AT1a Receptor Has Interacted with Angiotensin-converting Enzymes 2 mRNA Expression in Mouse Brainstem
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Abstract The involvement of salt-inducible kinase, a recently cloned protein serine/threonine kinase, in adrenal steroidogenesis was investigated. When Y1 mouse adrenocortical tumor cells were stimulated by ACTH, the cellular content of salt-inducible kinase mRNA, protein, and enzyme activity changed rapidly. Its level reached the highest point in 1–2 h and returned to the initial level after 8 h. The mRNA levels of cholesterol side-chain cleavage cytochrome P450 and steroidogenic acute regulatory protein, on the other hand, began to rise after a few hours, reaching the highest levels after 8 h. The salt-inducible kinase mRNA level in ACTH-, forskolin-, or 8-bromo-cAMP-treated Kin-7 cells, mutant Y1 with less cAMP-dependent PKA activity, remained low. However, Kin-7 cells, when transfected with a PKA expression vector, expressed salt-inducible kinase mRNA. Y1 cells that overexpressed salt-inducible kinase were isolated, and the mRNA levels of steroidogenic genes in these cells were compared with those in the parent Y1. The level of cholesterol side-chain cleavage cytochrome P450 mRNA in the salt-inducible kinase-overexpressing cells was markedly low compared with that in the parent, while the levels of Ad4BP/steroidogenic factor-1-, ACTH receptor-, and steroidogenic acute regulatory protein-mRNAs in the former were similar to those in the latter. The ACTH-dependent expression of cholesterol side-chain cleavage cytochrome P450- and steroidogenic acute regulatory protein-mRNAs in the salt-inducible kinase-overexpressing cells was significantly repressed. The promoter activity of the cholesterol side-chain cleavage cytochrome P450 gene was assayed by using Y1 cells transfected with a human cholesterol side-chain cleavage cytochrome P450 promoter-linked reporter gene. Addition of forskolin to the culture medium enhanced the cholesterol side-chain cleavage cytochrome P450 promoter activity, but the forskolin-dependently activated promoter activity was inhibited when the cells were transfected with a salt-inducible kinase expression vector. This inhibition did not occur when the cells were transfected with a salt-inducible kinase (K56M) vector that encoded an inactive kinase. The salt-inducible kinase’s inhibitory effect was also observed when nonsteroidogenic, nonAd4BP/steroidogenic factor-1 -expressing, NIH3T3 cells were used for the promoter assays. These results suggested that salt-inducible kinase might play an important role(s) in the cAMP-dependent, but Ad4BP/steroidogenic factor-1-independent, gene expression of cholesterol side-chain cleavage cytochrome P450 in adrenocortical cells.
Steroidogenic factor 1
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Angiotensin (A) II plays a critical role in vascular remodeling, and its action is mediated by type 1 AII receptor (AT1R). Recently, 15-deoxy-(Delta)(12,14)-prostaglandin J(2) and thiazolidinediones have been shown to be ligands for peroxisome proliferator-activated receptor (PPAR)-gamma and activate PPAR-gamma. In the present work, we have studied the effect of PPAR-gamma on AT1R expression in rat vascular smooth muscle cells (VSMCs). We observed that: 1) endogenous AT1R expression was significantly decreased by PPAR-gamma ligands both at messenger RNA and protein levels, whereas AT1R messenger RNA stability was not affected; 2) AII-induced increase of (3)H-thymidine incorporation into VSMCs was inhibited by PPAR-gamma ligands; 3) rat AT1R gene promoter activity was significantly suppressed by PPAR-gamma ligands, and PPAR-gamma overexpression further suppressed the promoter activity; 4) transcriptional analyses using AT1R gene promoter mutants revealed that a GC-box-related sequence within the -58/-34 region of the AT1R gene promoter was responsible for the suppression; 5) Sp1 overexpression stimulated AT1R gene transcription via the GC-box-related sequence, which was inhibited by additional PPAR-gamma overexpression; 6) electrophoretic mobility shift assay suggested that Sp1 could bind to the GC-box-related sequence whereas PPAR-gamma could not; 7) antibody supershift experiments using VSMC nuclear extracts revealed that protein-DNA complexes formed on the GC-box-related sequence, which were decreased by PPAR-gamma coincubation, were mostly composed of Sp1; and 8) glutathione S-transferase pull-down assay revealed a direct interaction between PPAR-gamma and Sp1. Taken together, it is suggested that activated PPAR-gamma suppresses AT1R gene at a transcriptional level by inhibiting Sp1 via a protein-protein interaction. PPAR-gamma ligands, thus, may inhibit AII-induced cell growth and hypertrophy in VSMCs by AT1R expression suppression and possibly be beneficial for treatment of diabetic patients with hypertension and atherosclerosis.
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LH receptor (LHR) expression in the ovary is regulated by the RNA binding protein, (LHR mRNA binding protein [LRBP]), which has been identified as being mevalonate kinase. This study examined the role of microRNA miR-122 in LRBP-mediated LHR mRNA expression. Real-time PCR analysis of ovaries from pregnant mare serum gonadotropin/human chorionic gonadotropin (hCG)-primed female rats treated with hCG to down-regulate LHR expression showed that an increase in miR-122 expression preceded LHR mRNA down-regulation. The expression of miR-122 and its regulation was confirmed using fluorescent in situ hybridization of the frozen ovary sections using 5′-fluorescein isothiocyanate-labeled miR-122 locked nucleic acid probe. The increased expression of miR-122 preceded increased expression of LRBP mRNA and protein, and these increases were followed by LHR mRNA down-regulation. Inhibition of protein kinase A (PKA) and ERK1/2 signaling pathways by H89 and UO126, respectively, attenuated the hCG-mediated up-regulation of miR-122 levels. This was also confirmed in vitro using human granulosa cells. These results suggest the possibility that hCG-mediated miR-122 expression is mediated by the activation of cAMP/PKA/ERK signaling pathways. Inhibition of miR-122 by injection of the locked nucleic acid-conjugated antagomir of miR-122 abrogated the hCG-mediated increases in LRBP protein expression. Because it has been previously shown that miR-122 regulates sterol regulatory element-binding proteins (SREBPs) and SREBPs, in turn, regulate LRBP expression, the role of SREBPs in miR-122-mediated increase in LRBP expression was then examined. The levels of active forms of both SREBP-1a and SREBP-2 were increased in response to hCG treatment, and the stimulatory effect was sustained up to 4 hours. Taken together, our results suggest that hCG-induced down-regulation of LHR mRNA expression is mediated by activation of cAMP/PKA/ERK pathways to increase miR-122 expression, which then increases LRBP expression through the activation of SREBPs.
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The renin-angiotensin system is an important regulator of blood pressure and volume homeostasis in mammals. Angiotensinogen, a precursor of the octapeptide angiotensin II and an effector of the renin-angiotensin system, is synthesized in numerous rat tissues. Angiotensinogen is expressed in an islet cell line (RIN 1056A) derived from a rat pancreatic tumor. Angiotensinogen mRNA detected by Northern analysis is abundant in the cell line and is approximately 200 bases longer than the mRNA isolated from rat liver, due to both a longer poly(A) tract and the use of a second polyadenylation site. Dexamethasone is a potent inducer of angiotensinogen mRNA, producing a progressive accumulation from 3 to 96 hr in culture (9-fold above control levels). The dexamethasone effect is competitively inhibited by the glucocorticoid antagonist RU486, and transcription rate assays using isolated nuclei indicate that the effect is primarily at the transcriptional level.
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Angiotensinogen gene expression has a broad tissue specificity. Whereas angiotensinogen mRNA is undetectable in normal rat pancreas, we have identified angiotensinogen mRNA in all tumors and cell lines derived from a rat islet cell line, RIN-r. A subclone with the highest angiotensinogen mRNA levels, 1056A, secreted N-glycosylated angiotensinogen. Angiotensinogen mRNA of 1056A cells was approximately 200 nucleotides longer than that of liver, and this was shown to be due to an extension of the 3'-untranslated region. Dexamethasone increased angiotensinogen mRNA levels approximately 9-fold above control, and this increase was linear over 110 h, indicating a half-life of greater than 55 h for angiotensinogen mRNA during dexamethasone induction. This effect of dexamethasone was inhibited by the glucocorticoid antagonist RU 38486. Dexamethasone increased angiotensinogen gene transcription approximately 5-fold in a nuclear run-on assay. These results demonstrate that dexamethasone induction of angiotensinogen mRNA levels in 1056A cells is due, at least in part, to a transcriptional response and that 1056A cells will be useful for the study of angiotensinogen gene regulation and the identification of glucocorticoid regulatory sequences.
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In both cell culture based model systems and in the failing human heart, β-adrenergic receptors (β-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3′-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by β-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the β-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (~50%) in the abundance of β1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of β2-AR mRNA was first described, exposure to β-AR agonist resulted in a significant increase in AUF1 mRNA and protein (~ 100%). Conversely, agonist exposure significantly decreased (~40%) β2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3′-untranslated region of the human β1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to β1-AR 3′-untranslated region mRNA. In both cell culture based model systems and in the failing human heart, β-adrenergic receptors (β-AR) undergo agonist-mediated down-regulation. This decrease correlates closely with down-regulation of its mRNA, an effect regulated in part by changes in mRNA stability. Regulation of mRNA stability has been associated with mRNA-binding proteins that recognize A + U-rich elements within the 3′-untranslated regions of many mRNAs encoding proto-oncogene and cytokine mRNAs. We demonstrate here that the mRNA-binding protein, AUF1, is present in both human heart and in hamster DDT1-MF2 smooth muscle cells and that its abundance is regulated by β-AR agonist stimulation. In human heart, AUF1 mRNA and protein was significantly increased in individuals with myocardial failure, a condition associated with increases in the β-adrenergic receptor agonist norepinephrine. In the same hearts, there was a significant decrease (~50%) in the abundance of β1-AR mRNA and protein. In DDT1-MF2 cells, where agonist-mediated destabilization of β2-AR mRNA was first described, exposure to β-AR agonist resulted in a significant increase in AUF1 mRNA and protein (~ 100%). Conversely, agonist exposure significantly decreased (~40%) β2-adrenergic receptor mRNA abundance. Last, we demonstrate that AUF1 can be immunoprecipitated from polysome-derived proteins following UV cross-linking to the 3′-untranslated region of the human β1-AR mRNA and that purified, recombinant p37AUF1 protein also binds to β1-AR 3′-untranslated region mRNA.
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