Steady-state Receptor mRNA Levels Research Articles

Protein kinase C activation stabilizes LDL receptor mRNA via the JNK pathway in HepG2 cells

LDL is the most abundant cholesterol transport vehicle in plasma and a major prognostic indicator of atherosclerosis. Hepatic LDL receptors limit circulating LDL levels, since cholesterol internalized by the liver can be excreted. As such, mechanisms regulating LDL receptor expression in liver cells are appealing targets for cholesterol-lowering therapeutic strategies. Activation of HepG2 cells with phorbol esters enhances LDL receptor mRNA levels through transcriptional and posttranscriptional mechanisms. Here, we show that 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced stabilization of receptor mRNA requires the activity of protein kinase C and is accompanied by activation of the major mitogen activated protein kinase pathways. Inhibitor studies demonstrated that receptor mRNA stabilization is independent of the extracellular signal-regulated kinase or p38(MAPK), but requires activation of the c-Jun N-terminal kinase (JNK). An essential role for JNK in stabilizing receptor mRNA was further confirmed through small interfering RNA (siRNA) experiments and by activating JNK through two protein kinase C-independent mechanisms. Finally, prolonged JNK activation increased steady-state levels of receptor mRNA and protein, and significantly enhanced cellular LDL-binding activity. These data suggest that JNK may play an important role in posttranscriptional control of LDL receptor expression, thus constituting a novel mechanism to enhance plasma LDL clearance by liver cells.

Transcriptional regulation by phytoestrogens in neuronal cell lines

Widespread epidemiological data support the notion that high isoflavone intake is safe and may provide health benefits similar to estrogen. Evidence from rodents shows that certain phytoestrogens can act as estrogen receptor (ER) ligands in the brain. This study sought to determine the estrogenic profile of food-borne phytoestrogens in neuronal cell lines using physiologically attainable concentrations. At sub-micromolar concentrations genistein, daidzein, and zearalenone stimulated ERα and ERβ-dependent transcription in Neuro2A cells co-transfected with ERs and simple and complex estrogen-response-element (ERE) containing promoters, although compounds were more active in the presence of ERβ. In SN56, neuronblastoma cells expressing endogenous ERs, only genistein mimicked estrogen regulation of progesterone receptor steady state mRNA levels. Unlike pharmaceutical SERMs, phytoestrogens did not stimulate an AP-1-dependent promoter. Micromolar concentrations of phytoestrogens did not antagonize physiological estrogen concentrations or antagonist activation of an AP-1-dependent promoter. These results demonstrate that food-borne phytoestrogens, particularly those found in soy, act as ERE-, but not AP-1-dependent transcriptional activators in neurons in the absence of estrogen, and dietary levels of these compounds do not act as antagonists to physiological estrogen concentrations.

Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density.

P2X receptors function as ATP-gated cation channels. The P2X(7) receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X(7) receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X(7) receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 microM ATP was sufficient for activation of nonselective pores by P2X(7) receptors. Comparison of P2X(7) receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X(7) receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X(7) receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X(7) receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.

Regulation of cerebrovascular prostaglandin E 2 (PGE 2) and PGF 2α receptors and their functions during development

The density of PGF2 alpha (FP) and PGE2 (EP) receptors on the cerebral microvasculature of the newborn is less than on that of the adult animal. High levels of prostaglandins in the newborn brain could be responsible for down-regulation of FP and EP receptors and their functions in the cerebral microvasculature. Cerebrovascular FP and EP receptor density, receptor-coupled second messenger production and cerebral vasoconstrictor responses to PGF2 alpha and PGE2 were studied in newborn pigs (1 to 2 days old) treated intravenously with ibuprofen (40 mg/kg every 6 hours for 48 hours) or saline, and compared with adults. Ibuprofen treatment in the newborn increased brain microvascular FP and EP receptor densities to levels found in that of adult pigs. Likewise brain microvessel inositol 1,4,5-triphosphate production in response to PGF2 alpha, fenprostalene (PGF2 alpha analog), PGE2, 17-phenyl trinor PGE2 (EP1 receptor subtype agonist) and M&B 28,767 (EP3 agonist) was greater in ibuprofen-treated newborn pigs; the latter was associated with increased vasoconstriction to these agents to levels comparable with those of adults. Steady-state levels of FP receptor mRNA in cerebral microvas-culature did not differ between saline-treated newborn, and ibuprofen-treated newborn and adult pigs. It is concluded that the low FP and EP receptor densities and receptor-coupled functions on newborn brain microvessels seem to be secondary to high levels of brain prostaglandins; the changes in receptor levels do not seem to be related to steady-state levels of receptor mRNA in brain microvessels.

Modulation of macrophage scavenger receptor transport by protein phosphorylation

The identification of three highly conserved phosphorylation sites in the cytoplasmic domain of each of the monomeric subunits of the macrophage scavenger receptor suggests that protein phosphorylation may regulate this receptor pathway. To investigate this, mouse peritoneal macrophages were pretreated with either the protein phosphatase inhibitor okadaic acid or the protein kinase inhibitor staurosporine to modulate cellular protein phosphorylation and their effects on the metabolism of acetyl-LDL were measured. Both okadaic acid and staurosporine inhibited the degradation of acetyl-low density lipoprotein (LDL) without affecting cellular lactic dehydrogenase (LDH) levels. The inhibition by okadaic acid was due to a 70% decrease in acetyl-LDL binding whereas post-receptor processing was minimally affected. Calyculin A, another serine/threonine phosphatase inhibitor, also reduced acetyl-LDL binding, whereas lithium chloride, an inositol phosphatase inhibitor, did not. Okadaic acid did not decrease steady state receptor mRNA levels nor decrease the number of total cellular receptors, consistent with a posttranslational mechanism of action. Interestingly, protease sensitivity studies showed that the receptors were still located on the cell surface. These studies suggest that okadaic acid inhibits acetyl-LDL binding by causing the redistribution of surface receptors into a sequestered compartment or inactivating the receptors. In contrast, staurosporine produced a paradoxical increase in receptor expression (30%) but slowed post-receptor processing (2.3-fold decrease). The latter was due to an inhibition of ligand internalization (2.9-fold decrease) via a protein kinase C-independent mechanism. Macrophage pinocytosis was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also slowed by staurosporine (38% decrease); however, this does not appear to account for the inhibition of scavenger receptor internalization. Direct receptor phosphorylation was also investigated and it was established that the receptor can be phosphorylated; however, changes in receptor function did not correlate with changes in the degree of receptor phosphorylation. Together these studies demonstrate that changes in cellular protein phosphorylation affect the expression, surface transport, and internalization of the macrophage scavenger receptor and suggest that the regulated phosphorylation/dephosphorylation of cellular proteins may be an important biochemical mechanism that controls normal processing of ligands by this receptor pathway.

Altered Cholesterol Trafficking in Herpesvirus-infected Arterial Cells: EVIDENCE FOR VIRAL PROTEIN KINASE-MEDIATED CHOLESTEROL ACCUMULATION

Herpesvirus infection of arterial smooth muscle cells has been shown to cause cholesteryl ester (CE) accumulation. However, the effects of human herpes simplex virus type 1 (HSV-1) infection on cholesterol binding and internalization, intracellular metabolism, and efflux have not been evaluated. In addition, the effects of viral infection on signal transduction pathways that impact upon cholesterol metabolism have not been studied. We show in studies reported herein that HSV-1 infection of arterial smooth muscle cells enhances low density lipoprotein (LDL) binding and uptake which parallels an increase in LDL receptor steady state mRNA levels and transcription of the LDL receptor gene. HSV-2 also increases CE synthesis and 3-hydroxy- 3-methylglutaryl-CoA reductase activity but concomitantly reduces CE hydrolysis and cholesterol efflux. Interestingly, this viral infection was associated with a time-dependent decrease in protein kinase A activity and an increase in viral-induced protein kinase (VPK) activity commensurate with the accumulation of esterified cholesterol. The relationship between increased VPK activity and alterations in CE accumulation in virally infected cells was explored using an HSV-1 VPK- mutant in which the portion of the HSV-1 genome encoding VPK had been deleted. Cholesteryl ester accumulation was significantly increased (> 50-fold) in HSV-1-infected cells compared to uninfected cells. However, the HSV-1 VPK- mutant had no significant effect on CE accumulation. The relationship between VPK activity and these alterations in cholesterol metabolism was further supported by the observation that staurosporine and calphostin C (protein kinase inhibitors) reduced protein kinase activity in HSV-1-infected cells. These results suggest several potential mechanisms by which alterations in kinase activities in response to HSV-1 infection of vascular cells may alter cholesterol trafficking processes that eventually lead to CE accumulation.

1,25-Dihydroxyvitamin D3 increases type 1 interleukin-1 receptor expression in a murine T cell line.

The biologically active metabolite of vitamin D3, 1,25 (OH)2 D3, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and 125I-interleukin 1 alpha, we show that 1,25 (OH)2 D3 (5,50 nM) enhanced 125I-interleukin 1 alpha binding up to almost 2-fold over control. This 1,25 (OH)2 D3 effect occurred in a dose-dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of 125I-interleukin 1 alpha binding data demonstrated that 1,25 (OH)2 D3 enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D3, 25 (OH) D3, also augmented 125I-interleukin 1 alpha binding but at steroid levels 2-3 log orders greater than 1,25 (OH)2 D3. This observation, combined with the presence of high-affinity 3H-1,25 (OH)2 D3 receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti-type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)2 D3-enhanced 125I-interleukin 1 alpha binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)2 D3 enhanced interleukin 1 receptor expression. Northern blots hybridized with a 32P-type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)2 D3 enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)2 D3 pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1 alpha). These findings further support 1,25 (OH)2 D3's role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.

Regulation of thyrotropin receptor gene expression in rat FRTL-5 thyroid cells.

TSH receptor mRNA levels in FRTL-5 thyroid cells are autoregulated at a transcriptional level by the same hormones required for the growth and function of the cells: TSH, insulin, and insulin-like growth factor-I (IGF-I). Thus, the ability of TSH, via its cAMP signal, to down-regulate steady state receptor mRNA levels is preceded by the action of TSH to decrease pre-mRNA levels in nuclear run-on assays to the same quantitative level as evident in Northern analyses. In contrast, the receptor mRNA half-life is shown not to change when down-regulation is reversed by withdrawing TSH in the presence or absence of actinomycin-D. Evidence is additionally provided that TSH receptor mRNA levels are increased by insulin, IGF-I, or calf serum in both Northern and run-on assays. This action cannot be duplicated by hydrocortisone and is evident at more than 20-fold lower concentrations of IGF-I than insulin. Moreover, insulin, IGF-I, and/or calf serum are required for the autoregulatory negative transcriptional regulation of the TSH receptor by TSH/cAMP, as is the case for thyroglobulin. This occurs despite the opposite actions of TSH/cAMP on the two genes, positive in the case of thyroglobulin and negative with TSH receptor. The positive and negative regulatory actions, respectively, of insulin/IGF-I and TSH on receptor gene expression are associated with coincident increases or decreases in cell surface receptors measured by [125I]TSH binding. The autoregulation additionally involves the interplay of a second cAMP-modulated regulatory factor, one which up-regulates TSH receptor mRNA levels rather than causing down-regulation. Thus, cycloheximide inhibits the transcriptional action of both TSH/cAMP and insulin/IGF-I/serum within 4 h, i.e. a rapidly synthesized protein is an intermediate in both cases. The presence of cycloheximide for as little as 1 h, however, uncovers the ability of TSH/cAMP to increase TSH receptor mRNA levels. This activity is the result of the action of a stable cAMP-induced activator which can be detected physiologically, i.e. in the absence of cycloheximide. For example, low levels of a cAMP analog (0.2 mM), as opposed to high levels (greater than 1 mM), can increase TSH receptor RNA levels. Low levels also accelerate the insulin/IGF-I-dependent return of receptor mRNA to normal levels after TSH withdrawal.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of thyrotropin receptor gene expression in rat FRTL-5 thyroid cells

TSH receptor mRNA levels in FRTL-5 thyroid cells are autoregulated at a transcriptional level by the same hormones required for the growth and function of the cells: TSH, insulin, and insulin-like growth factor-I (IGF-I). Thus, the ability of TSH, via its cAMP signal, to down-regulate steady state receptor mRNA levels is preceded by the action of TSH to decrease pre-mRNA levels in nuclear run-on assays to the same quantitative level as evident in Northern analyses. In contrast, the receptor mRNA half-life is shown not to change when down-regulation is reversed by withdrawing TSH in the presence or absence of actinomycin-D. Evidence is additionally provided that TSH receptor mRNA levels are increased by insulin, IGF-I, or calf serum in both Northern and run-on assays. This action cannot be duplicated by hydrocortisone and is evident at more than 20-fold lower concentrations of IGF-I than insulin. Moreover, insulin, IGF-I, and/or calf serum are required for the autoregulatory negative transcriptional regulation of the TSH receptor by TSH/cAMP, as is the case for thyroglobulin. This occurs despite the opposite actions of TSH/cAMP on the two genes, positive in the case of thyroglobulin and negative with TSH receptor. The positive and negative regulatory actions, respectively, of insulin/IGF-I and TSH on receptor gene expression are associated with coincident increases or decreases in cell surface receptors measured by [125I]TSH binding. The autoregulation additionally involves the interplay of a second cAMP-modulated regulatory factor, one which up-regulates TSH receptor mRNA levels rather than causing down-regulation. Thus, cycloheximide inhibits the transcriptional action of both TSH/cAMP and insulin/IGF-I/serum within 4 h, i.e. a rapidly synthesized protein is an intermediate in both cases. The presence of cycloheximide for as little as 1 h, however, uncovers the ability of TSH/cAMP to increase TSH receptor mRNA levels. This activity is the result of the action of a stable cAMP-induced activator which can be detected physiologically, i.e. in the absence of cycloheximide. For example, low levels of a cAMP analog (0.2 mM), as opposed to high levels (greater than 1 mM), can increase TSH receptor RNA levels. Low levels also accelerate the insulin/IGF-I-dependent return of receptor mRNA to normal levels after TSH withdrawal.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of β2-adrenergic receptors in keratinocytes: glucocorticoids increase steady-state levels of receptor mRNA in foetal rat keratinizing epidermal cells (FRSK cells)

Glucocorticoids increase the beta-adrenergic adenylate cyclase response of epidermal keratinocytes. Using FRSK cells, a cultured cell line of foetal rat keratinocytes, the regulatory mechanism of the beta-adrenergic augmentation effect was investigated. Treatment with dexamethasone (1 x 10(-6) M increase by 1.5-fold the beta-adrenergic adenylate cyclase response of FRSK cells. The effect was observed at 6 h incubation and remained for at least 48 h. The prostaglandin E-adenylate cyclase response was also increased 1.5-fold by glucocorticoid treatment. Neither the adenosine-adenylate cyclase response nor cholera toxin- or forskolin-induced cyclic AMP accumulations were altered. Northern blot hybridization showed that levels of the beta 2-adrenergic receptor mRNA increased within 3 h, while actin-, Gs-alpha, Gi-2 alpha, Gi-3 alpha mRNA levels were unchanged. Testosterone, 17 beta-oestradiol, and progesterone had no effect on either the beta 2-adrenergic adenylate cyclase response or the expression of beta 2-adrenergic receptor mRNA. The increase in the numbers of the beta-adrenergic receptors was visualized by immunofluorescence with an antibody specific for the beta 2-adrenergic receptor. Our results indicate that glucocorticoids regulate the beta 2-adrenergic adenylate cyclase response of FRSK cells through the enhanced expression of the receptor.

Hormonal regulation of LH receptor mRNA and expression in the rat ovary

Agonist-induced changes in expression and mRNA levels of luteinizing hormone (LH) receptors were compared during stimulation of ovarian follicular maturation and luteinization by gonadotropic hormones. Three major species of LH receptor mRNA, 5.8, 2.6 and 2.3 kb, were present throughout differentiation and changed similarly, the 5.8 kb species being consistently more abundant than the smaller forms. The increased expression of plasma-membrane LH receptors in preovulatory follicles and luteinized ovaries and their homologous down-regulation during follicular and luteal desensitization were closely correlated with the steady-state receptor mRNA levels. The reappearance of LH receptors following desensitization during the luteal stage was preceded by an increase in mRNA levels. These studies have demonstrated that the expression of LH receptors during follicular maturation, ovulation and desensitization is related to the prevailing levels of receptor mRNA in the ovary.

Role of an estrogen receptor-dependent mechanism in the regulation of estrogen receptor mRNA in MCF-7 cells.

We have previously demonstrated that regulation of estrogen receptor (ER) expression in MCF-7 breast cancer cells is a complex process involving transcriptional and posttranscriptional regulation by estradiol. Treatment of MCF-7 cells with estradiol results in the down-regulation of receptor expression; posttranscriptional suppression of receptor mRNA appears to be the predominant mechanism. To determine whether posttranscriptional regulation of ER gene expression is mediated by an ER-dependent mechanism independent of protein synthesis, we have used the competitive estrogen antagonist, 4-hydroxytamoxifen, and the inhibitor of protein synthesis, cycloheximide, to study regulation of ER mRNA by estradiol. 4-Hydroxytamoxifen had no effect on the steady-state level of receptor mRNA and effectively blocked the suppression of ER mRNA by estradiol. The metabolic inhibitor, cycloheximide, was unable to prevent the estrogen induced decrease in ER mRNA. These data provide evidence that the posttranscriptional suppression of ER expression through estradiol is mediated through the ER independent of protein synthesis. A study of the effects of estradiol on the steady-state levels of nuclear and cytoplasmic receptor mRNA suggest that posttranscriptional suppression is a nuclear event.

Regulation of beta-adrenergic receptors by "permissive" hormones: glucocorticoids increase steady-state levels of receptor mRNA.

Incubation of DDT1 MF-2 hamster vas deferens cells with glucocorticoids results in a marked increase in beta-adrenergic receptor (beta AR) number. The increase in receptor number was visualized by indirect immunofluorescence with antiserum specific for the beta AR and was verified by radioligand binding. The steady-state levels of beta AR mRNA were quantified in untreated (control) and glucocorticoid-treated cells by DNA-excess solution hybridization using a single-stranded probe corresponding to nucleotides +12 to 182 of the hamster beta 2AR cDNA coding region. The steady-state level increased from 0.37 pg of beta AR mRNA per microgram of total cellular RNA in untreated cells to 1.05 pg of beta AR mRNA per microgram of RNA in cells treated with dexamethasone (500 nM) for 2-4 hr. After this sharp transient peak, the steady-state level of receptor mRNA declined by 6 hr to a level approximately twice that of the untreated cells. Half-maximal effects were achieved at 20-40 nM dexamethasone. Testosterone (500 nM) and 17 beta-estradiol (500 nM), in contrast, did not alter the steady-state levels of beta AR mRNA. Actinomycin D, a potent inhibitor of transcription, abolished the dexamethasone-induced increase in beta AR mRNA, suggesting that the permissive hormone effect was exerted on gene transcription. The half-life of the receptor mRNA measured in the presence of actinomycin D was found to be 12 hr in both the untreated and the dexamethasone-treated cells. These studies provide a molecular explanation for the well-known regulation of GTP-binding protein (G-protein)-linked cell-surface receptors by permissive hormones.

Evidence that glucocorticoid response elements in the 5′-noncoding region of the hamster β2-adrenergic receptor gene are obligate for glucocoticoid regulation of receptor mRNA levels

Glucocorticoids and thyroid hormones regulate the expression of G-protein-linked receptors, typified by the β-adrenergic receptor. The influence of glucocorticoids on the steady-state levels of receptor and receptor mRNA were examined in Chinese hamster ovary (CHO) cells in culture. Incubation of wild-type CHO cells with dexamethasone, a potent glucocorticoid, elevated both receptor number (as measured by radioligand binding) and receptor mRNA levels (as measured by DNA-excess solution hybridization). Both responses were time- and dose-dependent. Stable transfectant CHO clones harboring the hamster β 2- adrenergic receptor cDNA under the control of the SV40 early promoter were investigated for their ability to respond to glucocorticoid stimulation. The cDNA employed in these studies displays glucocorticoid-response elements (GREs) in the coding and 3′- noncoding regions, but lacks GREs identified in the 5′-noncoding regions of the gene. Transfectant clones expressing 200 (X13), 600 (32E), or 1580 (32A) fmol of receptor/10 6 cells, as compared to wild-type CHO clones expressing 15 fmol/10 6 cells, displayed elevated steady-state levels of receptor mRNA. Incubating the transfectant CHO clones with glucocorticoids, however, failed to enhance the level of receptor or receptor mRNA derived from the expression vector employed in the transfection. These data demonstrate for wild-type CHO cells that glucocorticoids stimulate an increase in receptor mRNA and receptor expression, but that in CHO clones stably transfected with a cDNA lacking specifically the GREs in the 5′-noncoding region of the gene, the glucocorticoid-stimulated increase in β-adrenergic receptor mRNA is not evident.