©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Long Term Phorbol Ester Treatment Down-regulates the -Adrenergic Receptor in 3T3-F442A Adipocytes (*)

Bruno Fève (1)(§), France Piétri-Rouxel (2), Khadija El Hadri (1), Marie-Franoise Drumare (2), A. Donny Strosberg (2)

From the (1) INSERM Unité82, Hôpital Henri Mondor, 94010 Créteil, France and the (2) CNRS UPR 0415 and Université Paris VII, Institut Cochin de Génétique Moléculaire, 22 rue Méchain, 75014 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The role of protein kinase C (PKC) in the regulation of the -adrenergic receptor (-AR) gene was examined in murine 3T3-F442A adipocytes, which express this receptor subtype at a high level. We also investigated the involvement of this kinase in the modulation of -AR gene expression by insulin. Long term exposure of 3T3-F442A adipocytes to phorbol 12-myristate 13-acetate (PMA) decreased -AR mRNA content in a time- and concentration-dependent manner, with maximal changes observed at 6 h (6.5-fold decrease) and at 100 nM PMA. This inhibition was selective for -AR transcripts, since - and -AR mRNA content remained unchanged. Also, (-)-[I]cyanopindolol saturation and competition binding experiments on adipocyte membranes indicated that PMA induced an 2-fold decrease in -AR expression, while that of the two other subtypes was not affected. This correlated with a lower efficacy of -AR agonists to stimulate adenylyl cyclase. Conversely, long term exposure to PMA did not alter adenylyl cyclase activity in response to guanosine 5`- O-(3-thiotriphosphate) or forskolin. The inactive phorbol ester 4-phorbol 12,13-didecanoate did not repress -AR mRNA levels. Inhibition of -AR mRNA by PMA was suppressed by the PKC-selective inhibitor bisindolylmaleimide, and was not observed in PKC-depleted cells, indicating that PKC was involved in this response. mRNA turnover experiments showed that the half-life of -AR transcripts was not affected by long term PMA exposure. When 3T3-F442A adipocytes were pretreated with PMA for 24 h to down-regulate PKC, or with bisindolylmaleimide, the insulin-induced inhibition of -AR mRNA levels was reduced by 44-67%. These findings demonstrate that sustained PKC activation exerts a specific control of -AR gene expression and is involved, at least in part, in the modulation by insulin of this adrenergic receptor subtype.


INTRODUCTION

Catecholamines play a central role in the regulation of lipid metabolism in adipose cells. Through activation of -ARs,() they modulate cAMP-dependent processes, such as lipolysis, and the genetic control of the lipogenic or thermogenic pathways. The -AR, whose gene and cDNA have been characterized in human (1) and rodents (2, 3, 4) , appears to be the main functional subtype in rodent adipocytes (5) . The murine 3T3-F442A cell line, which undergoes adipose conversion in vitro, presents a coordinated expression of the three -AR subtypes. Overall, the -AR subtype, which is absent in 3T3 preadipocytes, represents more than 90% of total -ARs in mature adipocytes (6) . The 3T3-F442A cell line thus constitutes an appropriate in vitro model to investigate the physiology of the -AR in rodent adipocytes.

Catecholamine responsiveness of adipocytes can be controlled through several mechanisms, including the modulation of -AR number on the cell surface. Through heterologous regulation, a number of other hormone systems can influence, at the genetic level, -AR density and adrenergic sensitivity (7) . The complexity of these regulatory mechanisms is emphasized by the -AR subtype-selective modulation by several effectors. Thus glucocorticoids exert at a transcriptional level a differential regulation of the three -ARs in 3T3 adipocytes: while they enhance -AR expression, they strongly repress that of - and -ARs (8, 9, 10, 11) . Butyric acid, a short chain fatty acid, up-regulates - and -AR gene expression, but potently decreases that of the -AR (12) . These heterologous regulations are likely to contribute to adipocyte adaptation to environmental conditions.

Recently, we demonstrated that insulin specifically inhibits -AR gene expression, while that of - and -ARs remains unchanged (13) . This regulation is exerted at a transcriptional level and is followed by a decrease in -AR sites and coupling to the adenylyl cyclase system. It represents a mechanism for the long term regulation by insulin of cAMP-dependent biological processes in adipocytes and may be involved in the pathogenesis of nutritional disorders associated with hyperinsulinemia. So far, the intracellular signaling pathways leading to the heterologous regulation of the -AR by insulin are still unknown. One possibility is that insulin activates one or several isotypes of PKC (14) . PKC activation by insulin is involved in the stimulation of glucose transport (15) , lipogenesis (16) , protein synthesis (17) , c-Fos expression (18, 19) , and mitogen-activated protein kinase and protein phosphatase-1 activation (20) . However, due to the persistance of a normal insulin effect after PKC down-regulation, several authors have questioned the involvement of PKC in insulin action (for review, see Ref. 21). The role of PKC in mediating insulin effects may depend on a specific cellular environment or phenotype, on the diversity of PKC isotypes in a given cell (22) , or on the nature of the insulin-regulated process (23) .

The aim of the present study was to determine whether prolonged activation of PKC by phorbol esters modulates -AR subtype expression in 3T3-F442A adipocytes. In addition, we addressed the issue of the contribution of PKC to the specific transcriptional regulation of the -AR mRNA by insulin.


MATERIALS AND METHODS

Cell Culture

3T3-F442A cells were grown and differentiated as described (6) . At day 7 after confluence, more than 90% of the cells had the morphology of mature adipocytes. After two washes, cells were kept for 24 h in a defined medium consisting of Dulbecco's modified Eagle's medium/Ham's F-12 medium (2:1, v/v) and 0.1% bovine serum albumin. From there on the cells were maintained either in the absence or presence of PMA and/or the indicated drugs.

Pharmacological Experiments

Cell extracts were prepared as described previously (6) . Protein content was assayed (24) using bovine serum albumin as a standard.

Adenylyl cyclase activity (EC 4.6.1.1) was measured as described (6) . Unless when tested in the presence of GTPS or forskolin, assays were performed in the presence of 100 µM GTP.

Binding assays were carried out as previously mentioned (12) . Saturation experiments were performed with (-)-[I]CYP concentrations ranging from 5 to 4000 pM. Competition experiments were carried out at 250 pM (-)-[I]CYP. Nonspecific binding was determined in the presence of 10 µM (±)-bupranolol and was usually 15 ± 4% of total binding at 250 pM (-)-[I]CYP.

Data from saturation and competition binding experiments were analyzed with the EBDA and LIGAND programs (Biosoft-Elsevier, Cambridge, United Kingdom).

Chemicals

(-)-[I]CYP was obtained from Amersham and [-P]ATP from ICN Radiochemicals. CGP12177 and CGP20712A were generous gifts from Ciba-Geigy (Basel, Switzerland). ICI118551 and ICI201651 were provided by Imperial Chemical Industries (Macclesfield, United Kingdom), BRL37344 by Smith Kline Beecham Pharmaceuticals (Epsom, United Kingdom), and cyanopindolol by Sandoz (Basel, Switzerland). (±)-Bupranolol was donated by Schwarz Pharma (Monheim, Germany). ISO, forskolin, GTPS, PMA, dioctanoyl- sn-glycerol, 4-PDD, and insulin were Sigma products. GTP, bisindolylmaleimide, and actinomycin D were from Boehringer Mannheim.

RNA Analysis

Total RNA was extracted from 3T3-F442A adipocytes by the method of Cathala et al.(25) . RNA samples were electrophoresed through a 1.5%, 2.2 M formaldehyde gel, and transferred to nylon Nitran-plus membranes (Schleicher and Schuell). After RNA fixation, RNA content in each lane was assessed by methylene blue staining of ribosomal RNAs. Prehybridization was carried out at 60 °C for 30 min in the presence of 0.5 M sodium phosphate (pH 6.8), 7% SDS, 1% bovine serum albumin, and 1 mM EDTA (26) . Hybridization was performed in the same buffer in the presence of the heat-denaturated probe (2-3 10 cpm/ml). Membranes were washed twice for 30 min at 60 °C in 2 SSC (1 SSC: 150 mM NaCl, 15 mM sodium citrate), 0.1% SDS, then once in 0.1 SSC, 0.1% SDS for 15 min at 60 °C. Probes were labeled by random priming with [-P]dCTP (ICN Radiochemicals). The -AR probe is a 305-base pair amplification product of the cloned murine -AR gene (2) between a sense 5`-GCATGCTCCGTGGCCTCACGAGAA-3` and an antisense primer 5`-CCCAACGGCCAGTGGCCAGTCAGCG-3`.

For RT-PCR analysis of -AR expression, total RNA was digested for 15 min at 37 °C with 0.1 unit of RNase-free DNase I (RQ1 DNase, Promega) per µg of nucleic acid in 40 mM Tris-HCl (pH 7.9), 10 mM NaCl, 6 mM MgCl, 10 mM CaCl in the presence of 1 unit/µl ribonuclease inhibitor (RNAguard, Pharmacia). After phenol/chloroform extraction and ethanol precipitation, RNA (0.25-2 µg) was reverse-transcribed with MMLV RT (200 units/µg) (Life Technologies, Inc.) in the presence of 10 µM random hexanucleotides (Pharmacia), 1 unit/µg ribonuclease inhibitor, 400 µM of each dNTP in a final volume of 20 µl consisting of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl, and 10 mM dithiothreitol. After a 1-h incubation at 42 °C, MMLV RT was heat-inactivated. To ensure that subsequent amplification did not derive from contaminant genomic DNA, a control without MMLV RT was included for each RNA sample. cDNAs were denatured for 5 min at 94 °C and submitted to 25 (-AR and -actin) or 33 (- and -AR) cycles of amplification (1 cycle: 94 °C, 1 min; 60 °C, 2 min; 72 °C, 2 min) followed by a final extension of 7 min at 72 °C in a DNA thermal cycler 480 (Perkin Elmer). PCR was performed in a 25-µl reaction containing 1 unit of Taq polymerase (Bioprobe, France), 125 µM each dNTP, 5% formamide, 125 nM both sense and antisense oligonucleotides. The buffer consisted of 20 mM Tris-HCl (pH 8.55), 16 mM (NH)SO, 2.5 mM MgCl, and 150 µg/ml bovine serum albumin. Sequences of the sense and antisense oligonucleotides were: 5`-GGATCCAAGCTTTCGTGTGCACCGTGTGGGCC-3` and 5`-GGATCCAAGCTTAGGA-AACGGCGCTCGCAGCTGTCG-3` for the -AR; 5`-GCCTGCTGACCAAGAATAAG-GCC-3` and 5`-CCCATCCTGCTCCACCT-3` for the -AR; 5`-ATGGCTCCGTGGCCT-CAC-3` and 5`-CCCAACGGCCAGTGGCCAGTCAGCG-3` for the -AR; 5`-GAGACC-TTCAACACCCC-3` and 5`-GTGGTGGTGAAGCTGTAGCC-3` for -actin. These oligonucleotides were derived from the sequences of the corresponding genes and cDNAs (2, 27, 28, 29) . Amplification products had expected sizes of 286, 329, 308, and 236 base pairs for -, -, and -ARs and -actin, respectively. They were separated on a 2% agarose gel and visualized by ethidium bromide staining. In mature adipocytes, amplification of -AR and -actin cDNAs with 25 cycles was linear up to 100 and 50 ng of RNA, respectively. - and -AR cDNA amplification with 33 cycles was linear up to 200 and 400 ng of RNA, respectively. With this prerequisite, we were thus able to compare the relative levels of each transcript between the different culture conditions.

For quantitation, autoradiograms of the Northern blots or ethidium bromide-stained gels of PCR products were analyzed by video densitometric scanning (Vilber Lourmat Imaging).

Statistical Analysis

Results are presented as means ± S.E. The level of significance between groups was assessed using either paired or unpaired Student's t test.


RESULTS

Effects of Long Term PMA Treatment on -AR Expression

To evaluate the effects of activation of PKC on -AR gene expression, 3T3-F442A adipocytes were treated with 300 nM PMA for 1-72 h, and -AR mRNA abundance was examined by RT-PCR analysis. While no significant change in the level of -actin amplification product could be detected during PMA exposure, we observed a decrease in -AR mRNA transcript as early as 2 h (Fig. 1) after the onset of phorbol ester treatment, with a maximal effect occurring by 6 h (6.5 ± 1.0-fold decrease, p < 0.005). Thereafter, with regards to PKC down-regulation induced by a prolonged exposure to a high dose of PMA (30) , -AR mRNA levels began to increase and returned almost to control levels within 24 h. In addition, after an initial 30-min treatment by PMA, cells were extensively washed and maintained for a further 6-h period without the phorbol ester. This procedure had no effect on -AR mRNA levels (not shown), indicating that a sustained PKC activation was required for -AR mRNA regulation. We also tested the time course of -AR mRNA content during exposure to dioctanoyl- sn-glycerol, a cell permeable analog of diacylglycerol (100 µg/ml every 30 min). No effect of this compound was detectable after a 60- or 90-min treatment, but -AR transcripts were decreased by 30 ± 5% at 2 h ( p < 0.002, n = 6).


Figure 1: Time-dependent down-regulation by PMA of -AR mRNA expression in 3T3-F442A adipocytes. 3T3-F442A adipocytes maintained in a serum-free medium were exposed or not to PMA (300 nM) for the indicated times. Total RNA from control and PMA-treated cells were prepared at each time point. Samples were digested by DNase I and treated or not with reverse transcriptase to verify that subsequent PCR amplification performed with a mixture of -AR and -actin specific primers did not derive from contaminating genomic DNA. -AR and -actin cDNA amplification was carried out in nonsaturating conditions (25 cycles, 25 ng of RNA). PCR products were resolved on a 2% agarose gel stained with ethidium bromide, and analyzed by video densitometric scanning. -Actin mRNA levels were used to standardize -AR mRNA content. Results for PMA-treated cells are expressed as the percentage of the level detected in control adipocytes and represented the mean ± S.E. of three independent experiments. *, p < 0.005, PMA-treated versus control adipocytes.



The dose dependence of -AR mRNA repression by PMA was studied by both Northern blotting and RT-PCR analyses. With these two approaches, down-regulation of the -AR mRNA was detectable at a PMA concentration of 10 nM (Fig. 2). Repression of the -AR transcript was maximal between 0.1 and 1 µM, the half-maximal action being between 15 and 20 nM. The major mRNA species of 2.3 kilobases in size and the two minor species of 2.8 and 4.4 kilobases appeared equally affected by PMA treatment.


Figure 2: Effect of PMA concentration on -AR mRNA levels. 3T3-F442A adipocytes were exposed for 6 h to the indicated concentrations of PMA. Total RNA was extracted and analyzed by Northern hybridization with a specific -AR probe, or by RT-PCR as described in the legend to Fig. 1. Panel A shows a representative autoradiogram of a Northern blot analysis. The sizes (in kilobases) of -AR transcripts are indicated in the left margin, and positions of 28 and 18 S ribosomal RNAs are shown in the right margin. Panel B corresponds to a typical RT-PCR experiment. Positions of -AR and -actin PCR products are given on the left. The sizes of molecular weight markers (in base pairs) are indicated on the right. In panel C is represented the mean ± S.E. of three independent experiments of Northern () or RT-PCR () analysis. Results are represented as the percentage of the level detected in control adipocytes. EC values for PMA-induced down-regulation of -AR mRNA were 18.3 ± 3.0 and 15.5 ± 9.4 nM PMA in Northern and RT-PCR analyses, respectively. *, p < 0.01; **, p < 0.001, PMA-treated versus control adipocytes.



Analysis of -AR subtype gene expression by RT-PCR showed that PMA selectively inhibited -AR mRNA levels (Fig. 3). For this purpose we ensured that -, -, and -AR cDNA amplification was performed in nonsaturating conditions, as indicated by the stoichiometry between the amount of RNA and the level of the corresponding amplification product. While a 6-h exposure of the cells to 300 nM PMA caused a 3.3-4-fold decline in -AR mRNA content, this treatment did not influence the abundance of - and -AR transcripts.


Figure 3: Selective down-regulation of -AR mRNA levels by PMA. 3T3-F442A were treated ( filled bars) or not ( empty bars) with PMA (300 nM) for 6 h. Total RNA was extracted, digested with DNase I, then treated or not with MMLV RT. cDNA derived from various amounts of total RNA (indicated in ng under each column) were then amplified in the presence of Taq polymerase and primers specific for -, -, or -ARs. The resulting PCR products were analyzed as described in legend to Fig. 1. Results are expressed as the percentage of the -, -, or -AR mRNA content observed in control adipocytes with the maximal indicated amount of RNA (200, 400, and 100 ng of RNA for -, - and -ARs, respectively). They represented the mean ± S.E. of three to five separate experiments. *, p < 0.05; **, p < 0.01, PMA-treated versus control adipocytes.



To determine the functional consequences of the specific down-regulation of -AR mRNA levels by PMA, adenylyl cyclase activity in response to CGP12177 was measured on membranes from control or PMA-treated cells. CGP12177 is a /-AR antagonist, but has agonistic properties at the -AR site (2, 31) , so that this compound allows to address specifically -AR coupling. Exposure of 3T3-F442A adipocytes to PMA provoked a time- and dose-dependent decline in adenylyl cyclase activity in response to a maximal dose (100 µM) of CGP12177. As compared to the kinetics of the PMA-induced decrease in -AR mRNA levels, the inhibitory effect of the phorbol ester on -AR responsiveness was slightly delayed. A significant decline in CGP12177-stimulated adenylyl cyclase activity was detectable after a 6-h exposure to PMA (Fig. 4 A) and was maximal after a 12-h treatment (2.9-fold decrease, p < 0.02). Thereafter, we observed an increased adenylyl cyclase responsiveness to CGP12177, with a restoration of the control adenylyl cyclase sensitivity within 48 h. Inhibition of CGP12177-stimulated adenylyl cyclase activity was observed at 30 nM PMA and was maximal at 300 nM, giving a half-maximal concentration of 44 ± 11 nM (Fig. 4 B). In control and PMA-exposed (300 nM for 12 h) adipocytes, adenylyl cyclase activity was also measured in response to maximal concentrations of various effectors: forskolin, GTPS, ISO, and the -AR agonists CGP12177, ICI201651, and CYP. As illustrated in , chronic PMA exposure specifically altered -AR responsiveness. By contrast, GTPS- and forskolin-stimulated adenylyl cyclase activities were not affected by phorbol ester treatment. This result strongly suggested that the PMA-induced decrease in -AR sensitivity was a receptor- rather than a G-protein- or adenylyl cyclase-mediated event.


Figure 4: Time and dose dependence of PMA-induced decrease in CGP12177-stimulated adenylyl cyclase activity. A, adenylyl cyclase activity in response to a maximal concentration (100 µM) of CGP12177 was determined in membranes from adipocytes treated or not by PMA (300 nM) for the indicated times. Results of PMA-treated cells are expressed as the percentage of CGP12177 stimulated over basal adenylyl cyclase activity measured in control cells. B, adenylyl cyclase activity in response to a maximal concentration (100 µM) of the -AR agonist CGP12177 was measured in membranes from adipocytes exposed for 12 h to the mentioned concentrations of PMA. Results are expressed as CGP12177-stimulated over basal adenylyl cyclase activity (in pmol cAMP/min/mg of protein). The figures represent the mean ± S.E. of three independent experiments performed in triplicate. *, p < 0.02; **, p < 0.01, PMA-treated versus control adipocytes.



We also confirmed that the decrease in -AR mRNA and -AR coupling to the adenylyl cyclase system corresponded to specific regulation of -AR sites. (-)-[I]CYP saturation binding experiments were performed in membranes from control and PMA-treated (300 nM for 12 h) 3T3-F442A adipocytes. In control cells, the density of -ARs (low-affinity class) was 544.2 ± 59.4 fmol/mg, while that of - and -ARs (high-affinity class) was only 11.3 ± 2.3 fmol/mg (). In PMA-exposed cells, we observed an 2-fold reduction in -AR population (278.9 ± 50.6 fmol/mg), whereas the number of high-affinity binding sites remained unaffected. No significant difference in the Kvalues of the two binding classes for (-)-[I]CYP could be detected between treated and untreated cells. Furthermore, competition curves of -, - and -AR selective ligands against (-)-[I]CYP allowed estimation of the relative proportions of the three -AR subtypes in control and PMA-treated adipocytes (I). These experiments confirmed that PMA induced a 2-fold decrease in the -AR population which corresponds to the high affinity sites for the -AR selective compound BRL37344. In agreement with RT-PCR analysis of - and -AR mRNA levels, analysis of the displacement curves of (-)-[I]CYP by the -AR selective antagonist CGP20712A or by the -AR selective antagonist ICI118551 indicated that the density of - and -ARs was not modified by PMA treatment.

PKC Involvement in the Effect of PMA on -AR mRNA

A series of experiments were designed to determine whether the effects of PMA on -AR gene expression were mediated by activation of PKC. The biologically inactive phorbol ester 4-PDD did not modify -AR mRNA content (Fig. 5 A), thereby excluding a nonspecific effect of the phorbol moiety. Also, 3T3-F442A adipocytes were exposed for 24 h to a high dose (1 µM) of PMA to down-regulate PKC; after this initial treatment, a subsequent exposure to PMA (100 nM) for 6 h did not modify -AR mRNA levels (Fig. 5 B). We also studied the reversibility of the PMA-induced decrease in -AR mRNA content by a recently characterized PKC inhibitor, bisindolylmaleimide, which acts as a competitive inhibitor with respect to ATP binding site on the catalytic moiety of the kinase (32) . In contrast with other PKC inhibitors, this novel compound displays a high selectivity for classical isoforms of PKC as compared to several other protein kinases. Pretreatment of 3T3-F442A adipocytes with increasing concentrations of bisindolylmaleimide completely inhibited the PMA-induced down-regulation of -AR mRNA (Fig. 6), with an IC value of 52.1 ± 11.9 nM. Cell exposure to bisindolylmaleimide alone did not affect -AR gene expression.


Figure 5: Effects of 4-PDD and PKC down-regulation on -AR mRNA levels. 3T3-F442A adipocytes were exposed or not (control, C) for 6 h to PMA (100 nM) or to the biologically inactive phorbol ester 4-PDD (100 nM) ( panel A). Adipocytes pretreated for 24 h by 1 µM PMA to down-regulate PKC (desensitized, DSTZ) were then treated ( PMA) or not (control, C) for 6 h with 100 nM PMA ( panel B). Total RNA was prepared from 3T3-F442A adipocytes cultured under these various conditions. Samples were digested with DNase I, then treated or not by MMLV RT. cDNAs were amplified (25 cycles) in the presence of Taq polymerase and specific primers for -AR and -actin. cDNA content in the PCR assay corresponded to initial amounts of DNase I-treated RNA of 25 ng. PCR products (one-fifth volume) were separated on a 2% agarose gel, visualized by ethidium bromide staining, and analyzed by video densitometric scanning. -AR mRNA levels were normalized to -actin mRNA content and are expressed as the percentage of the level detected in control adipocytes. The values represent the mean ± S.E. of three to four independent experiments. *, p < 0.001, PMA-treated versus control adipocytes.




Figure 6: Effect of the selective PKC inhibitor bisindolylmaleimide on PMA-induced decrease in -AR mRNA levels. 3T3-F442A adipocytes were pretreated or not for 30 min with the indicated concentrations of bisindolylmaleimide. Thereafter, cells were treated for 6 h with or without 100 nM PMA. Total RNA was prepared, digested with DNase I and treated or not with MMLV RT. cDNA amplification, analysis of PCR products, and expression of the results were carried out as described in the legend to Fig. 5. The figure shows the mean ± S.E. of three independent experiments performed in cells exposed () or not () to PMA. *, p < 0.005; **, p < 0.02, PMA-treated versus control cells.



Effect of PMA on -AR mRNA Turnover

The PMA-induced decrease in -AR mRNA levels resulted either from decreased rate of gene transcription, accelerated rate of RNA degradation, or a combination of both. We therefore examined the turnover of the -AR mRNA in adipocytes exposed to an inhibitor of transcription (actinomycin D) in the absence or presence of PMA. The disappearance of the -AR transcripts was then assayed by Northern analysis over a 4-h period (Fig. 7). The rate of receptor mRNA decay in PMA-treated adipocytes ( t = 84 ± 10 min) was not different from that observed in control cells ( t = 86 ± 6 min), strongly suggesting that PMA caused a decrease in -AR gene expression by inhibiting gene transcription, rather than by a decrease in mRNA stability.


Figure 7: Effect of PMA on -AR mRNA stability. 3T3-F442A adipocytes were treated (, dashed line) or not (, solid line) by 300 nM PMA for 2 h, then actinomycin D (5 µg/ml) was added to control or PMA-exposed cell cultures. At the indicated times of actinomycin treatment, total RNA was extracted and Northern analysis was performed as described under ``Materials and Methods.'' Autoradiograms were analyzed by video densitometric scanning. The half-life of -AR mRNA was calculated by linear estimation from the best fit of each line on the logarithmic plot. The figure represents the mean ± S.E. of three separate experiments.



Evaluation of the Role of PKC in Insulin Action on -AR mRNA

We have recently shown that insulin selectively down-regulates -AR transcripts in 3T3-F442A adipocytes by a transcriptional mechanism (13) . To further test the hypothesis that insulin could modulate -AR gene expression through the PKC signaling pathway, 3T3-F442A adipocytes were maintained with or without the hormone in the absence or presence of the selective PKC inhibitor bisindolylmaleimide. RT-PCR analysis indicated that bisindolylmaleimide partially inhibited the decline in -AR mRNA levels caused by insulin (Fig. 8). When cells were exposed to the PKC-selective inhibitor prior to insulin addition, the decrease in -AR mRNA content induced by 1 or 5 nM of the hormone was reduced by 44 and 49%, respectively. In addition, after initial down-regulation of PKC by a 24-h exposure to a high dose (1 µM) of PMA, we evaluated the effect of insulin on -AR mRNA content in these desensitized cells. While insulin provoked a clear decrease in -AR gene expression in nondesensitized cells, the initial down-regulation of PKC partially prevented its modulating action which was reduced by 67% (Fig. 9), thus providing additional evidence for the role of PKC in -AR mRNA regulation by insulin.


Figure 8: Effect of bisindolylmaleimide on insulin-induced decrease in -AR mRNA levels. 3T3-F442A adipocytes were treated or not with 1 or 5 nM insulin ( INS) for 6 h in the absence or the presence of the PKC inhibitor bisindolylmaleimide ( BIM) (0.5 µM added 30 min prior to insulin). RNA extraction, RT-PCR, and expression of the results were performed as described in the legend to Fig. 5. The results represent the mean ± S.E. of five independent experiments. **, p < 0.001, insulin-treated cells versus control cells. *, p < 0.01, insulin- and bisindolylmaleimide-treated cells versus control cells. , p < 0.005, insulin- and bisindolylmaleimide-treated cells versus insulin-(alone) treated cells.




Figure 9: Effect of insulin on -AR mRNA expression after chronic incubation with PMA. 3T3-F442A adipocytes were untreated or treated (desensitized, DSTZ) for 24 h with 1 µM PMA to down-regulate PKC. Thereafter, desensitized or nondesensitized cells were exposed or not for 4 h to 10 nM insulin ( INS). The control ( C) condition corresponded to cells neither treated by insulin nor PMA. Total RNA was isolated, digested with DNase I, and treated (+) or not (-) with MMLV RT. cDNA amplification and analysis of the results were carried out as described in the legend to Fig. 5. Panel A shows a typical ethidium bromide-stained gel of PCR products. Sizes (in base pairs) of the molecular weight markers are indicated in the right margin, while the positions of -AR and -actin PCR products are shown in the left margin. Panel B represents the mean ± S.E. of seven separate experiments. *, p < 0.001, insulin-treated versus control adipocytes. #, p < 0.001, desensitized cells treated with insulin ( DSTZ + INS) versus insulin-treated cells. , p < 0.05, desensitized cells treated with insulin ( DSTZ + INS) versus desensitized cells ( DSTZ).




DISCUSSION

In our study, several observations support the proposed role of PKC in the mediation of the phorbol ester-induced decrease in -AR mRNA content: (i) the high sensitivity to PMA contrasts with the lack of effect of the inactive phorbol ester 4-PDD, in agreement with recently published results (33) . (ii) PKC down-regulation abolishes the subsequent inhibitory action of PMA on -AR mRNA levels. (iii) The effect of PMA is suppressed by a selective PKC inhibitor, bisindolylmaleimide, with IC values consistent with its potency to inhibit classical PKC subtypes (32) . In contrast to most reported PKC inhibitors that are poorly selective, such as H-7 (34) or staurosporine (35) , bisindolylmaleimide displays high selectivity for PKC, as compared to other kinases (32) .

Earlier studies have reported that in several cell types, phorbol esters cause an attenuation of -AR-mediated cAMP production (Ref. 36 and references herein), while others have described a potentiation of this response (Refs. 37 and 38, and references herein). Phosphorylation of the -AR is at the molecular basis of the phorbol ester-provoked attenuation in catecholamine responsiveness (39, 40) . The PKA/PKC consensus phosphorylation site, located in the third intracellular loop, is involved in rapid desensitization by receptor uncoupling from Gs (41, 42, 43) . Nakada et al.(36) have also suggested that the nature of the -AR subtype may determine the ability of PKC activation to uncouple the receptor from adenylyl cyclase activation. Interestingly, the PKA/PKC phosphorylation sites are lacking in the -AR, and this absence is likely to contribute to the resistance of this subtype to undergo rapid agonist-promoted desensitization (44, 45) . The present study establishes the fact that PKC activation allows a long term desensitization of the -AR through a process of down-regulation. A sustained activation of PKC provokes a specific inhibition in -AR gene expression, whereas - and -AR mRNA levels remain unchanged. An accelerated receptor turnover may also be involved in -AR down-regulation, although this seems unlikely in view of our previous results (46) . In 3T3 adipocytes, the regulation of -AR responsiveness by activation of PKC could involve the complex and sequential contribution of both transcriptional and post-translational processes that differentially affect the three -AR subtypes. Acutely, phosphorylation of - and -ARs (36) by PKC uncouples these subtypes from G-protein, while a more prolonged PKC activation decreases -AR gene and protein expression.

Transcriptional and/or post-transcriptional mechanisms are involved in the control of gene expression by PKC. In our study, the absence of any significant effect of PMA on -AR mRNA stability indicates that PKC activity regulates the transcription rate of the -AR gene. Several mechanisms at the basis of this transcriptional modulation can be considered. Studies performed in various cell types, including adipocytes (30) , have shown that PKC activation leads to the transcriptional induction of the cellular immediate-early response gene c- fos. c-Fos and c-Jun proteins heterodimers, or c-Jun homodimers are components of the transcription factor AP-1. AP-1, alone or in combination with other transcription factors (47, 48) , modulates gene activity through interaction with a specific DNA recognition sequence. Alternatively, the PMA-induced increase in AP-1 activity could involve post-translational modifications of pre-existing AP-1. Boyle et al.(49) have demonstrated that activation of PKC results in dephosphorylation of c-Jun and coincides with increased AP-1 binding and trans-activating activity. Furthermore, the PKC signaling pathway controls the nuclear translocation of transcription factors. The transcription factor NF-B provides a prototypical example of an ``anchorage-release'' signal transduction mechanism to the nucleus. In a resting state, NF-B is present in an inactive form in the cytoplasm by association with an inhibitory protein known as IB. IB phosphorylation by a variety of kinases, including PKC, leads to the dissociation of the complex and nuclear translocation of NF-B that can bind to its target elements (48) . Specific consensus sequences for AP-1 and NF-B are present in the promoter of the human, mouse, and rat -AR genes (50, 51) , but no data are currently available to suggest a functional role for these sequences. It is conceivable that these and other mechanisms controlling gene transcription do not function independently of one another. Signal cross-talk could occur at every level of the transduction pathways. In this regard it has been reported that NF-B and AP-1 can physically interact to synergize DNA-binding and biological function (52) .

We have previously reported (13) that insulin can down-regulate -AR gene expression, primarily through a transcriptional mechanism. This sharp insulin-induced decrease of the main -AR subtype of rodent adipocytes inhibits -AR responsiveness and has potential consequences on all cAMP-dependent biological processes of adipocytes. Thus it is of considerable interest to identify the intracellular transduction pathways responsible for such a regulation. Our results suggest that modulation of -AR mRNA levels by insulin could involve PKC-dependent and -independent mechanisms. Several of our data indicate that the down-regulation of -AR gene expression by insulin occurs partially through the PKC signaling pathway. PMA provokes a decrease in -AR mRNA amounts comparable to that caused by insulin (13) . Both the phorbol ester and insulin induce a specific modulation of -AR gene expression. Depletion of cellular PKC levels by prolonged exposure to PMA, or pretreatment with a PKC selective inhibitor alter the effects of insulin on -AR transcripts. Also, PMA and insulin appear to control -AR gene expression primarily at the transcriptional level. Taken together, these data confirm that PKC is involved in the repression of the -AR gene by insulin. However, other results suggest that PKC is not a unique and obligatory step in the control by the peptidic hormone of this adrenergic receptor. While insulin induces a rapid decrease in -AR mRNA content (13) , PMA- or diacylglycerol analog-induced down-regulation of these transcripts was significantly detectable only after 2 h. Moreover, PKC depletion or pretreatment with bisindolylmaleimide reversed only 50% of the insulin effect on -AR transcripts. Different explanations could account for this partial reversibility of insulin action after PKC depletion or blockade. One possibility is that the extent of PKC down-regulation after a chronic exposure to phorbol esters varies with cell type. In this regard, it is noteworthy that in 3T3-L1 adipocytes, there is a relative lack of effectiveness of this procedure of PKC depletion (21) . However, the absence of PMA effect on -AR transcripts in PKC-depleted adipocytes does not favor this hypothesis. Alternatively, insulin could regulate gene expression by activation of PKC isotypes which are less responsive to PMA. More recently discovered PKC subtypes, such as the and isoforms, appear to be more or completely resistant to down-regulation by phorbol esters (53, 54, 55) and might be preferentially activated by insulin in some cell types (56, 57) . In rat adipocytes, it is thus documented that chronic phorbol ester treatment differentially affects PKC isoform depletion (58) . Whatever the phenotype of PKC isozymes in mature 3T3 adipocytes, our study leaves open the possibility that activation of atypical PKC isotypes by non-diacylglycerol activating ligands plays a role in the control by insulin of -AR gene expression. Thus, it has been reported that phosphatidylinositol 3,4,5-trisphosphate produced by phosphatidylinositol 3-kinase, an enzyme of the insulin receptor signaling system, is able to activate the atypical PKC (59) . Finally, the persistance of a significant insulin effect on -AR mRNA after PKC depletion or selective blockade could also reflect the dual involvement of PKC-dependent and -independent pathways. The similarity between the effects of insulin and PMA may be related to the convergence at a common point of cellular events caused by these two effectors, for example, at the level of transcription. This hypothesis is supported by the characterization in the phosphoenolpyruvate carboxykinase promoter of an insulin-responsive sequence that coincides with the phorbol ester recognition site (60) .

In summary, the results of the present study indicates that sustained PKC stimulation specifically down-regulates -AR gene expression in 3T3-F442A adipocytes. In addition, the effect of insulin on this adrenergic receptor subtype could be mediated, at least in part, through this intracellular signaling pathway. The decrease of the main -AR subtype of rodent adipocytes and of catecholamine responsiveness induced by a prolonged PKC activation may have important consequences on cAMP-dependent biological processes of adipocytes, such as the positive control of lipolysis or thermogenesis, or the negative modulation of lipogenesis.

  
Table: PMA selectively inhibits adenylyl cyclase activity stimulated by -adrenergic agonists

Membranes were obtained from 3T3-F442A adipocytes treated or not with PMA (300 nM) for 12 h. Adenylyl cyclase activity in response to an optimal concentration (100 µM) of each indicated effector was determined. Results are expressed as effector-stimulated over basal adenylyl cyclase activity and represented the mean ± S.E. of 4-13 independent experiments. Basal adenylyl cyclase activity was 12.7 ± 1.0 and 14.6 ± 1.0 pmol of cAMP/min/mg of protein in control and PMA-treated cells, respectively.


  
Table: Characterization of (-)-[I]CYP binding sites in membranes from control and PMA-treated 3T3-F442A adipocytes

Membranes from control and PMA-treated (300 nM for 12 h) 3T3-F442A adipocytes were tested in (-)-[I]CYP saturation binding experiments using a wide range of concentrations (5-4000 pM) of the radioligand. Scatchard analysis of the data with the EBDA/LIGAND program was used to calculate the Kand B values of the high (- and -ARs) and the low (-AR) affinity sites for (-)-[I]CYP. Results are expressed as mean ± S.E. of five separate experiments. The percentage of each affinity binding class is indicated in parentheses after the B value.


  
Table: Competition of (-)-[I]CYP against -AR subtype-selective ligands in membranes from control and PMA-exposed cells

Membranes were prepared from control and PMA-exposed (300 nM for 12 h) 3T3-F442A adipocytes. Competition binding experiments were performed at 250 pM (-)-[I]CYP in the absence or the presence of various concentrations of CGP20712A, ICI118551, or BRL37344. Data from displacement of (-)-[I]CYP binding by these subtype-selective ligands were used to calculate the Kvalues for each affinity component. The corresponding B values were derived from total -AR density drawn from (-)-[I]CYP saturation experiments (see Table I) taking into account the percentage of each affinity component (indicated in parentheses after the B values) obtained from competition experiments. Results are expressed as mean ± S.E. of three independent experiments.



FOOTNOTES

*
This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale, the Centre National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer, the Ligue Nationale contre le Cancer, the Fondation pour la Recherche Médicale, the Ministère de l'Enseignement Supérieur et de la Recherche, the Université Paris VII, and the Bristol-Myers-Squibb Company. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-49-81-36-69; Fax: 33-1-48-98-04-69.

The abbreviations used are: -AR(s), -adrenergic receptor(s); BRL37344, sodium-4-{2-[2-hydroxy-2-(3-chloro-phenyl)ethylamino]propyl}phenoxyacetate sesquihydrate ( RR.SS distereoisomer); CGP12177, (±)-4-(3- t-butylamino-2-hydroxypropoxy)-benzimidazole-2-one; CGP20712A, (±)-(2-(3-carbamoyl-4-hydroxyphenoxy)-ethylamino)-3-(4-(1-methyl-4-trifluormethyl-2-imidazolyl)-phenoxy)-2-propanol methane sulfonate; (-)-[I-CYP, (-)-[I]cyanopindolol; GTPS, guanosine 5`- O-(3-thiotriphosphate); ICI118551, erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropylaminobutan-2-ol; ICI201651, ( R)-4-(2-hydroxy-3-phenoxypropylamino-ethoxy)- N-(2-methoxyethyl)phenoxyacetamide; ISO, (-)-isoproterenol; kb, kilobase(s); MMLV RT, Moloney murine leukemia virus reverse transcriptase; 4-PDD, 4-phorbol 12,13-didecanoate; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; PMA, 4-phorbol 12-myristate 13-acetate; RT-PCR, reverse transcriptase-polymerase chain reaction; Taq polymerase, Thermus aquaticus polymerase.


ACKNOWLEDGEMENTS

We thank J. L. Guillaume, Dr. D. Lacasa, Dr. S. Cazaubon, Dr. S. Marullo, and Dr. B. Manning for helpful discussions and for critical reading of the manuscript.


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