Successive occupancy by immediate early transcriptional factors of the tyrosine hydroxylase gene TRE and CRE sites in PACAP-stimulated PC12 pheochromocytoma cells

Abstract-We previously showed that CGP 42112 (an angiotensin type 2 [AT 2 ] agonist) markedly reduces catecholamine biosynthesis by decreasing cGMP production mediated by AT 2 , a subtype of Ang II receptor that is dominantly expressed in cultured porcine chromaffin cells. To elucidate the relationship of the 2 types of Ang II receptors, angiotensin type 1 (AT 1 ) and AT 2 , in the synthesis of catecholamine in adrenal medullary cells, we have examined the effect of Ang II plus CV-11974 (an AT 1 antagonist that selectively simulates AT 2 stimulation) and the effect of Ang II plus PD 123319 (an AT 2 antagonist that selectively simulates AT 1 stimulation) on catecholamine synthesis. We found that Ang II reduced cGMP production via AT 2 , in a similar manner to that found with CGP 42112. Stimulation of AT 1 significantly upregulated protein kinase C activity. Tyrosine hydroxylase (TH) is a rate-limiting enzyme involved in the biosynthesis of catecholamine, and this catecholamine synthesis depends both on TH enzyme activity and on the levels of TH protein after TH gene transcription. We found that AT 2 stimulation significantly inhibited TH enzyme activity, whereas AT 1 stimulation significantly upregulated TH enzyme activity. The stimulatory effect of AT 1 was completely inhibited by Ro-32-0432 (a protein kinase C inhibitor) and PD 98059 (a MAP kinase kinase-1 [MEK-1] inhibitor). Pretreatment of cells with either 8-Br-cGMP (a membrane-permeable cGMP analog) or Zaprinast (a phosphodiesterase inhibitor) abolished the inhibitory effect of AT 2 on TH enzyme activity, indicating that the stimulatory effect of AT 2 may be mediated through a reduction in cGMP concentration. Similar to the effect on TH enzyme activity, AT 2 stimulation significantly reduced TH mRNA and protein levels and net catecholamine content below basal levels, whereas AT 1 stimulation increased them. We confirmed these findings by gel mobility shift assay. Our results show that stimulation of AT 2 reduces catecholamine biosynthesis via a decrease in cGMP levels. In contrast, stimulation of AT 1 stimulates catecholamine biosynthesis through activation of PKC. Thus, we conclude that AT 1 and AT 2 have counter-regulatory roles in the synthesis of catecholamine in adrenal medullary chromaffin cells. Key Words: receptors, angiotensin II Ⅲ tyrosine hydroxylase Ⅲ cyclic GMP Ⅲ angiotensin antagonist Ⅲ catecholamines T here are 2 major subtypes of Ang II receptor, type 1 (AT 1 ) and type 2 (AT 2 ). 1 Most of the stimulatory effects of Ang II, such as those on the cardiovascular system and on fluid volume homeostasis, are mediated by AT 1 . AT 1 is linked with activation of phospholipase C and with a subsequent increase in [Ca 2ϩ ] i and protein kinase C (PKC) activity. 1 In contrast, the physiological role of and signal transduction pathways connected with AT 2 are not established. In adults, AT 2 expression is restricted to only a few organs, such as adrenal medulla. 2 It has been shown that stimulation of AT 2 induces a decrease in intracellular cGMP leve...

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“…17 An expanded Methods section can be found in an online data supplement available at http://www.hypertensionaha.org.…”

Section: Gel Mobility Shift Assaymentioning

confidence: 99%

Angiotensin II Type 2 Receptor Counter-Regulates Type 1 Receptor in Catecholamine Synthesis in Cultured Porcine Adrenal Medullary Chromaffin Cells

et al. 2002

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“…The activation of PNMT through the sympatho-adrenal system can occur via the release of acetylcholine and PACAP from the splanchnic nerve [5]. Neurotransmitters such as acetylcholine, serotonin, and the peptide neurotransmitter PACAP have been shown to induce PNMT via the protein kinase A (PKA) and protein kinase C (PKC) pathways [10, 11]. In addition, both acetylcholine and PACAP activate signaling cascades that regulate transcription factors expressed exclusively in adrenergic cells such as the early growth response transcription factor 1 (Egr-1) and promote PNMT transcription [12].…”

Section: Introductionmentioning

confidence: 99%

Role of Reactive Oxygen Species in the Neural and Hormonal Regulation of the PNMT Gene in PC12 Cells

Crispo

Ansell

Ubriaco

et al. 2011

Oxidative Medicine and Cellular Longevity

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The stress hormone, epinephrine, is produced predominantly by adrenal chromaffin cells and its biosynthesis is regulated by the enzyme phenylethanolamine N-methyltransferase (PNMT). Studies have demonstrated that PNMT may be regulated hormonally via the hypothalamic-pituitary-adrenal axis and neurally via the stimulation of the splanchnic nerve. Additionally, hypoxia has been shown to play a key role in the regulation of PNMT. The purpose of this study was to examine the impact of reactive oxygen species (ROS) produced by the hypoxia mimetic agent CoCl2, on the hormonal and neural stimulation of PNMT in an in vitro cell culture model, utilizing the rat pheochromocytoma (PC12) cell line. RT-PCR analyses show inductions of the PNMT intron-retaining and intronless mRNA splice variants by CoCl2 (3.0- and 1.76-fold, respectively). Transient transfection assays of cells treated simultaneously with CoCl2 and the synthetic glucocorticoid, dexamethasone, show increased promoter activity (18.5-fold), while mRNA levels of both splice variants do not demonstrate synergistic effects. Similar results were observed when investigating the effects of CoCl2-induced ROS on the neural stimulation of PNMT via forskolin. Our findings demonstrate that CoCl2-induced ROS have synergistic effects on hormonal and neural activation of the PNMT promoter.

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“…Like the upstream AP-1 site, the CRE in the core promoter is highly conserved throughout mammals and is recognized by several bZIP transcription factors (Sabban, 1997; Liu et al, 1999; Yukimasa et al, 1999; Suzuki et al, 2002). A putative PITX3 binding site partially overlaps the CRE (Cazorla et al, 2000; Kim et al, 2003b).…”

Section: Resultsmentioning

confidence: 99%

“…The strong conservation of the CRE is consistent with reports showing mutation of this site blocks reporter gene expression in all adult brain catecholaminergic regions (Trocmé et al, 1998). CREB is the canonical bZIP protein that recognizes the CRE, but the specific bZIP proteins bound to the TH promoter CRE in vivo are likely cell-type dependent since Fos, Jun and ATF family proteins can also bind this site (Sabban, 1997; Liu et al, 1999; Yukimasa et al, 1999; Suzuki et al, 2002). Partially overlapping the CRE is a putative and conserved PITX3 site.…”

Section: Discussionmentioning

confidence: 99%

Nucleotide sequence conservation of novel and established cis-regulatory sites within the tyrosine hydroxylase gene promoter

et al. 2014

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Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis and its gene proximal promoter ( < 1 kb upstream from the transcription start site) is essential for regulating transcription in both the developing and adult nervous systems. Several putative regulatory elements within the TH proximal promoter have been reported, but evolutionary conservation of these elements has not been thoroughly investigated. Since many vertebrate species are used to model development, function and disorders of human catecholaminergic neurons, identifying evolutionarily conserved transcription regulatory mechanisms is a high priority. In this study, we align TH proximal promoter nucleotide sequences from several vertebrate species to identify evolutionarily conserved motifs. This analysis identified three elements (a TATA box, cyclic AMP response element (CRE) and a 5′-GGTGG-3′ site) that constitute the core of an ancient vertebrate TH promoter. Focusing on only eutherian mammals, two regions of high conservation within the proximal promoter were identified: a ∼250 bp region adjacent to the transcription start site and a ∼85 bp region located approximately 350 bp further upstream. Within both regions, conservation of previously reported cis-regulatory motifs and human single nucleotide variants was evaluated. Transcription reporter assays in a TH -expressing cell line demonstrated the functionality of highly conserved motifs in the proximal promoter regions and electromobility shift assays showed that brain-region specific complexes assemble on these motifs. These studies also identified a non-canonical CRE binding (CREB) protein recognition element in the proximal promoter. Together, these studies provide a detailed analysis of evolutionary conservation within the TH promoter and identify potential cis-regulatory motifs that underlie a core set of regulatory mechanisms in mammals.

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Successive occupancy by immediate early transcriptional factors of the tyrosine hydroxylase gene TRE and CRE sites in PACAP-stimulated PC12 pheochromocytoma cells

Cited by 12 publications

References 29 publications

Angiotensin II Type 2 Receptor Counter-Regulates Type 1 Receptor in Catecholamine Synthesis in Cultured Porcine Adrenal Medullary Chromaffin Cells

Angiotensin II Type 2 Receptor Counter-Regulates Type 1 Receptor in Catecholamine Synthesis in Cultured Porcine Adrenal Medullary Chromaffin Cells

Role of Reactive Oxygen Species in the Neural and Hormonal Regulation of the PNMT Gene in PC12 Cells

Nucleotide sequence conservation of novel and established cis-regulatory sites within the tyrosine hydroxylase gene promoter

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