Protein kinase C-mediated down-regulation of beta1-adrenergic receptor gene expression in rat C6 glioma cells.

In the current study, we investigated the mechanism by which protein kinase C (PKC) regulates the expression of beta1-adrenergic receptor (beta1AR) mRNA in rat C6 glioma cells. Exposure of the cells to 4beta-phorbol-12-myristate-13-acetate (PMA), an activator PKC, resulted in a down-regulation of both beta1AR binding sites and mRNA levels in a time- and concentration-dependent manner. This effect was not observed with phorbol esters that do not activate PKC and was blocked by bisindolylmaleimide, a specific PKC inhibitor. Activation of PKC did not reduce the half-life of beta1AR mRNA but significantly decreased the activity of the beta1AR promoter, as determined by reporter analysis. A putative response element, with partial homology to a consensus cAMP response element, was identified by mutation analysis of the promoter at positions -343 to -336, relative to the translational start site. Mutation of this putative regulatory element, referred to as a beta1AR-PKC response element, completely blocked the PKC-mediated down-regulation of beta1AR promoter activity. Gel mobility shift analysis detected two specific bands when C6 cell extracts were incubated with a labeled DNA probe containing the beta1AR-PKC response element sequence. Formation of one of these bands was inhibited by an oligonucleotide probe containing a consensus CRE and disrupted by an antibody for cAMP response element binding protein. Based on these studies, we propose that the PKC-induced down-regulation of beta1AR gene transcription in C6 cells is mediated in part by a cAMP response element binding protein-dependent mechanism acting on a novel response element.

Endogenous beta 3- but not beta 1-adrenergic receptors are resistant to agonist-mediated regulation in human SK-N-MC neurotumor cells.

Although there is considerable interest in the regulation of the different beta-adrenergic receptor (AR) subtypes, most previous studies have utilized stably transfected cells expressing recombinant receptors under the control of viral promoters. Human SK-N-MC neurotumor cells appear to be novel, since they express both endogenous beta 1AR and beta 3AR based on radioligand binding and on functional response. Saturation binding of either the hydrophilic ligand (-)-[3H]CGP-12177 or the more hydrophobic (-)-[125I]iodocyanopindolol indicated the presence of two populations of binding sites with high and low affinities. With either ligand, the beta 1AR antagonist CGP-20712A preferentially inhibited binding to the high-affinity sites. This is consistent with the latter representing beta 1AR whereas the low-affinity sites represent beta 3AR. Both subtypes appeared to be functional on the basis of isoproterenol stimulation of cyclic adenosine monophosphate (cAMP) in intact cells and adenylyl cyclase activity in cell membranes in the absence and presence of CGP-20712A. SK-N-MC-IXC cells, derived by twice subcloning the parental cells, also expressed both beta AR subtypes, indicating that they co-exist in the same cell. SK-N-MC cells exposed to isoproterenol exhibited a rapid sequestration and a slower downregulation of beta 1AR. The latter subtype also underwent desensitization, as indicated by a rightward shift to less sensitivity in the EC50 for isoproterenol stimulation of adenylyl cyclase activity. In contrast, the beta 3AR subtype was resistant to agonist-mediated sequestration, downregulation, and desensitization. Thus, when endogenously expressed in the same cell line, human beta 1AR and beta 3AR display differences in their ability to be regulated by agonist.

Independent and coordinate regulation of beta 1- and beta 2-adrenergic receptors in rat C6 glioma cells.

Rat C6 glioma cells have both beta 1- and beta 2-adrenergic receptors in approximately 7:3 ratio. When the cells were exposed to the beta-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [3H] CGP-12177 to intact cells. Using the beta 1-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both beta 1 and beta 2 receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of approximately 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [125I]iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous beta 1-and beta 2-adrenergic receptors in the same cell line.

Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action.

Cholera toxin (CT) consists of a pentameric B subunit which binds to ganglioside GM1 on the cell surface and an A subunit which activates adenylylcyclase. The latter process involves the reduction of A to the A1 peptide which ADP-ribosylates the stimulatory G protein, Gs of adenylylcyclase. There is a distinct lag phase between toxin binding and activation of adenylylcyclase. Little is known about the events during this lag including where A1 is generated and how it gains access to Gs on the cytoplasmic side of the plasma membrane. We explored the effects of several inhibitors of intracellular trafficking on the response of human SK-N-MC neurotumor and Caco-2 intestinal tumor cells to CT. Whereas chloroquine or monensin had little or no effect on CT stimulation of cyclic AMP accumulation, brefeldin A (BFA) totally inhibited the response to CT in a time- and dose-dependent and reversible manner. BFA was effective when added at the same time as CT and had an IC50 of 30 ng/ml. BFA did not alter cell surface GM1 as cells treated with BFA for 30 min bound as much 125I-CT as control cells. Furthermore, BFA inhibited CT stimulation of GM1-treated rat glioma C6 cells. BFA treatment did not affect beta-adrenergic agonist stimulation of cyclic AMP. In addition, adenylylcyclase was activated by A1 peptide and NAD+ to the same extent in membranes from control and BFA-treated cells, or when BFA was added directly to the assay. Whereas control cells generated small amounts of A1 from bound CT with time, no A1 was detected in BFA-treated cells. BFA treatment did not prevent the internalization of CT but did inhibit its degradation. BFA is known to disrupt the organization of the Golgi complex, resulting in inhibition of protein transport from the endoplasmic reticulum and redistribution of Golgi enzymes to the endoplasmic reticulum. BFA also prevents the formation of non-clathrin-coated vesicles from Golgi membranes and thus vesicular transport between Golgi cisternae. We confirmed that BFA caused the morphological disruption of the Golgi apparatus in Caco-2 cells. The data support a role for a functional Golgi apparatus with its associated vesicular routing in CT action.

Brefeldin A inhibits protein synthesis in cultured cells.

The fungal metabolite brefeldin A (BFA) is known to disrupt the Golgi apparatus resulting in redistribution of Golgi proteins to the endoplasmic reticulum and inhibition of protein secretion. BFA was found to inhibit protein synthesis in rat glioma C6 cells by up to 70% between 0.1 and 1 microgram/ml. Inhibition was both time-dependent and reversible. BFA inhibited protein synthesis to varying degrees in a number of other cell lines but not in BFA-resistant marsupial kidney cells. The same concentrations of BFA which inhibited protein synthesis, also blocked the inhibitory effects of Pseudomonas exotoxin and ricin on BFA-sensitive cells. BFA, however, was unable to block the inhibition of protein synthesis by the toxins in the resistant marsupial kidney cells.