamento antifibrose de pacientes DMD, tendo como alvo os mediadores da citocina, TGF-beta, e o tratamento com antiinflamatórios, podem limitar a degeneração muscular e contribuir para a melhora do curso da doença. O presente estudo teve como objetivo observar os possíveis efeitos adversos na fisiologia renal, por meio de avaliação bioquímica sanguínea e da pressão arterial, verificando a viabilidade do uso do Losartan (um inibidor de TGF-beta) nos cães afetados pela distrofia muscular. Foram utilizados quatro cães adultos, sendo dois machos e duas fêmeas. Utilizou-se a dose de 50mg de Losartan, administrada via oral, uma vez ao dia. Os exames clínicos, bem como alterações na função renal, o nível do potássio sérico e a pressão arterial não evidenciaram reação adversa durante todo o período do experimento. O uso de Losartan, por um período de 9 semanas, mostrou-se como uma terapia segura para o tratamento antifibrótico em cães adultos, não afetando a função renal ou pressão arterial dos animais.

AT1-receptor mediated vascular damage in myocardium, kidneys and liver in rats

The systemic aspect of vascular damage induced by angiotensin II (ANG II) has been poorly explored in the literature. Considering the presence of ANG II and its specific receptor AT1, in several organs, all tissues might be potentially affected by its effects. The aims of this study were: To evaluate the early histological changes in the heart, liver and kidneys, produced by ANG II infusion, to evaluate the protective effect of losartan. Wistar rats were distributed into three groups: control (no treatment), treated with ANG II, and treated with ANG II + losartan. ANG II was continuously infused over 72 hours by subcutaneous osmotic pumps. Histological sections of the myocardium, kidneys and liver were stained and observed for the presence of necrosis. There were ANG II-induced perivascular inflammation and necrosis of the arteriolar wall in the myocardium, kidney, and liver by, which were partially prevented by losartan. There was no significant correlation between heart and kidney damage. Tissue lesion severity was lower than that of vascular lesions, without statistical difference between groups. ANG II causes vascular injury in the heart, kidneys and liver, indicating a systemic vasculotoxic effect; the mechanisms of damage/protection vary depending on the target organ; perivascular lesions may occur even when anti-hypertensive doses of losartan are used.

Toll-like receptor 4 (TLR4) expression in human and murine pancreatic beta-cells affects cell viability and insulin homeostasis

Background: Toll-like receptor 4 (TLR4) is widely recognized as an essential element in the triggering of innate immunity, binding pathogen-associated molecules such as Lipopolysaccharide (LPS), and in initiating a cascade of pro-inflammatory events. Evidence for TLR4 expression in non-immune cells, including pancreatic beta-cells, has been shown, but, the functional role of TLR4 in the physiology of human pancreatic beta-cells is still to be clearly established. We investigated whether TLR4 is present in beta-cells purified from freshly isolated human islets and confirmed the results using MIN6 mouse insulinoma cells, by analyzing the effects of TLR4 expression on cell viability and insulin homeostasis. Results: CD11b positive macrophages were practically absent from isolated human islets obtained from nondiabetic brain-dead donors, and TLR4 mRNA and cell surface expression were restricted to beta-cells. A significant loss of cell viability was observed in these beta-cells indicating a possible relationship with TLR4 expression. Monitoring gene expression in beta-cells exposed for 48h to the prototypical TLR4 ligand LPS showed a concentration-dependent increase in TLR4 and CD14 transcripts and decreased insulin content and secretion. TLR4-positive MIN6 cells were also LPS-responsive, increasing TLR4 and CD14 mRNA levels and decreasing cell viability and insulin content. Conclusions: Taken together, our data indicate a novel function for TLR4 as a molecule capable of altering homeostasis of pancreatic beta-cells.

Interleukin-22 forms dimers that are recognized by two interleukin-22R1 receptor chains

Interleukin-22 (IL-22) is a class 2 cytokine whose primary structure is similar to that of interleukin 10 (IL-10) and interferon-gamma (IFN-gamma). IL-22 induction during acute phase immune response indicates its involvement in mechanisms of inflammation. Structurally different from IL-10 and a number of other members of IL-10 family, which form intertwined inseparable V-shaped dimers of two identical polypeptide chains, a single polypeptide chain of IL-22 folds on itself in a relatively globular structure. Here we present evidence, based on native gel electrophoresis, glutaraldehyde cross-linking, dynamic light scattering, and small angle x-ray scattering experiments, that human IL-22 forms dimers and tetramers in solution under protein concentrations assessable by these experiments. Unexpectedly, low-resolution molecular shape of IL-22 dimers is strikingly similar to that of IL-10 and other intertwined cytokine dimeric forms. Furthermore, we determine an ab initio molecular shape of the IL-22/IL-22R1 complex which reveals the V-shaped IL-22 dimer interacting with two cognate IL-22R1 molecules. Based on this collective evidence, we argue that dimerization might be a common mechanism of all class 2 cytokines for the molecular recognition with their respective membrane receptor. We also speculate that the IL-22 tetramer formation could represent a way to store the cytokine in nonactive form at high concentrations that could be readily converted into functionally active monomers and dimers upon interaction with the cognate cellular receptors.