The role of JUN in the regulation of PRKCC-mediated STAR expression and steroidogenesis in mouse Leydig cells

Manna, Pulak R.; Stocco, Douglas M.

doi:10.1677/jme-08-0077

Cited by 24 publications

(37 citation statements)

References 59 publications

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“…Anti-STAT3 shRNA (5Ј-CCAATTGTGATGCCTCCTT-3Ј), 26 anti-BAK1 shRNA (5Ј-TGCCTACGAACTCTTCACCTT-3Ј), 27 anti-JUN shRNAs (5Ј-GATGGAAACGACCTTCTA-3Ј and 5Ј-CAGTAACCCTAA-GATCCTA-3Ј referred to as shRNA#1 and shRNA#2, respectively) 28,29 lentiviral transgene plasmids pdcH1-shRNA-SR and pdcH1-Gl4-SR (shRNA-control) were cloned as previously described. 30 All shRNAlentiviral constructs encode RFP (red fluorescent protein) as a reporter gene.…”

Section: Dna Constructs Oligonucleotides and Site-directed Mutagenesismentioning

confidence: 99%

“…RNA22 allows identification of miRNA/mRNA binding even in the presence of nucleotide mismatches within the seed sequence. For gene-specific RNAi, we lentivirally expressed published shRNAs 28,29 predicted to target both c-JUN and JUND (shRNA#1) or c-JUN only (shRNA#2; Figure 7A). As shown in Figure 7B, miR-125b reduced expression of both c-JUN and JUND by approximately 50% and 60%, respectively.…”

Section: Mir-125b Regulates Expression Of C-jun and Jundmentioning

confidence: 99%

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Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways

Surdziel¹,

Cabanski

Dallmann³

et al. 2011

Blood

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MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression by sequence-specific targeting of multiple mRNAs. Although lineage-, maturation-, and disease-specific miRNA expression has been described, miRNAdependent phenotypes and miRNAregulated signaling in hematopoietic cells are largely unknown. Combining functional genomics, biochemical analysis, and unbiased and hypothesis-driven miRNA target prediction, we show that lentivirally over-expressed miR-125b blocks G-CSF-induced granulocytic differentiation and enables G-CSF-dependent proliferation of murine 32D cells. In primary lineage-negative cells, miR-125b over-expression enhances colonyformation in vitro and promotes myelopoiesis in mouse bone marrow chimeras. We identified Stat3 and confirmed Bak1 as miR-125b target genes with approximately 30% and 50% reduction in protein expression, respectively. However, genespecific RNAi reveals that this reduction, alone and in combination, is not sufficient to block G-CSF-dependent differentiation. STAT3 protein expression, DNAbinding, and transcriptional activity but not induction of tyrosine-phosphorylation and nuclear translocation are reduced upon enforced miR-125b expression, indicating miR-125b-mediated reduction of one or more STAT3 cofactors. Indeed, we identified c-Jun and Jund as potential miR-125b targets and demonstrated reduced protein expression in 32D/ miR-125b cells. Interestingly, gene-specific silencing of JUND but not c-JUN partially mimics the miR-125b over-expression phenotype. These data demonstrate coordinated regulation of several signaling pathways by miR-125b linked to distinct phenotypes in myeloid cells. IntroductionMicroRNAs (miRNAs) represent an emerging class of noncoding single-stranded RNAs of approximately 22 nucleotides 1,2 that play an important role in posttranscriptional regulation of gene expression. miRNAs are processed from primary primiRNA transcripts to pre-miRNAs and mature miRNAs in a multistep process. Mature miRNAs are incorporated into and subsequently recruit a multi-protein effector complex RISC (RNA-induced silencing complex) to complementary miRNAbinding sites located preferentially within the 3ЈUTR of target mRNAs. Sequence-specific binding of RISC results in reduced mRNA translation and/or degradation through RNA interference (RNAi). 3 The interaction between a miRNA and its target mRNAs usually requires complementarity only within so-called seed sequence (miRNA nucleotides 2-8). Hence, a single miRNA has the potential to regulate hundreds of proteins 4-6 but resulting target protein repression is typically relatively mild. 5,6 Thereby, the ratio of regulatory RNAs to target mRNAs may modulate the silencing activity with a negative correlation between target abundance and target down-regulation. 7 To identify miRNA target genes, several prediction programs based on the hybridization energy of complementary miRNA/mRNAs sequences have been described. [8][9][10][11][12] However, the use of these programs is error-prone and identification of miRNA tar...

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Section: Dna Constructs Oligonucleotides and Site-directed Mutagenesismentioning

confidence: 99%

Section: Mir-125b Regulates Expression Of C-jun and Jundmentioning

confidence: 99%

Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways

Surdziel¹,

Cabanski

Dallmann³

et al. 2011

Blood

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“…Transfection with PKC␣, -␦, and -and PKD small interfering RNAs (siRNAs) was performed using X-tremeGENE siRNA transfection reagent (Roche), using the procedures described previously (38,41). Silencer negative control, and the PKC␣ (no.…”

Section: Cells Plasmids Transfections and Luciferase Assaysmentioning

confidence: 99%

“…Immunoblotting studies were carried out using total cellular protein (25,26,41). Equal amounts of total protein (20 -30 g) were solubilized in sample buffer and loaded onto either 8 or 10% SDS-PAGE (Mini Protean II System; Bio-Rad Laboratories, Inc., Hercules, CA).…”

Section: Immunoblottingmentioning

confidence: 99%

The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

2011

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Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKC␣, -␦, -, and -/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKC␣, ␦, and and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a Ϫ83/Ϫ67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the steroidogenic machinery in mouse Leydig cells. (Endocrinology 152: 313-325, 2011)

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“…Recently, several groups have reported that the ERK1/2, JNK and p38 MAP kinases are involved in the regulation of testosterone production [15][16][17] in response to a variety of extracellular factors, including LH, 18 interleukin-1a 15 and human chorionic gonadotropin.…”

Section: Introductionmentioning

confidence: 99%

Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat Leydig cells

Yao

Liu²,

Gu³

et al. 2011

Asian J Androl

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Gonadotropin-releasing hormone (GnRH) is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase (MAPK) pathways are involved in GnRH agonist (GnRHa)-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3b-hydroxysteroid dehydrogenase (3b-HSD) and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay (RIA). The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3b-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L 21 after 24 h, activation of ERK1/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERK1/2, the upregulation of 3b-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3b-HSD gene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.

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The role of JUN in the regulation of PRKCC-mediated STAR expression and steroidogenesis in mouse Leydig cells

Abstract: Activator protein 1

Cited by 24 publications

References 59 publications

Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways

Enforced expression of miR-125b affects myelopoiesis by targeting multiple signaling pathways

The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat Leydig cells

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