Andrea Acquier

MAPK Phosphatase-1 (MKP-1) Expression Is Up-Regulated by hCG/cAMP and Modulates Steroidogenesis in MA-10 Leydig Cells

MAP kinases (MAPKs), such as ERK1/2, exert profound effects on a variety of physiological processes. In steroidogenic cells, ERK1/2 are involved in the expression and activation of steroidogenic acute regulatory protein, which plays a central role in the regulation of steroidogenesis. In MA-10 Leydig cells, LH and chorionic gonadotropin (CG) trigger transient ERK1/2 activation via protein kinase A, although the events that lead to ERK1/2 inactivation are not fully described. Here, we describe the hormonal regulation of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates MAPKs, in MA-10 cells. In our experiments, human CG (hCG)/cAMP stimulation rapidly and transiently increased MKP-1 mRNA levels by a transcriptional action. This effect was accompanied by an increase in protein levels in both nuclear and mitochondrial compartments. In cells transiently expressing flag-MKP-1 protein, hCG/cAMP promoted the accumulation of the recombinant protein in a time-dependent manner (10-fold at 1 h). Moreover, hCG/cAMP triggered ERK1/2-dependent MKP-1 phosphorylation. The blockade of cAMP-induced MAPK kinase/ERK activation abated MKP-1 phosphorylation but only partially reduced flag-MKP-1 protein accumulation. Together, these results suggest that hCG regulates MKP-1 at transcriptional and posttranslational level, protein phosphorylation being one of the mechanisms involved in this regulation. Our study also demonstrates that MKP-1 overexpression reduces the effects of cAMP on ERK1/2 phosphorylation, steroidogenic acute regulatory gene promoter activity, mRNA levels, and steroidogenesis, whereas MKP-1 down-regulation by small interfering RNA produces opposite effects. In summary, our data demonstrate that hCG regulates MKP-1 expression at multiple stages as a negative feedback regulatory mechanism to modulate the hormonal action on ERK1/2 activity and steroidogenesis.

Activation of the alternative pathway of complement during the acute phase of typical haemolytic uraemic syndrome

Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopenia and acute renal failure. We studied the activation state of classical and alternative pathways of complement during the acute phase of Shiga toxin‐associated HUS by performing a prospective study of 18 patients and 17 age‐matched healthy controls to evaluate C3, C3c, C4, C4d, Bb and SC5b‐9 levels. SC5b‐9 levels were increased significantly in all patients at admission compared to healthy and end‐stage renal disease controls, but were significantly higher in patients presenting with oliguria compared to those with preserved diuresis. C3 and C4 levels were elevated significantly at admission in the non‐oliguric group when compared to controls. No significant differences were found for C4d values, whereas factor Bb was elevated in all patients and significantly higher in oliguric patients when compared to both controls and non‐oliguric individuals. A positive and significant association was detected when Bb formation was plotted as a function of plasma SC5b‐9 at admission. Bb levels declined rapidly during the first week, with values not significantly different from controls by days 3 and 5 for non‐oligurics and oligurics, respectively. Our data demonstrate the activation of the alternative pathway of complement during the acute phase of Stx‐associated HUS. This finding suggests that complement activation may represent an important trigger for the cell damage that occurs during the syndrome.

Cisplatin inhibits testosterone synthesis by a mechanism that includes the action of reactive oxygen species (ROS) at the level of P450scc

Cisplatin (Cs) is a chemotherapeutic agent able to generate reactive oxygen species (ROS) which are linked to several side effects of the drug. Even when it is known that Cs produces Leydig cell dysfunction, it is unknown whether this particular side effect is mediated by ROS. The aim of this study was to evaluate the in vitro effects of Cs on testosterone production and the participation of ROS in this effect. We demonstrate that Cs promotes the generation of ROS in a time-, and concentration-dependent fashion, not only in mouse testicular interstitial cells but also in MA-10 Leydig cells. Also, Cs inhibits testosterone synthesis in a concentration-dependent fashion (5-50 μM for 4 h) and to a similar extent, in cells exposed to human chorionic gondadotropin hormone (hCG), to an analog of the second messenger cAMP (8Br-cAMP) or to a freely diffusible cholesterol analog (22R-hydroxycholesterol). However, this treatment does not inhibit the conversion of pregnenolone to testosterone. These data suggest that Cs exerts its inhibitory action on testosterone synthesis by an action at the level of P450scc. We also demonstrated that an antioxidant impairs the inhibitory effect of Cs on the conversion of the cholesterol analog into pregnenolone and that Cs does not change the expression level of P450scc mRNA. Therefore, it is concluded that Cs inhibits testosterone synthesis by a mechanism that includes the inhibition of P450scc by ROS.

Oxidative stress status during the acute phase of haemolytic uraemic syndrome

BACKGROUND Haemolytic uraemic syndrome (HUS) is characterized by haemolytic anaemia, thrombocytopaenia and acute renal failure. The aim of this study was to investigate the levels of oxidative stress (OS) during the acute phase of HUS. METHODS This prospective study included 18 patients diagnosed with D + HUS, 6 age-matched healthy controls and 29 children with end-stage renal disease (ESRD) not caused by HUS under regular haemodialysis. Plasma lipid peroxidation and non-enzymatic antioxidant defences were measured as thiobarbituric acid-reactive substances (TBARs) and total reactive antioxidant potential (TRAP), respectively, during hospitalization and in control individuals. RESULTS TBARs were significantly higher in both oliguric and non-oliguric patients at admission (1.8 ± 0.1; 1.7 ± 0.2 μM) and discharge (1.5 ± 0.1; 1.0 ± 0.1 μM) vs controls (0.5 ± 0.1 μM, P < 0.01) following disease progression. Maximal TBARs values differed significantly between oliguric and non-oliguric groups (4.5 ± 0.9 vs 2.4 ± 0.3 μM, P < 0.01) and were significantly higher (P < 0.05) than those found in ESRD patients (1.63 ± 0.1). TRAP values were significantly higher at admission and when the disease was fully established (measured here as highest TBARs record) vs controls (675 ± 51, 657 ± 60 and 317 ± 30 μM Trolox, P < 0.01), and were similar to control values at discharge (325 ± 33 μM Trolox). CONCLUSIONS We demonstrate here increased levels of OS during the acute phase of HUS, with peak plasma lipid peroxidation values well above those registered in ESRD individuals, and suggest a connection between OS and the clinical course of HUS.

MAP kinase phosphatase-3 (MKP-3) is transcriptionally and post-translationally up-regulated by hCG and modulates cAMP-induced p21 expression in MA-10 Leydig cells

Luteinizing hormone (LH) activates ERK1/2, MAP kinases (MAPKs) necessary for its action on steroidogenesis and cell proliferation, and also induces MAPK phosphatase-1 (MKP-1), which rapidly dephosphorylates nuclear ERK1/2. MKP-3 is a cytoplasmic ERK-phosphatase up-regulated by proliferative stimuli. MKP-3 also dephosphorylates transcription factor FOXO1, promoting its transport to the nucleus. Here we analyzed MKP-3 expression in MA-10 Leydig cells and demonstrated that LH receptor (LHR) activation with human gonadotropin hormone (hCG) and an analog of its second messenger, 8Br-cAMP, up-regulates MKP-3 by transcriptional and post-translational mechanisms. It is known that FOXO1 drives the expression of the cell cycle inhibitor p21. Since the activation of this transcription factor by MKP-3 has been reported, we assessed the effect of shRNA against MKP-3 on p21mRNA levels. 8Br-cAMP increased these levels (2-fold at 2h) and MKP-3 down-regulation reduced this effect. Our work demonstrates that LH/hCG tightly up-regulates MKP-3 which in turn, dephosphorylates ERK1/2 and drives p21 expression. These events could contribute to counteract hormonal action on cell proliferation.

Association between Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis in subgingival plaque and clinical parameters, in Argentine patients with aggressive periodontitis

BACKGROUND Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) have been associated with aggressive (AgP) and chronic periodontitis. OBJECTIVE The aim of this study was to evaluate the levels of Aa and Pg in gingival crevicular fluid (GCF) of patients with AgP and its relation with clinical parameters. DESIGN Sixteen females and fourteen males with clinical diagnosis of AgP aged 17-23 years and their matchs controls, were included in this study. Clinical recording concerning probing pocket depth, clinical attachment level, plaque index and gingival bleeding index were performed at baseline, 30 and 60 days after baseline. After clinical examination GCF samples were analyzed for Aa and Pg with a real-time polymerase chain reaction technique. Patients group was treated with a combined of mechanical and oral antibiotic therapy (doxycycline 100 mg/day, during 21 days). A multivariate analysis was used to determine the relationship between Aa and Pg counts with clinical parameters. RESULTS GCF from all subjects was positive for Aa and PG. In controls Pg concentration was higher than Aa (Pg: 42,420 ± 3,034 copies/ml; Aa: 66.6 ± 5.4 copies/ml p < 0.001) while in patients both microbes showed the same concentration (Aa: 559,878 ± 39,698 Pg: 572,321 ± 58,752). A significant and positive correlation was observed between counts of Aa and Pg (R square: 0.7965, p < 0.0001). Female showed more counts/ml. Aa might be closely associated with clinical parameters while Pg did not. At 30 and 60 days Aa counts in patients were similar to controls while Pg counts were equal to baseline. However, in spite of Pg presence a clinical improvement was observed in all patients. CONCLUSIONS In our population the presence of Aa may be associated with AgP while Pg may be in GCF as an opportunistic pathogen which might caused disease when the ecological balance was favorable.

Comparison of salivary levels of mucin and amylase and their relation with clinical parameters obtained from patients with aggressive and chronic periodontal disease

Objective Salivary mucin and amylase levels are increased in patients with chronic periodontitis (CP). Due to the fact that aggressive periodontitis (AgP) not only differs from chronic periodontitis in terms of its clinical manifestation, the aim of this study was to compare salivary mucin and amylase levels and their relation to the clinical parameters of patients with aggressive periodontitis with that of patients with chronic periodontitis. Material and Methods Eighty subjects were divided into two groups: 20 patients with AgP and their 20 matched controls and 20 patients with CP and their 20 matched controls, based on clinical attachment loss (CAL), probing pocket depth (PPD) and bleeding on probing (BOP). Whole unstimulated saliva was obtained and mucin, amylase and protein were determined by colorimetric methods. Pearson’s correlation analysis was used to determine the relationship between salivary mucin, amylase and protein levels and the clinical parameters. Results Salivary mucin, amylase and protein levels were increased in patients with AgP and CP but there were no differences between them or between control groups. Pearson’s correlation analysis, determined in the entire subjects studied, showed a positive and significant correlation of mucin, amylase and proteins with CAL and PPD and a negative correlation with the flow rate. When Pearson’s correlation analysis was carried out in each group separately, Fisher’s z transformation showed no significant difference between both groups. Conclusion Comparison of the salivary levels of mucin, amylase and protein and their relationship with clinical parameters of AgP patients with that of CP patients revealed no differences between both groups.

MAPK Phosphatase-2 (MKP-2) Is Induced by hCG and Plays a Role in the Regulation of CYP11A1 Expression in MA-10 Leydig Cells

MAPKs such as ERK1/2 are dephosphorylated, and consequently inactivated, by dual specificity phosphatases (MKPs). In Leydig cells, LH triggers ERK1/2 phosphorylation through the action of protein kinase A. We demonstrate that, in MA-10 Leydig cells, LH receptor activation by human chorionic gonadotropin (hCG) up-regulates MKP-2, a phosphatase that dephosphorylates ERK1/2, among other MAPKs. After 2 hours, hCG and 8-bromo-cAMP (8Br-cAMP) significantly increased MKP-2 mRNA levels (3-fold), which declined to basal levels after 6 hours. MKP-2 protein accumulation exhibited a similar kinetic profile. In cells transiently expressing flag-MKP-2 protein, hCG/8Br-cAMP stimulation promoted the accumulation of the chimera (2.5-fold after 3 h of stimulation). Pharmacologic and biochemical approaches showed that the accumulation of flag-MKP-2 involves a posttranslational modification that increases MKP-2 half-life. MKP-2 down-regulation by a short hairpin RNA (MKP-2 shRNA) raised the levels of phosphorylated ERK1/2 reached by 8Br-cAMP stimulation. This effect was evident after 180 min of stimulation, which suggests that MKP-2 down-regulates the late phase of cAMP-induced ERK1/2 activity. Also, MKP-2 down-regulation by MKP-2 shRNA increased the stimulatory effect of 8Br-cAMP on both promoter activity and messenger levels of CYP11A1, which encodes for the steroidogenic enzyme P450scc and is induced by LH/hCG through protein kinase A and ERK1/2 activities. Our findings demonstrate, for the first time, that LH/hCG tightly regulates MKP-2 expression, which modulates the induction of CYP11A1 by 8Br-cAMP. MKP-2 up-regulation might control ERK1/2 activity in a specific temporal frame to modulate the expression of a finite repertory of ERK-dependent genes.

Modulation of albumin-induced endoplasmic reticulum stress in renal proximal tubule cells by upregulation of mapk phosphatase-1.

High amounts of albumin in urine cause tubulointerstitial damage that leads to a rapid deterioration of the renal function. Albumin exerts its injurious effects on renal cells through a process named endoplasmic reticulum (ER) stress due to the accumulation of unfolded proteins in the ER lumen. In addition, albumin promotes phosphorylation and consequent activation of MAPKs such as ERK1/2. Since ERK1/2 activation promoted by albumin is a transient event, the aims of the present work were to identify the phosphatase involved in their dephosphorylation in albumin-exposed cells and to analyze the putative regulation of this phosphatase by albumin. We also sought to determine the role played by the phospho/dephosphorylation of ERK1/2 in the cellular response to albumin-induced ER stress. MAP kinase phosphatase-1, MKP-1, is a nuclear enzyme involved in rapid MAPK dephosphorylation. Here we present evidence supporting the notion that this phosphatase is responsible for ERK1/2 dephosphorylation after albumin exposure in OK cells. Moreover, we demonstrate that exposure of OK cells to albumin transiently increases MKP-1 protein levels. The increase was evident after 15 min of exposure, peaked at 1 h (6-fold) and declined thereafter. In cells overexpressing flag-MKP-1, albumin caused the accumulation of this chimera, promoting MKP-1 stabilization by a posttranslational mechanism. Albumin also promoted a transient increase in MKP-1 mRNA levels (3-fold at 1 h) through the activation of gene transcription. In addition, we also show that albumin increased mRNA levels of GRP78, a key marker of ER stress, through an ERK-dependent pathway. In line with this finding, our studies demonstrate that flag-MKP-1 overexpression blunted albumin-induced GRP78 upregulation. Thus, our work demonstrates that albumin overload not only triggers MAPK activation but also tightly upregulates MKP-1 expression, which might modulate ER stress response to albumin overload.

Reactive oxygen species mediate dopamine-induced signaling in renal proximal tubule cells

Intrarenally‐produced dopamine (DA) induces a large increase in urinary sodium excretion mainly due to the inhibition of tubular sodium reabsorption. We aimed to study the participation of reactive oxygen species (ROS) in DA signaling pathway in proximal tubule cells. Our results show that DA increased ROS production in OK cells and indicate the mitochondria as the main source of ROS. DA also increased ERK1/2, superoxide dismutase (SOD) and transcription factor κB (NF‐κB) activity. These findings suggest that DA generates mitochondria‐derived ROS that activate ERK1/2 and subsequently NF‐κB and SOD activity at concentrations that exert a physiological regulation of renal function.