Natalia Gomez

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.

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.

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action.

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.

Synthesis, structural characterization, and pro-apoptotic activity of 1-indanone thiosemicarbazone platinum(II) and palladium(II) complexes: potential as antileukemic agents.

In the search for alternative chemotherapeutic strategies against leukemia, various 1‐indanone thiosemicarbazones, as well as eight novel platinum(II) and palladium(II) complexes, with the formula [MCl2(HL)] and [M(HL)(L)]Cl, derived from two 1‐indanone thiosemicarbazones were synthesized and tested for antiproliferative activity against the human leukemia U937 cell line. The crystal structure of [Pt(HL1)(L1)]Cl.2MeOH, where L1=1‐indanone thiosemicarbazone, was solved by X‐ray diffraction. Free thiosemicarbazone ligands showed no antiproliferative effect, but the corresponding platinum(II) and palladium(II) complexes inhibited cell proliferation and induced apoptosis. Platinum(II) complexes also displayed selective apoptotic activity in U937 cells but not in peripheral blood monocytes or the human hepatocellular carcinoma HepG2 cell line used to screen for potential hepatotoxicity. Present findings show that, in U937 cells, 1‐indanone thiosemicarbazones coordinated to palladium(II) were more cytotoxic than those complexed with platinum(II), although the latter were found to be more selective for leukemic cells suggesting that they are promising compounds with potential therapeutic application against hematological malignancies.

Activity on Trypanosoma cruzi, erythrocytes lysis and biologically relevant physicochemical properties of Pd(II) and Pt(II) complexes of thiosemicarbazones derived from 1-indanones.

American trypanosomiasis or Chagas disease, caused by the protist parasite Trypanosoma cruzi (T. cruzi), is a major health concern in Latin America. In the search for new bioactive compounds, eight Pd(II) and Pt(II) complexes of thiosemicarbazones derived from 1-indanones (HL) were evaluated as potential anti-T. cruzi compounds. Their unspecific cytotoxicity was determined on human erythrocytes. Two physicochemical features, lipophilicity and redox behavior, that could be potentially relevant for the biological activity of these complexes, were determined. Crystal structure of [Pd(HL1)(L1)]Cl·CH(3)OH, where HL1=1-indanone thiosemicarbazone, was solved by X-ray diffraction methods. Five of the eight metal complexes showed activity against T. cruzi with IC(50) values in the low micromolar range and showed significantly higher activity than the corresponding free ligands. Four of them resulted more active against the parasite than the reference antitrypanosomal drug Nifurtimox. Anti-T. cruzi activity and selectivity towards the parasite were both higher for the Pd(II) compounds than for the Pt(II) analogues, showing the effect of the metal center selection on the biological behavior. Among both physicochemical features tested for this series of compounds, lipophilicity and redox behavior, only the former seemed to show correlation with the antiproliferative effects observed. Metal coordination improved bioactivity but lead to an increase of mammalian cytotoxicity. Nevertheless, some of the metal complexes tested in this work still show suitable selectivity indexes and deserve further developments.

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.

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.

Dual Role of cAMP in the Transcriptional Regulation of Multidrug Resistance-Associated Protein 4 (MRP4) in Pancreatic Adenocarcinoma Cell Lines

Cyclic AMP represents one of the most studied signaling molecules and its role in proliferation and differentiation processes has been well established. Intracellular cAMP levels are tightly regulated where the MRP4 transporter plays a major role. In the present study, we sought to establish whether cAMP modulated MRP4 expression in pancreatic adenocarcinoma cell lines. Quantitative PCR and western blot studies showed that cAMP-increasing agents enhanced MRP4 transcripts and protein levels in PANC-1 cells. Reporter luciferase experiments carried out in pancreatic AR42J cells showed that intracellular cAMP up-regulates MRP4 through an Epac2- and Rap1- mediated mechanism whereas extracellular cAMP reduced MRP4 promoter activity by a MEK/ERK-mediated pathway. Present results show that cAMP regulates MRP4 promoter activity, and further indicate that the balance between intracellular and extracellular cAMP levels determines MRP4 expression.

G2/M Cell Cycle Arrest and Tumor Selective Apoptosis of Acute Leukemia Cells by a Promising Benzophenone Thiosemicarbazone Compound

Anti-mitotic therapies have been considered a hallmark in strategies against abnormally proliferating cells. Focusing on the extensively studied family of thiosemicarbazone (TSC) compounds, we have previously identified 4,4’-dimethoxybenzophenone thiosemicarbazone (T44Bf) as a promising pharmacological compound in a panel of human leukemia cell lines (HL60, U937, KG1a and Jurkat). Present findings indicate that T44Bf-mediated antiproliferative effects are associated with a reversible chronic mitotic arrest caused by defects in chromosome alignment, followed by induced programmed cell death. Furthermore, T44Bf selectively induces apoptosis in leukemia cell lines when compared to normal peripheral blood mononuclear cells. The underlying mechanism of action involves the activation of the mitochondria signaling pathway, with loss of mitochondrial membrane potential and sustained phosphorylation of anti-apoptotic protein Bcl-xL as well as increased Bcl-2 (enhanced phosphorylated fraction) and pro-apoptotic protein Bad levels. In addition, ERK signaling pathway activation was found to be a requisite for T44Bf apoptotic activity. Our findings further describe a novel activity for a benzophenone thiosemicarbazone and propose T44Bf as a promising anti-mitotic prototype to develop chemotherapeutic agents to treat acute leukemia malignancies.