Parallel Up-Regulation of Catecholamine Biosynthetic Enzymes by Dexamethasone in PC12 Cells

Abstract: We sought to investigate whether dexamethasone produces a coordinated, time-dependent effect on all enzymes in the catecholamine biosynthetic pathway in PC12 cells. The levels of mRNAs of tyrosine hydroxylase (TH), aromatic L-amino acid decarboxylase (AADC), and dopamine γ-hydroxylase (DBH) were examined at 0, 6, 12, 24, and 48 h after dexamethasone (5 μ M ) treatment to PC12 cells. The levels of all enzyme mRNAs steadily increased for 24 h, although the increase of AADC mRNA content was slow. The increased mRNA levels of TH and AADC were maintained at 48 h, whereas the level of DBH mRNA was sharply decreased at 48 h. The maximally induced mRNA levels were ∼5.0-, 2.4-, and 7.0-fold higher than the control levels of TH, AADC, and DBH, respectively. The elevation of enzyme activities was detected later than the increase in levels of mRNAs. The maximal activities of TH, AADC, and DBH were reached between 48 and 72 h with 3.6-, 1.8-, and 8.0-fold increases, respectively. Low, but detectable, phenylethanolamine N -methyltransferase (PNMT) activity was observed in PC12 cells, and dexamethasone increased its activity 5.6-fold at 72 h. The PNMT mRNA was easily detected by northern blot analysis after exposure for 24 h to dexamethasone. The data suggest that, in PC12 cells, dexamethasone up-regulates all catecholamine biosynthetic enzyme genes in a parallel fashion.     

Regulation of vasopressin messenger RNA levels in the small cell lung carcinoma cell line GLC-8: interactions between glucocorticoids and second messengers.

The role of glucocorticoids and second messenger systems in the regulation of the vasopressin (VP) gene was studied in the human small cell lung carcinoma cell line GLC-8. Small cell lung carcinoma GLC-8 cells express VP mRNA and contain both glucocorticoid and mineralocorticoid receptors. Treatment with the synthetic glucocorticoid dexamethasone when added alone at 10(-8) M had no effect on the VP mRNA level and decreased the level by 30% at 10(-6) M. However, the effect of dexamethasone changed to positive when cells were simultaneously treated with cAMP-enhancing agents. VP mRNA levels, which were elevated by 1.5- to 2-fold by the cAMP-enhancing agents alone, increased a further 1.5- to 3-fold by dexamethasone. Thus, the combined effect of dexamethasone and cAMP stimulation was a 3- to 7.5-fold increase in VP mRNA levels. Long term treatment with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) reduced the VP mRNA level by 75%. The TPA-suppressed VP mRNA levels could be up-regulated about 6-fold by simultaneous treatment with 8-bromo-cAMP. Dexamethasone did not alter the TPA-suppressed VP mRNA levels. These results indicate that both cAMP and protein kinase-C pathways as well as glucocorticoid receptors are involved in the regulation of VP mRNA levels and that these factors interact. This leads to a negative or positive response of VP gene expression to glucocorticoids in a state-dependent manner. The interactions may be of significance in a physiological context and relate to the different regulation of VP-expressing systems in the brain.     

Rat neuropeptide Y precursor gene expression. mRNA structure, tissue distribution, and regulation by glucocorticoids, cyclic AMP, and phorbol ester

Rat brain neuropeptide Y precursor (prepro-NPY) cDNA clones were isolated and sequenced in order to study regulation of the prepro-NPY gene. Rat prepro-NPY (98 amino acid residues) contains a 36-residue NPY sequence, followed by a proteolysis/amidation site Gly-Lys-Arg, followed by a 30-residue COOH-terminal sequence. The strong evolutionary conservation of rat and human sequences of NPY (100%) and COOH-terminal peptide (93%) suggests that both peptides have important biological functions. In the rat central nervous system, prepro-NPY mRNA (800 bases) is most abundant in the striatum and cortex and moderately abundant in the hippocampus, hypothalamus, and spinal cord. The rat adrenal, spleen, heart, and lung have significant levels of prepro-NPY mRNA. Regulation of the prepro-NPY mRNA abundance was studied in several rodent neural cell lines. PC12 rat pheochromocytoma and N18TG-2 mouse neuroblastoma cells possess low basal levels of prepro-NPY mRNA, while NG108-15 hybrid cells possess high levels. Treatment of PC12 cells with a glucocorticoid such as dexamethasone or elevation of cAMP by forskolin increased the prepro-NPY mRNA level 2-3-fold or 3-10-fold, respectively. In N18TG-2 cells dexamethasone and forskolin synergistically increased prepro-NPY mRNA 7-fold. Treatment of PC12 cells with the protein kinase C activator phorbol 12-myristate 13-acetate alone elevated prepro-NPY mRNA marginally, but the phorbol ester plus forskolin elicited 20-70-fold increases, which were further enhanced to over 200-fold by dexamethasone and the calcium ionophore A23187. These results indicate that NPY gene expression can be positively regulated by synergistic actions of glucocorticoids, cAMP elevation, and protein kinase C activation.     

Hormonal induction of hepatic mitochondrial ornithine/citrulline transporter mRNA

The urea cycle, which involves enzymes located in both the mitochondrion and cytoplasm, requires transport of ornithine and citrulline across the mitochondrial membrane by the ornithine/citrulline antiporter ORNT1. Expression of the urea cycle enzymes can change dramatically in response to hormones, but it is not known whether ORNT1 expression also is hormonally regulated. This study therefore tested the hypothesis that ORNT1 mRNA levels in hepatocytes are induced by cAMP and glucocorticoid as are the urea cycle enzyme mRNAs. ORNT1 mRNA was rapidly induced by a cAMP analog and dexamethasone in cultured rat hepatocytes and there was a strong synergistic response to a combination of these agents. Ongoing protein synthesis was required for induction of ORNT1 mRNA by dexamethasone but not by cAMP, suggesting that the dexamethasone response required an accessory factor. Thus, hormonal regulation of ORNT1 mRNA in hepatocytes is coordinated with that of mRNAs encoding the urea cycle enzymes.     

Distribution and regulation of the prohormone convertases PC1 and PC2 in the rat pituitary.

PC1 and PC2 are enzymes involved in the activation of prohormones via the cleavage of pairs of basic amino acids. The expression levels of each of these enzymes were evaluated in the rat anterior and neurointermediate pituitary lobes by in situ hybridization and Northern gel analysis and after various pharmacological manipulations. All intermediate lobe melanotrophs expressed high levels of PC2 mRNA and lower levels of PC1 mRNA. PC1 mRNA was highly expressed throughout the anterior lobe; however, appreciable PC2 mRNA levels were also found. Based on colocalization studies, anterior lobe corticotrophs were found to express PC1 mRNA, but very little PC2 mRNA. Neurointermediate lobe levels of PC1, PC2, and POMC mRNA increased 2- to 6-fold in rats treated with haloperidol, while they decreased to 10-25% of their control values after bromocriptine treatment. These results indicate that in the intermediate lobe, dopamine is involved in the regulation of PC1 and PC2. In the anterior lobe, haloperidol had a strong effect on PC2 mRNA, increasing its levels by 8- to 12-fold compared to the control value, while PC1 mRNA was unaffected. Both PC1 and PC2 mRNA levels were increased 5- to 9-fold in animals made hypothyroid by treatment with 6-n-propyl-2-thiouracil. Adrenalectomy had no significant effect on anterior lobe PC1 mRNA levels. However, both PC1 and PC2 mRNA levels were responsive to dexamethasone treatment in the AtT-20 cell lines. Our results indicate that dopamine, thyroid hormones, and corticosteroids are involved in PC1 and/or PC2 gene expression. These data are also consistent with the role of PC1 and PC2 as prohormone-processing enzymes.     

Regulation of cytosolic aspartate aminotransferase mRNAs in the Fao rat hepatoma cell line by dexamethasone, insulin and cyclic AMP

Glucocorticoid hormones increase the activity of cytosolic aspartate aminotransferase (cAspAT) in the Fao rat hepatoma cell line. Maximal increase (6-10-fold) was observed 48 h following the addition of the glucocorticoid agonist dexamethasone at a concentration of 0.1 microM. The effect of dexamethasone was specific since it was not mimicked by sex steroids and was inhibited by the glucocorticoid antagonist RU 486. Insulin (0.1 microM) inhibited by more than 50% the induction of cAspAT by glucocorticoids. The cAMP analog, 8-bromoadenosine 3',5'-monophosphate (Br8cAMP, 0.5 mM), potentiated the effect of dexamethasone (2-3-fold) and partially relieved the inhibitory effect of insulin on the induction by dexamethasone. Both insulin and Br8-cAMP had no significant effect on basal activity. The mitochondrial isoenzyme was insensitive to the various hormonal treatments. Northern blot analysis revealed the presence of two major (2.1-kb and 1.8-kb) and one minor (4-kb) mRNA species hybridizing with a rat cAspAT probe. The regulation of these mRNAs by glucocorticoids, insulin and cAMP correlated with the variation of the cAspAT activity, suggesting that these hormones act at the pretranslational level. We compared the regulation of cAspAT mRNAs with those of tyrosine aminotransferase mRNA. Both were similarly increased by dexamethasone but the latter was also increased by cAMP even in the absence of the glucocorticoid agonist. In addition, the increase in tyrosine aminotransferase mRNA was inhibited by cycloheximide whereas the increase in cAspAT mRNAs was not. These results show that there are significant differences in the regulation of cAspAT and tyrosine aminotransferase by glucocorticoids and other hormones, although both enzymes probably contribute to the same metabolic pathway.     

Synergistic effect of prostaglandin F2alpha and cyclic AMP on glucose transport in 3T3-L1 adipocytes

The combined effect of prostaglandin F2alpha (PGF2alpha) and cAMP on glucose transport in 3T3-L1 adipocytes was examined. In cells pretreated with PGF2alpha and 8-bromo cAMP for 8 h, a synergy between these two agents on glucose uptake was found. Insulin-stimulated glucose transport, on the other hand, was only slightly affected. The synergistic effect of these two agents was suppressed in the presence of cycloheximide and actinomycin D. In concord, immunoblot and Northern blot analyses revealed that GLUT1 protein and mRNA levels were both increased in cells pretreated with both PGF2alpha and 8-bromo cAMP, greater than the additive effect of each agent alone. The synergistic action of PGF2alpha with 8-bromo cAMP to enhance glucose transport was inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor. In addition, in cells depleted of diacylglycerol-sensitive PKC by prolonged treatment with 4beta-phorbol 12beta-myristate 13alpha-acetate, a PKC activator, the synergistic effects of PGF2alpha and 8-bromo cAMP on glucose transport and GLUT1 mRNA accumulation were both abolished. Taken together, these results indicate that PGF2alpha may act with cAMP in a synergistic way to increase glucose transport, probably through enhanced GLUT1 expression by a PKC-dependent mechanism.     

Glucocorticoid regulation of phenylethanolamine N-methyltransferase in vivo.

In vivo, supraphysiological doses of glucocorticoids are required to restore adrenal medullary phenylethanolamine N-methyltransferase (PNMT, E.C. 2.1.1.28) activity after hypophysectomy. However, in vitro, phenylethanolamine N-methyltransferase gene expression appears normally glucocorticoid-responsive. To explore this paradox, rats were given dexamethasone or the type II-specific glucocorticoid RU28362 (1-1000 micrograms/day), and adrenal phenylethanolamine N-methyltransferase activity and mRNA levels were determined. At low doses (1-30 micrograms/day), neither steroid altered mRNA whereas at higher doses (100-1000 micrograms/day), mRNA rose 10- to 20-fold, with dexamethasone approximately 3 times as potent as RU28362. In contrast, enzyme activity fell with low doses of either steroid, consistent with suppression of ACTH and endogenous steroidogenesis. At higher doses of RU28362, enzyme activity remained low and unchanged despite increased mRNA expression, whereas higher doses of dexamethasone progressively restored the enzyme to normal. These findings suggest 1) that glucocorticoid regulation of phenylethanolamine N-methyltransferase activity occurs largely independent of gene expression; 2) that glucocorticoid effects on enzyme activity are primarily indirect, probably through cosubstrate regulation and/or enzyme stabilization; and 3) that these effects are not mediated via a classical (type II) glucocorticoid receptor mechanism, given the high doses of dexamethasone and corticosterone required and the inability of RU28362 to mimic the effects of these less selective steroids.     

Glucocorticoids and Nerve Growth Factor Differentially Modulate Cell Adhesion Molecule L1 Expression in PC12 Cells

Abstract: The differential expression of the cell adhesion molecule L1 by chromaffin cells has recently been suggested to be responsible for the segregation of chromaffin cells into homotypic catecholaminergic groups in the adrenal gland. The present study was undertaken to test the hypothesis that glucocorticoids, which increase in the adrenal gland during development, could be responsible for the repression of L1 in adrenergic chromaffin cells. PC12 cells were used as the experimental model, and relative L1 protein and mRNA levels were examined after treating the cells with glucocorticoids or NGF. Analysis of western blots indicated that glucocorticoids decreased the L1 protein levels by one-half, whereas NGF increased L1 protein levels ∼2.3-fold. In addition, the glucocorticoids inhibited both the NGF induction of the neurite outgrowth and the increase in L1 expression. Analysis of the mRNA levels by PCR and northern blots indicated that glucocorticoids reduced the L1 mRNA, whereas NGF increased the level of L1 mRNA. Maximal inhibition of L1 expression was observed at concentrations of 10⁻⁷ M dexamethasone, and the decrease occurred during the second day of treatment. The effects of dibutyryl cyclic AMP and phorbol ester on the glucocorticoid and NGF regulation of L1 protein were also examined. This is the first report indicating that L1 expression can be down-regulated by glucocorticoids. The results support the hypothesis that during development the repression of L1 in adrenergic chromaffin cells may be, in part, linked to the increase in glucocorticoid levels in the adrenal gland.     

Tissue-specific regulation of angiotensinogen mRNA accumulation by dexamethasone

The regulation of angiotensinogen gene expression in response to adrenalectomy and dexamethasone treatment was examined in multiple rat tissues. Angiotensinogen mRNA as quantitated by slot blot hybridization utilizing an angiotensinogen cRNA probe was most abundant in the liver with levels in the brain, kidney, and adrenal of 50, 25, and 10%, respectively. No angiotensinogen mRNA was detected in testes or heart. Although no change in the quantity of angiotensinogen mRNA was found following adrenalectomy and maintenance on 0.9% saline, dexamethasone treatment of both normal and adrenalectomized rats resulted in a time-dependent and tissue-specific accumulation of angiotensinogen mRNA. In normal animals, the hepatic response to treatment was a 4.5-fold increase in angiotensinogen mRNA by 8 h which remained 2.4-fold above basal levels by 24 h. Angiotensinogen mRNA levels in the brains of normal rats treated with dexamethasone increased only 60% by 6 h and returned to basal levels by 24 h. In contrast to the increases seen in brain and liver, angiotensinogen mRNA derived from kidney did not significantly change following dexamethasone treatment. In adrenalectomized animals, the hepatic response to dexamethasone was similar to normal animals with a 3.7-fold increase by 6 h. The accumulation in brain was greater in these animals compared to normals and increased 3-fold by 8 h. Finally, dexamethasone did not significantly increase levels in the kidney. These results clearly demonstrate glucocorticoid regulation of angiotensinogen mRNA levels in liver and brain. In contrast, the kidney, an organ known to contain glucocorticoid receptors, does not respond with increased angiotensinogen mRNA levels following glucocorticoid stimulation. These studies provide the first evidence for tissue-specific differences in the control of angiotensinogen mRNA.     

Effects of PAMP on mRNAs coding for catecholamine-synthesizing enzymes in PC12 cells.

Proadrenomedullin N-terminal 20 peptide (PAMP) is a novel hypotensive peptide found in the N-terminal portion of the precursor of adrenomedullin (AM). Although PAMP and AM originate from the same precursor and exert both a potent hypotensive action, they seem to control blood pressure through different mechanisms. To gain new insight into the anticholinergic actions of PAMP, we determined the effects of PAMP on the tyrosine hydroxylase (TH)- and dopamine beta-hydroxylase (DBH) mRNA expression in the rat pheochromocytoma cell line PC12 stimulated by nicotine. PAMP (> or =1 microM) significantly inhibited the nicotine-induced increases of TH- and DBH mRNA expression in a concentration-dependent manner. Also, PAMP at the concentrations (> or =1 microM) significantly inhibited nicotine-induced cyclic adenosine monophosphate (cAMP) production. These results indicate that the anticholinergic hypotensive actions of PAMP can be explained, at least in part, by its inhibition of the expression of mRNAs coding for catecholamine-synthesizing enzymes, and that the inhibitory effect is mediated by the cAMP/protein kinase A pathway.     

Regulation of Carboxypeptidase E by Membrane Depolarization in PC12 Pheochromocytoma Cells: Comparison with mRNAs Encoding Other Peptide- and Catecholamine-Biosynthetic Enzymes

PC12 cells, a rat pheochromocytoma cell line, have been found to express carboxypeptidase E (CPE) enzymatic activity and CPE, furin, and peptidylglycine alpha-amidating monooxygenase (PAM) mRNAs. PC12 cells secrete CPE activity in response to depolarization induced by 50 mM KCl. Short-term (1- to 3-h) treatments of PC12 cells with KCl stimulates the secretion of CPE but does not appear to stimulate the synthesis of new CPE protein, based on the measurement of CPE activity and incorporation of [35S]-Met into CPE. Also, CPE mRNA is not altered by 2-h treatments with KCl. In contrast, prolonged treatment (24-48 h) of PC12 cells with 50 mM KCl continues to stimulate the secretion of CPE activity, without altering the cellular level of CPE. Levels of CPE mRNA are significantly elevated after long-term treatment of the cells with KCl, with increases of 35% after 5 h and 55-75% after 24 to 72 h of treatment. The level of PAM mRNA is also elevated approximately 70% after 24 h of stimulation with KCl. In contrast, the mRNA levels of furin and dopamine beta-hydroxylase (DBH) do not change on treatment of PC12 cells with KCl. These findings indicate that long-term depolarization, which leads to a prolonged stimulation of PC12 cells to secrete CPE, also stimulates the synthesis of CPE and PAM but not furin or DBH.