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.
SummaryHaemolytic 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.
Previous studies propose 20-hydroxyeicosatetraenoic acid (20-HETE), a major arachidonic acid metabolite of cytochrome P-450 (CYP), as a possible mediator of Na(+)-K(+)-ATPase inhibition by dopamine (DA). The aim of this study was to investigate the intracellular mechanisms involved in this effect and to elucidate the DA receptor associated with the 20-HETE pathway in the rat kidney. DA (10(-5) M) inhibited Na(+)-K(+)-ATPase activity in microdissected tubular segments to 59.4 +/- 3.8% of control activity. This response was suppressed by the CYP4A inhibitor 17-octadecynoic acid (10(-6) M), which had no effect per se, thus confirming the participation of CYP arachidonic acid metabolites in DA-induced Na(+)-K(+)-ATPase inhibition. We next examined whether 20-HETE is involved in the signaling pathways triggered by either D(1) or D(2) receptors. Neither fenoldopam nor quinpirole (D(1) and D(2) agonists, respectively, both 10(-5) M) modified Na(+)-K(+)-ATPase activity when tried alone. However, coincubation of a threshold concentration of 20-HETE (10(-9) M) with fenoldopam resulted in a synergistic inhibition of Na(+)-K(+)-ATPase activity (66 +/- 2% of control activity), while 20-HETE plus quinpirole had no effect. Furthermore, 20-HETE (10(-9) M) synergized with forskolin (10(-5) M) and with the diacylglycerol analog 1-oleoyl-2-acetoyl-sn-glycerol (OAG; 10(-11) M; 62.0 +/- 5.3 and 69.9 +/- 2.0% of control activity, respectively), indicating a cooperative role of 20-HETE with the D(1)-triggered pathways. In line with these results, no additive effect was observed when OAG and 20-HETE were combined at concentrations which per se produced maximal inhibition (10(-6) M). These results demonstrate that the inhibition of Na(+)-K(+)-ATPase activity by DA in the proximal tubule may be the result of the synergism between 20-HETE and the D(1) signaling pathway.
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.
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