scholarly journals The Lutropin/Choriogonadotropin Receptor-Induced Phosphorylation of the Extracellular Signal-Regulated Kinases in Leydig Cells Is Mediated by a Protein Kinase A-Dependent Activation of Ras

2003 ◽  
Vol 17 (11) ◽  
pp. 2189-2200 ◽  
Author(s):  
Takashi Hirakawa ◽  
Mario Ascoli
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Leydig Cells ◽  
Primary Cultures ◽  
Nucleotide Exchange ◽  
Camp Phosphodiesterase ◽  
Human Choriogonadotropin ◽  
Extracellular Signal ◽  
Guanosine Triphosphatase ◽  
Kinase A

Abstract The pathways involved in activation of the ERK1/2 cascade in Leydig cells were examined in MA-10 cells expressing the recombinant human LH receptor (hLHR) and in primary cultures of rat Leydig cell precursors. In MA-10 cells expressing the recombinant hLHR, human choriogonadotropin-induced activation of ERK1/2 is effectively inhibited by overexpression of a cAMP phosphodiesterase (a manipulation that blunts the human choriogonadotropin-induced cAMP response), by addition of H89 (a selective inhibitor of protein kinase A), or by overexpression of the heat-stable protein kinase A inhibitor, but not by overexpression of an inactive mutant of this inhibitor. Stimulation of hLHR did not activate Rap1, but activated Ras in an H89-sensitive fashion. Addition of H89 to MA-10 cells that had been cotransfected with a guanosine triphosphatase-deficient mutant of Ras almost completely inhibited the hLHR-mediated activation of ERK1/2. We also show that 8-bromo-cAMP activates Ras and ERK1/2 in MA-10 cells and in primary cultures of rat Leydig cells precursors in an H89-sensitive fashion, whereas a cAMP analog 8-(4-chloro-phenylthio)-2′-O-methyl-cAMP (8CPT-2Me-cAMP) that is selective for cAMP-dependent guanine nucleotide exchange factor has no effect. Collectively, our results show that the hLHR-induced phosphorylation of ERK1/2 in Leydig cells is mediated by a protein kinase A-dependent activation of Ras.

Rexinoid-Triggered Differentiation and Tumor-Selective Apoptosis of AML by Protein Kinase A-Mediated De-Subordination.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2235-2235
Author(s):  
Ettore Mariano Schiavone ◽  
Lucia Altucci ◽  
Mariacarla De Simone ◽  
Felicetto Ferrara ◽  
Aurélie Rossin ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Ex Vivo ◽  
Therapeutic Option ◽  
Hdac Inhibitors ◽  
Death Receptor ◽  
Phosphodiesterase Inhibitors ◽  
Camp Phosphodiesterase ◽  
Tumor Selective ◽  
Kinase A

Abstract Apart from PML-RARα acute promyelocytic leukemia all other acute myeloid leukemias (AML) are unresponsive to retinoid differentiation therapy. However, in our study we show that elevating the levels of cyclic AMP (cAMP) confers onto retinoid X receptor (RXR)-selective agonists (“rexinoids”) the ability to induce terminal granulocyte differentiation and apoptosis of all-trans retinoic acid-resistant and insensitive AML cell lines and patients’ AML blasts. Protein kinase A activation leads to co-repressor release from the RAR subunit of the RAR-RXR heterodimer, resulting in “de-subordination” of otherwise silent RXR, which acquires transcriptional competence in response to cognate ligands. Rexinoid-cAMP induction of endogenous RARβ is blunted in mouse embryo fibroblasts lacking RARs, but re-introduction of exogenous RARα re-establishes responsiveness, thus confirming that the RARα-RXR heterodimer is the rexinoid mediator. The apoptogenic effect of this treatment involves enhanced expression of the death receptor DR5 and its cognate ligand, the tumor necrosis factor-related apoptosis inducing ligand (TRAIL), both of which are known to induce apoptosis in a tumor cell-selective manner and lead to the activation of initiator caspases. Immunohistochemistry confirmed induction of TRAIL and DR5 in AML patient blasts cultured “ex vivo”. AML patients’ blasts responded to rexinoid-cAMP combination treatment with induction of maturation and apoptosis, independent of karyotype, immunophenotype, and FAB classification. Clonogenic assays revealed complete inhibition of blast clonogenicity in four out of five tested samples. Indeed, it is known that cAMP levels can be elevated also by treating cells with 3′, 5′-cAMP phosphodiesterase inhibitors (PDEi’s). These observation and the clinical availability of the corresponding drugs provide a rationale for initiating clinical studies addressing the efficacy of combinatorial PDEi-rexinoid therapy in AML patients. Together with our recent finding that a very promising class of epigenetic anti-tumor drugs operates through activation of TRAIL expression (Nat Med2005, 11(1):7784), the possibility to target both the ligand (TRAIL) by HDAC inhibitors and the cognate receptors (DR4, DR5) by the above described rexinoid crosstalk may represent a promising therapeutic option which might lead, despite the genetic, morphologic, and clinical variability of AML, to a novel therapeutic option for AML patients by inducing a tumor-selective death pathway.

scholarly journals Inhibition of Thromboxane A Synthase Activity Enhances Steroidogenesis and Steroidogenic Acute Regulatory Gene Expression in MA-10 Mouse Leydig Cells

Endocrinology ◽  
2007 ◽  
Vol 149 (2) ◽  
pp. 851-857 ◽  
Author(s):  
XingJia Wang ◽  
Xiangling Yin ◽  
Randolph B. Schiffer ◽  
Steven R. King ◽  
Douglas M. Stocco ◽  
...  
Keyword(s):  
Rna Interference ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Leydig Cells ◽  
Steroid Hormone ◽  
Mrna Levels ◽  
Star Protein ◽  
Steroidogenic Acute Regulatory ◽  
Star Gene ◽  
Kinase A

The cyclooxygenase-2 (COX2)-dependent inhibition of Leydig cell steroidogenesis has been demonstrated. To understand the mechanism for this effect of COX2, the present study examined the role of an enzyme downstream of COX2, namely thromboxane A synthase (TBXAS), in steroidogenesis. Inhibition of TBXAS activity with the inhibitor furegrelate induced a concentration-dependent increase in cAMP-induced steroidogenic acute regulatory (StAR) protein in MA-10 mouse Leydig cells. The increase in StAR protein occurred concomitantly with a significant increase in steroid hormone production. Similar results were obtained in StAR promoter activity assays and RT-PCR analyses of StAR mRNA levels, suggesting that inhibition of TBXAS activity enhanced StAR gene transcription. These observations were corroborated when TBXAS expression was specifically inhibited by RNA interference. Although the RNA interference reduced mRNA levels of TBXAS, it increased StAR mRNA levels, StAR protein, and steroidogenesis. Additional studies indicated that inhibition of TBXAS activity reduced DAX-1 protein, a repressor in StAR gene transcription. In the absence of cAMP, inhibition of TBXAS activity did not induce a significant increase in steroid hormone and StAR protein. However, addition of a low level of cAMP analogs dramatically increased steroidogenesis. Lastly, inhibition of protein kinase A activity essentially abolished the steroidogenic effect of the TBXAS inhibitor. Thus, the results from the present study suggest that a minimal level of protein kinase A activity is required for the steroidogenic effect of the TBXAS inhibitor and that inhibition of TBXAS activity or its expression increase the steroidogenic sensitivity of MA-10 mouse Leydig cells to cAMP stimulation.

scholarly journals Protein Kinase A-Dependent Phosphorylation Modulates β1Pix Guanine Nucleotide Exchange Factor Activity through 14-3-3β Binding

2007 ◽  
Vol 28 (5) ◽  
pp. 1679-1687 ◽  
Author(s):  
Ahmed Chahdi ◽  
Andrey Sorokin
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Guanine Nucleotide Exchange Factor ◽  
Small Gtpases ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Exchange Factor ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Kinase A

ABSTRACT β1Pix is a guanine nucleotide exchange factor (GEF) for the small GTPases Rac and Cdc42 which has been shown to mediate signaling pathways leading to cytoskeletal reorganization. In the present study, we show that the basal association between endogenous βPix and endogenous 14-3-3β was increased after forskolin stimulation and significantly inhibited by protein kinase A inhibitor. However, forskolin stimulation failed to increase the interaction between 14-3-3β and a β1Pix mutant that is insensitive to protein kinase A phosphorylation, β1Pix(S516A, T526A). We present evidence indicating that forskolin-induced binding of 14-3-3β to β1Pix results in inhibition of Rac1 GTP loading in 293 cells and in vitro. Furthermore, we show that deletion of 10 amino acid residues within the leucine zipper domain is sufficient to block β1Pix homodimerization and 14-3-3β binding and modulates β1Pix-GEF activity. These residues also play a crucial role in β1Pix intracellular localization. These results indicate that 14-3-3β negatively affects the GEF activity of dimeric β1Pix only. Altogether, these results provide a mechanistic insight into the role of 14-3-3β in modulating β1Pix-GEF activity.

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