Calcium and protein kinase C regulation of the glucocorticoid receptor in mouse corticotrope tumor cells

Pergamon

0960-0760(93)E0032-3

J. Steroid Biochem. Molec. Biol. Vol. 48, No. 4, pp. 337-345, 1994 Copyright (c~ 1994 Elsevier Science L t d Printed in Great Britain. All rights reserved 0960-0760/94 $6.00 + 0.00

C a l c i u m and P r o t e i n Kinase C R e g u l a t i o n of the G l u c o c o r t i c o i d R e c e p t o r in M o u s e C o r t i c o t r o p e T u m o r Cells Karen E. S h e p p a r d Molecular Physiology Laboratory, Baker Medical Research Institute, Prahran, Vic. 3181, Australia

T h e effect o f i n c r e a s i n g i n t r a c e l l u l a r f r e e c a l c i u m a n d a c t i v a t i n g p r o t e i n k i n a s e C o n g l u c o c o r t i c o i d r e c e p t o r ( G R ) e x p r e s s i o n w a s i n v e s t i g a t e d in A t T - 2 0 cells, a m o u s e c o r t i c o t r o p e t u m o r cell line. T r e a t m e n t o f A t T - 2 0 cells w i t h t h e c a l c i u m i o n o p h o r e A23187 i n d u c e d a r a p i d t i m e - a n d d o s e d e p e n d e n t d e c r e a s e in [ 3 H ] d e x a m e t h a s o n e ( [ 3 H ] D E X ) b i n d i n g w h e n m e a s u r e d in i n t a c t cells. B i n d i n g fell to 16~/o o f c o n t r o l f o l l o w i n g 3 h o f t r e a t m e n t w i t h 10 g M A23187. I n c o n t r a s t , A23187 d i d n o t a c u t e l y e f f e c t s t e a d y s t a t e l e v e l s o f G R m R N A , a l t h o u g h l e v e l s fell to 76 + 1% o f c o n t r o l a f t e r 8-15 h o f t r e a t m e n t . S c a t c h a r d a n a l y s i s o f A23187 t r e a t e d c u l t u r e s d e m o n s t r a t e d a d e c r e a s e in G R b i n d i n g c a p a c i t y b u t n o c h a n g e in a f f i n i t y f o r [3H]DEX. A c u t e i n h i b i t i o n o f p r o t e i n s y n t h e s i s w i t h c y c l o h e x i m i d e h a d n o effect o n [ 3 H ] D E X b i n d i n g , s u g g e s t i n g t h a t t h e c a l c i u m - d e p e n d e n t d e c r e a s e w a s n o t s i m p l y d u e to i n h i b i t i o n o f G R p r o t e i n s y n t h e s i s . I n c o n t r a s t to t h e A23187 i n d u c e d d e c r e a s e in [ 3 H ] D E X b i n d i n g in i n t a c t cells, w h e n b i n d i n g w a s m e a s u r e d in c y t o s o l e x t r a c t f r o m A23187 t r e a t e d c u l t u r e s t h e r e w a s n o d e c r e a s e . T h e s e d a t a s u g g e s t t h a t t h e A23187 i n d u c e d d e c r e a s e in G R b i n d i n g in w h o l e cells is n o t d u e to a d e c r e a s e in G R p r o t e i n b u t r e v e r s i b l e c o n v e r s i o n o f t h e r e c e p t o r to a n o n - b i n d i n g f o r m . I n d u c i n g c a l c i u m i n f l u x o n l y t h r o u g h L - t y p e v o l t a g e - d e p e n d e n t c a l c i u m c h a n n e l s w i t h B A Y K8644 a l s o d e c r e a s e d [ 3 H ] D E X b i n d i n g a t A t T - 2 0 cells, t h o u g h t h e effect w a s less t h a n t h a t i n d u c e d b y A23187. A l t h o u g h a c t i v a t i o n o f p r o t e i n k i n a s e C w i t h p h o r b o l e s t e r t r a n s i e n t l y i n c r e a s e s i n t r a c e l l u l a r f r e e c a l c i u m in A t T - 2 0 cells, w h e n cells w e r e t r e a t e d f o r 0.5 to 22 h w i t h p h o r b o l 1 2 - m y r i s t a t e 1 3 - a c e t a t e , t h e r e w a s n o a c u t e fall in [ 3 H ] D E X b i n d i n g , a n d o n l y a s m a l l d e c r e a s e f o l l o w i n g 16 h o f t r e a t m e n t . T h e s e d a t a d e m o n s t r a t e t h a t s u s t a i n e d i n c r e a s e s in i n t r a c e l l u l a r c a l c i u m in c o r t i c o t r o p e s c a n i n d u c e a r a p i d a n d m a r k e d d e c r e a s e in G R b i n d i n g . T h e m e c h a n i s m is p o s t - t r a n s l a t i o n a l a n d i n v o l v e s t h e r e v e r s i b l e c o n v e r s i o n o f t h e r e c e p t o r to a n o n - b i n d i n g f o r m . I n a d d i t i o n , t h e c e l l u l a r m i l i e u is c l e a r l y i m p o r t a n t in c o n f e r r i n g n o n - b i n d i n g s t a t u s o n G R s i n c e o n c e t h e cell is d i s r u p t e d G R b i n d i n g is r e s t o r e d .

J. Steroid Biochem. Molec. Biol., Vol. 48, No. 4, pp. 337-345, 1994

INTRODUCTION

G R has also been reported [12-15]; although the m e c h anisms involved are unknown, the glucocorticoid effect T h e glucocorticoid receptor (GR) is a ligand-activated is probably not a direct genomic one, considering that nuclear transcription factor. In its non-activated state a complete glucocorticoid response element sequence is it is found in the cytoplasm, and upon ligand binding absent within a 3 kilobase 5' flanking sequence of the a series of events takes place leading to translocation of G R gene [16]. In addition to ligand-induced changes the receptor-ligand complex to the nucleus where it in G R expression, various neurotransmitters [17-19], regulates gene transcription. By altering the level of other steroids [20] and cyclic nucleotides [21-24] have functional G R the cellular glucocorticoid response can also been reported to regulate cellular levels of G R in be modified [1-5]. M a n y groups have demonstrated a variety of cell types. down-regulation of G R expression by ligand; the Most hormone receptors rely on second messenger mechanism(s) involved occur in a cell specific m a n n e r systems to relay their intracellular signal. Classical and include decreases in G R gene transcription [6, 7], steroid receptors are unusual in that they are localized G R m R N A [8-10] and the half-life of G R protein in either the cytoplasm or the nucleus of a cell, and are [7, 10, 11]. Glucocorticoid induced up-regulation of themselves part of their intracellular signal transduction pathway. T h u s , in addition to down-regulation of Received 2 Apr. 1993; accepted 9 Nov. 1993. GR, interactions between the receptor and various 337

338

Karen E. Sheppard

intracellular pathways can potentially play an important role in the overall cellular response to glucocorticoids. Interactions between the phospholipid- and c A M P - d e p e n d e n t pathways have been extensively reported in m a n y cells [25-28], though little is known about the possibility of cross-talk between G R and other intracellular signal pathways. Recently the transcription factors c-Fos and c-Jun, which are the end point of m a n y signal transduction pathways, were shown to associate with G R and inhibit its action [29-31]. In addition, the oncogene product of v-mos was demonstrated to impair glucocortioid function by affecting nuclear retention and reutilization of the receptor [32]. Furthermore, rapid inhibition of the glucocorticoid response by corticotropin-releasing factor (CRF) and adenylate cyclase/protein kinase A (PKA) pathway has been demonstrated in corticotropes [33, 34]. T h e inability to demonstrate a rapid C R F - i n d u c e d decrease in G R binding [24] suggests that G R has not been down-regulated by C R F but has been converted into a non-functional form. T h e aim of the present study is to determine the possible interaction between G R and intracellular pathways that are involved in regulating expression of P O M C in A t T - 2 0 cells, a mouse corticotrope t u m o r cell line. In these cells P O M C gene transcription and/or A C T H release are inhibited by glucocorticoids, and stimulated by C R F through the adenylate cyclase/ P K A pathway, arginine vasopressin (AVP) through phospholipid/protein kinase C ( P K C ) pathway, and various factors that increase intracellular free calcium (for reviews see [35-39]). We have previously demonstrated a C R F - and c A M P - m e d i a t e d decrease in G R biosynthesis in A t T - 2 0 cells [24]. T h e present study explores the potential regulation of ART-20 G R expression by P K C activation and intracellular calcium. We report that increasing intracellular free calcium either with a calcium ionophore or more specifically through voltage-dependent calcium channels rapidly decreases the level of G R binding in A t T - 2 0 cells; in contrast, activation of P K C with phorbol ester has little effect on G R expression in these cells. EXPERIMENTAL

Cell culture and treatment AtT-20D16_16 mouse corticotrope t u m o r cells were grown at 37°C under 95% 02 and 5% CO2 on 60 m m tissue culture plates in Dulbecco's modified Eagles m e d i u m ( D M E M ) , supplemented with 10% fetal calf serum (FCS), 5% horse serum (HS), penicillin (100U/ml), streptomycin (100#g/ml) and fungizone (0.25 #g/ml). Cells were treated and harvested 4-7 days after plating, and serum starved 1 6 - 2 4 h prior to treatment. A23187 ( 1 0 m M stock in Me2SO ), BAY K8644 ( 1 0 m M stock in methanol), phorbol 12-myristate 13-acetate (PMA: 1 0 # M stock in 0.2% Me2SO), or cycloheximide ( 1 0 m M stock in water) were added directly to media and the same volume of Me2SO or methanol added to the respective control.

At the end of the incubation period cells were collected by scraping into D M E M , centrifuged (200g for 7 m i n ) and then resuspended in D M E M or T M D buffer (10 m M Tris, 100 m M NaMoO4, 1 m M dithiothreitol, p H 7.4) for determination of G R binding capacity, or A T buffer ( 1 0 r a m Tris, p H 8 ; 3 m M CaC12; 2 m M MgC12; 0 . 5 m M dithiothreitol; 0.15% Triton X-100) containing 0 . 3 M sucrose for R N A extraction. Media were collected from cultures prior to cell harvesting and analyzed for immunoreactive /~-endorphin by a previously described R I A [40]. For all assays, each plate represents a single determination, thus " n " represents the n u m b e r of plates per data point. All data are expressed as mean + SEM.

Preparation of cells for [3H]dexamethasone ([3H]DEX) binding assays For intact cell and cytosol extract binding assays cells were collected into D M E M and pelleted by centrifugation at 200g for 7 min. Cell pellets were resuspended in D M E M for intact cell binding assays and in T M D buffer for cytosol extract binding. In all binding assays G R levels were quantified by a single saturating dose of [3H]DEX (30 nM), and non-specific binding was determined in the presence of a 200-fold excess of unlabeled D E X . For Scatchard analysis concentrations of [3H]DEX ranged over dilutions from 28 to 1.7 n M and non-specific binding was determined at each dose of tracer.

[SH]DEX binding in intact cells Cell pellets resuspended in D M E M were added to pre-gassed (5% CO2, 95% 02) glass tubes containing [3H]DEX with or without a 200-fold excess of D E X . T u b e s were then covered in parafilm and incubated at room temperature for 60 min at which time receptor binding had reached equilibrium (data not shown). Following the incubation 3 ml of ice cold D M E M was added and the cells pelleted by centrifugation (200g for 5 rain at 4°C); this was repeated 3 times. For Scatchard analysis, washed [3H]DEX labeled cells were taken up in a total volume of 1.2ml of 1.0 M perchloric acid and placed in glass scintillation vials. D N A was hydrolyzed by incubating vials at 68°C for 30 min, after which 0.2 ml of the solubilized nucleotides were taken and quantified by the diphenylamine assay [41] and the remaining 1.0 ml counted. Herring sperm D N A was used for a standard curve and specific [3H]DEX binding was then normalized to D N A recovered. T o determine [3H]DEX binding in the nuclei and cytoplasm of [3H]DEX labeled cells a nuclear/ cytoplasmic binding assay adapted from Spelsberg et al. [42] was employed. Briefly, washed [3H]DEX labeled cells were taken up into 0.5 ml of cold solution B [ 1 . 0 M sucrose, 10% (v/v) glycerol, 0.2% (v/v) T r i t o n X-100, 10 m M KC1 and 50 m M Tris, p H 7.4], layered over 0.3 ml of solution C [1.4 M sucrose, 10% (v/v) glycerol, 0.2% (v/v) T r i t o n X-100, 10 m M KC1 and 50 m M Tris, p H 7.4] and centrifuged for 20 min at

Calcium/PKC and GR Regulation 6000g and 4°C. T h e supernatant was either discarded, or collected and added to 15 ml of scintillation fluid for the determination of cytoplasmic binding. T h e nuclear pellet was taken up in solution D [10% (v/v) glycerol and 50 m M Tris, p H 7.4] and placed on nitrocellulose filters (2.5 cm diameter, 0.45/am pore size: Millipore) under vacuum. Filters were dried at 110°C for 10 min. Dried filters were placed in 20 ml scintillation vials containing 1.2 ml of 1.0 M perchloric acid. D N A was hydrolyzed and quantified as described for whole cell preparation. Scintillation fluid (15 ml) was added to the remaining 1 ml for counting.

[~H]DEX binding in cytosol extract Cells resuspended in T M D buffer were h o m ogenized by hand (Teflon-glass homogenizer) and the homogenate centrifuged (105,000g for 40 min at 4°C) to yield cytosol. Cytosol extracts (150/al) were incubated (25°C for 9 0 m i n ) with 50/al of T M D buffer containing [3H]DEX with or without D E X (200-fold excess). T o separate r e c e p t o r - b o u n d from free steroid, an equal volume of equilibrated hydroxylapatite slurry was added to each tube and shaken periodically for 3 0 m i n at 4°C. T h e mixture was then centrifuged (1000g for 3 m i n at 4°C), supernatant decanted and the hydroxylapatite pellet resuspended in 3 ml of wash buffer (1% T w e e n 80, 5 m M Na2HPO4, 1 0 m M Tris and 1.5 m M N a 2 - E D T A , p H 7.4, 4°C). T h i s sequence was repeated 3 times. Washed hydroxylapatite pellets were extracted with 2.0 ml ethanol at room temperature for 3 0 m i n , centrifuged (1000g for 5 m i n ) and the supernatant added directly to 10 ml of scintillation fluid and counted. A sample of the cytosol was taken and protein determined by the Bradford method [43]. For determination of D N A , the ultracentrifuge pellet was resuspended in 1.7 ml T E buffer ( 1 0 m M Tris and 1.0 m M N a 2 - E D T A p H 7.4, 4°C), perchloric acid (7 M) was then added to give a final concentration of 1 M. D N A was hydrolyzed by incubating at 68°C for 30 min, after which the tubes were centrifuged to removed debris and the solubilized nucleotides were taken and quantified by the diphenylamine assay [41]. Cytosol extract binding was then normalized to cytosolic protein. Normalization of cytosol extract binding to D N A reflected the same results observed when binding was normalized to protein (data not shown).

Cellular protein determination Following 3 h treatment with A23187 (1/aM), cells were collected into T E buffer containing 1% T r i t o n - X , vortexed, freeze-thawed twice, and then centrifuged (18,000g for 5 min). T h e supernatant was then assayed for protein by the L o w r y method [44].

R N A isolation and quantification Following treatment cells were pelleted and resuspended in ice-cold A T buffer containing 0.3 M sucrose. Cytoplasmic R N A was then isolated as described previously [24]. Briefly, nuclei were separated from the cytoplasmic fraction by layering disrupted cells

339

over A T buffer containing 0.4 M sucrose and centrifuging at 900g for 10 min. T h e cytoplasmic fraction (supernatant) was then proteinase K digested, phenol/ chloroform extracted, and cytoplasmic R N A precipitated with isopropanol. F o r cytoplasmic R N A quantification, samples were reconstituted in T E buffer and R N A concentration determined by absorbance at 260 nm.

Recombinant clones All R N A probes were synthesized with the Promega transcription system (Riboprobe Gemini system II: Promega, W I ) , to a specific activity of 0.5-1.0 × 10 9 c p m / # g R N A . As described previously [24], a 216 basepair c D N A fragment corresponding to a portion of the D N A binding region of the mouse G R was used to synthesize both sense R N A and radiolabeled antisense R N A probes.

Solution hybridization/RNA protection assay G R m R N A levels were quantified using a solution hybridization/S1 nuclease protection assay, described previously [14]. In brief, in vitro synthesized sense R N A and cytoplasmic R N A were hybridized to a 32P-antisense R N A probe for 18 h at 65°C, followed by $1 nuclease digestion (500-700 U) for 60 min at 56°C. Samples were then phenol/chloroform extracted, isopropanol precipitated, reconstituted in T E buffer and run on a 5% non-denaturing polyacrylamide gel. Specific m R N A - and sense RNA-32p-antisense R N A hybrid bands were excised from the dried gel and counted by liquid scintillation spectrophotometry. G R m R N A levels were determined by comparison to the standard curve and normalized to the total amount of cytoplasmic R N A loaded.

Statistics Statistical analysis was by one-way analysis of variance followed by Fisher P L S D test. All data are expressed as mean + SEM. RESULTS

A 2 3 1 8 7 time course A t T - 2 0 cells were treated for various time periods with the calcium ionophore A23187 (1 # M ) . Following treatment the level of specific nuclear localized [3H]DEX binding was determined by the nuclear/ cytoplasmic binding assay and normalized to D N A recovered, or G R m R N A was measured and normalized to cytoplasmic RNA. As illustrated in Fig. I(A), A23187 induced a rapid time-dependent decrease in [3H]DEX binding which fell to 57 + 2% of control by 3.5 h and remained at this level at the 7 h (59 + 1%) and 1 4 h (53 + 2 % ) time points. A slight decrease (10-14%) in the D N A recovered from A23187 treated cultures was observed at all time points [Fig. I(B)]. G R m R N A levels decreased by 24% + 1 at 8 h and remained at this level at the 15 h time point [22% + 4: Fig. 1(C)]. For all A23187 time points examined there

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Fig. 1. Effect o f A23187 on G R b i n d i n g (A), D N A r e c o v e r e d (B), a n d G R m R N A levels (C) in A t T - 2 0 cells. Cell c u l t u r e s w e r e i n c u b a t e d f o r t h e i n d i c a t e d t i m e s w i t h 1.0 p M A23187. F o l l o w i n g t r e a t m e n t , e i t h e r [3H]DEX n u c l e a r localized b i n d ing w a s m e a s u r e d a n d n o r m a l i z e d to t o t a l D N A r e c o v e r e d (A), o r G R m R N A levels w e r e d e t e r m i n e d a n d n o r m a l i z e d to c y t o p l a s m i c R N A (C). At t i m e zero, levels o f G R b i n d i n g , D N A r e c o v e r e d , a n d G R m R N A w e r e t a k e n as 100% a n d t h e r e m a i n i n g p o i n t s p r e s e n t e d as a p e r c e n t a g e t h e r e o f . Each point r e p r e s e n t s the m e a n + SEM; n = 4 plates per time p o i n t . *P < 0.05 c o m p a r e d w i t h c o n t r o l .

was no consistent change in A t T - 2 0 cytoplasmic R N A levels recovered (data not shown).

Fig. 2. C o m p a r i s o n o f A 2 3 1 8 7 - m e d i a t e d d e c r e a s e in G R b i n d ing in c y t o p l a s m (A) a n d n u c l e i (B) o f A t T - 2 0 cells. At T-20 cells w e r e t r e a t e d w i t h A23187 (3 p M ) f o r 3 h, cells w e r e t h e n collected a n d i n c u b a t e d w i t h [3H]DEX + D E X . F o l l o w i n g t h e i n c u b a t i o n specific b i n d i n g w a s m e a s u r e d in t h e n u c l e u s a n d c y t o p l a s m as d e s c r i b e d in ' E x p e r i m e n t a l ' . All d a t a w e r e n o r m a l i z e d to t h e D N A r e c o v e r e d in t h e n u c l e a r f r a c t i o n . E a c h p o i n t r e p r e s e n t s m e a n + SEM: n = 5: *P < 0.05.

nuclear [3H]DEX binding in a dose-dependent manner. Significant inhibition by A23187 was observed at 1/~M, at which dose [3H]DEX binding fell to 45 + 2°/o of control and at the highest dose of A23187 (10 # M ) , [3H]DEX binding was 16 + 1% of control. As an

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Nuclear and cytoplasmic localization of [3H]DEX binding Granted that non-specific binding in the cytoplasmic fraction is 50-70% of total cytoplasmic binding (compared with 5 - 1 5 % in the nuclear fraction), binding in cytoplasm was not routinely measured. However, the A23187 induced decrease in binding observed in the nuclear fraction (41% of control) was also seen in the cytoplasm (39% of control: Fig. 2). T h e ratio of cytoplasmic to nuclear binding in control and A23187 treated cells was 1:2.2 and 1:2.3, respectively, suggesting that this treatment does not alter the ability of activated G R to translocate to the nucleus.

A23187 dose-response T o study the dose-related effect of A23187 on G R binding, A t T - 2 0 cells were incubated for 3 h with a range of concentrations of A23187 from 0.1 to 1 0 # M . As depicted in Fig. 3(A), A23187 decreased

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Calcium/PKC and GR Regulation indication of the toxicity of the doses of A23187 the D N A recovered at each dose is p l o t t e d in Fig. 3(B). As seen in the time course e x p e r i m e n t A23187 from 3 to 10/~M i n d u c e d a small decrease in D N A recovered, suggesting that a small n u m b e r of cells had detached from the tissue culture plate d u r i n g A23187 treatment.

Scatchard analysis T h e decrease in [3H]DEX b i n d i n g following A23187 t r e a t m e n t p r e c e d e d and was greater than the timed e p e n d e n t decrease in G R m R N A , suggesting that A23187 has a p r e d o m i n a n t p o s t - t r a n s c r i p t i o n a l action on G R expression. T o further dissect the possible m e c h a n i s m by which calcium is decreasing G R b i n d i n g in these cells, the affinity of G R for D E X and the n u m b e r of b i n d i n g sites were assessed by Scatchard analysis in A23187 treated (1/~M for 3 h ) and nontreated cultures. As depicted in Fig. 4(B), in a whole cell b i n d i n g assay the affinity of [3H]DEX for G R is u n c h a n g e d in A23187 treated cultures. In nontreated cultures: K d = 6.3 n M and Bmax = 88 fmol, and in A23187 treated; K d = 7.3 n M and Bmax = 51 fmol.

G R binding: comparision of cytosol extract to intact cells T w o other possible m e c h a n i s m s by which A23187 could decrease G R b i n d i n g are either by decreasing receptor n u m b e r a n d / o r converting the receptor to a

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[3H]DEX Bound ( DPM / xl00 ) Fig. 4. S a t u r a t i o n c u r v e (A) a n d S c a t c h a r d a n a l y s i s ( B ) o f [ ~ H ] D E X b i n d i n g in c o n t r o l a n d A23187 t r e a t e d c u l t u r e s . A t T - 2 0 c e l l s w e r e i n c u b a t e d w i t h 1 . 0 p M A23187 f o r 3 h. F o l l o w i n g t h e i n c u b a t i o n c e l l s w e r e r e m o v e d a n d w h o l e cell [3H]DEX binding determined. The concentration of radiolig a n d r a n g e d f r o m 1.7 to 28 n M a n d n o n - s p e c i f i c b i n d i n g w a s d e t e r m i n e d at e a c h d o s e o f t r a c e r . T h e b i n d i n g site c o n c e n t r a t i o n ( B m a x ) , d i s s o c i a t i o n c o n s t a n t (Ka), a n d c o r r e l a t i o n c o e f f i c i e n t ( r ) f o r c o n t r o l c u l t u r e s a r e nmax = 88 f m o l , K a = 6 . 3 n M , a n d r = 0.90; a n d f o r A23187 t r e a t e d c u l t u r e s , Bma x 51 fmol, K a = 7.3 n M , a n d r = 0.93. =

341

Table 1. Comparison of [3H]DEX binding in cytosol extract and intact cells following A23187 treatment [~H]DEX binding Whole cells Control A23187

Cytosol extract

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1O0% 91 + 6°/o

1O0% 32 + 5%

1O0% 38 + 6%

AtT-20 cells were treated for 3 h with either A23187 (3 pM) or DMSO (Control). Following treatment cells were collected and either incubated with tracer and nuclear and cytoplasmic binding measured, or cytosol was prepared and this was then incubated with tracer. Cytosol extract binding was normalized to cytosolic protein and nuclear/ cytoplasmic binding to DNA. Results are expressed as a percentage of their respective control. Mean+ SEM: n = 4 for cytosol extract, and n = 3 for whole cells. n o n - b i n d i n g form. T o further examine the A23187 i n d u c e d decrease [3H]DEX b i n d i n g observed in intact cells, b i n d i n g was measured in a cytosol extract. A t T - 2 0 cells were treated for 3 h with 3 p M A23187. F o l l o w i n g t r e a t m e n t either cytosol extract was p r e p a r e d which was then incubated with [3H]DEX + D E X and specific b i n d i n g d e t e r m i n e d by the h y d r o x y lapatite m e t h o d , or intact cells were incubated with [3H]DEX + D E X and specific b i n d i n g m e a s u r e d in the nuclei and cytoplasm by the n u c l e a r / c y t o p l a s m i c assay. In contrast to the p o t e n t A23187 i n d u c e d decrease in b i n d i n g in both cytoplasm and nuclei (32 and 38% of control, respectively), in cytosol extract there was only a slight decrease in [3H]DEX b i n d i n g ( T a b l e 1). T o verify that both types of b i n d i n g assays (intact cell vs cytosol extract) can reflect the same result when there is a change in G R protein levels, G R b i n d i n g in cytosol extract was m e a s u r e d in C R F treated and n o n - t r e a t e d cells. C R F has been previously d e m o n s t r a t e d to decrease G R b i n d i n g in intact A t T - 2 0 cells [24], this decrease being partly due to a decrease in G R m R N A and p r e s u m a b l y G R protein. W h e n [3H]DEX b i n d i n g was m e a s u r e d in cytosol extract from C R F treated cells (10 n M for 24 h) there was a 30% decrease in b i n d i n g (data not shown). T h i s decrease is similar in m a g n i t u d e to that observed in intact cells ( 3 0 - 5 0 % ) following C R F t r e a t m e n t [24]. In a d d i t i o n differences in G R b i n d i n g levels between various cell lines are similar in both types of b i n d i n g assays (data not shown). T h e s e data suggest that the difference between the intact cell b i n d i n g assay and b i n d i n g in cytosol extract from A23187 treated cells is not a reflection of the different b i n d i n g assays, but rather that in intact cells A23187 t r e a t m e n t converts the receptor to a n o n - b i n d i n g form.

Cycloheximide treatment and cellular protein levels F o l l o w i n g 3 h of A23187 t r e a t m e n t (1 p M ) there was no significant change in total cellular protein; total protein in control cultures was 100 + 4 # g and in A23187 treated 94 + 4 p g (mean + S E M , n = 3). In addition, inhibition of p r o t e i n synthesis with cycloheximide (30 p M ) for 3 h did not m i m i c the A23187

342

Karen E. Sheppard

m e d i a t e d decrease in [ 3 H ] D E X b i n d i n g ; with [ 3 H ] D E X b i n d i n g i n c o n t r o l c u l t u r e s t a k e n as 100 + 2 % , foll o w i n g 3 0 / ~ M c y c l o h e x i m i d e t r e a t m e n t b i n d i n g was 88 + 4 % ( m e a n + S E M : n = 3). BAY

K8644

treatment

T o f u r t h e r e x a m i n e the specificity of the c a l c i u m i n d u c e d decrease in G R b i n d i n g B A Y K8644, w h i c h p r o m o t e s extracellular c a l c i u m influx via L - t y p e v o l t a g e - d e p e n d e n t c a l c i u m c h a n n e l s [45], was a p p l i e d to A t T - 2 0 cells. W h e n cells were treated for 3 h with either 0.1 or 1.0/~M of B A Y K 8 6 4 4 there was a decrease in [ 3 H ] D E X b i n d i n g to 81 + 3 % a n d 76 + 3 % of control, respectively [Fig. 5(A)], with n o c o n c u r r e n t change in D N A recovered [Fig. 5(B)]. I n a d d i t i o n , B A Y K 8 6 4 4 i n d u c e d a 3-fold increase in the release of f l - e n d o r p h i n from the same cells [Fig. 5(C)], in a g r e e m e n t with p r e v i o u s studies d e m o n s t r a t i n g p e p tide release via L - t y p e c a l c i u m c h a n n e l s in A t T - 2 0 cells [46]. PMA

Table 2. Time course of P M A cells

Time (h) Control PMA Control PMA Control PMA Control PMA Control PMA

0.5 1.5 4.0 16 22

treatment of A t T - 2 0

Specific[3H]DEX fl-Endorphin binding (%) (%) 100 + 2 103 + 1 100 + 8 92 + 3 100 + 3 97 + 3 100 +_2 87 + 3* 100 + 1 89 + 3

NT NT 100 + 4 140 + 15" 100 +_7 147 + 17" NT NT NT NT

AtT-20 cell were treated with 100nM PMA for the indicated times. Following treatment nuclear [3H]DEX binding was determined and normalized to DNA recovered. In addition, media were collected and assayed for immuno-reactive fl-endorphin. Data are expressed as a percentage of control and represent the mean _+SEM. *P < 0.05 compared to respective time control. (NT = not tested).

treatment

P M A activates P K C a n d stimulates c a l c i u m influx in A t T - 2 0 cells [47]. T o assess the possible role activation

A 100

8o o

o

60

'~

40 20

B

<

5O 4O 30 20

a

0

-~

O.

-.

C

A <:

of P K C has on G R in A t T - 2 0 cells, cells were treated with P M A ( 1 0 0 n M ) for various time periods f r o m 0.5 h to 22 h a n d the level of n u c l e a r localized [3H]DEX b i n d i n g d e t e r m i n e d a n d n o r m a l i z e d to D N A recovered. I n c o n t r a s t to the decrease in G R b i n d i n g d e m o n s t r a t e d following an A23187 or B A Y K 8 6 4 4 i n d u c e d c a l c i u m influx, there was no rapid change following P M A t r e a t m e n t , a l t h o u g h there was a small b u t significant decrease in b i n d i n g following 16 h of t r e a t m e n t ( T a b l e 2). I m m u n o r e a c t i v e f l - e n d o r p h i n levels i n the m e d i a from the P M A treated cells increased b y 4 0 % after 1.5 h of P M A t r e a t m e n t a n d 47°/0 after 4 h ( T a b l e 2), i n d i c a t i n g that the cells had been stimulated by PMA. DISCUSSION

600-

Z 500400"

~300" ¢ "F 200" O 100c I,u

0"

Control

-0.1BM-1.0~_MBAY K8644

Fig. 5. Effect of BAY K8644 on GR binding (A), DNA r e c o v e r e d (B), and ~-endorphin secretion (C) in AtT-20 cells. AtT-20 cells were incubated for 3 h with the indicated concentrations of BAY K8644. Following the incubation, cells were collected and nuclear localized [3H]DEX binding determined, and media collected and analyzed for secreted 18-endorphin; both binding and 18-endorphin were normalized to DNA recovered. Data are expressed as mean _+SEM: n = 4. *P < 0.05.

I n the p r e s e n t s t u d y we have d e m o n s t r a t e d that i n c r e a s i n g i n t r a c e l l u l a r free c a l c i u m either with a c a l c i u m i o n o p h o r e or m o r e specifically via L - t y p e c a l c i u m c h a n n e l s can acutely lower the level of G R b i n d i n g in A t T - 2 0 cells. T h e o b s e r v a t i o n that the c a l c i u m - i n d u c e d decrease in [ 3 H ] D E X b i n d i n g occurs earlier a n d is greater t h a n the decrease in G R m R N A w o u l d suggest that the initial loss of b i n d i n g is not due to a decrease in t r a n s c r i p t i o n , n o r in steady state G R m R N A levels. A c a l c i u m i n d u c e d decrease in the affinity of the G R for D E X has p r e v i o u s l y b e e n o b s e r v e d in cytosol p r e p a r a t i o n s from rat h e p a t o m a cells [48]. I n these studies, a d d i t i o n of m i c r o m o l a r c o n c e n t r a t i o n s of CaC12 to the cytosol p r e p a r a t i o n decreased G R b i n d i n g by decreasing the affinity of D E X for the G R [48]. I n the light of this o b s e r v a t i o n we c o m p a r e d the affinity of D E X for G R in u n t r e a t e d a n d A23187 treated A t T - 2 0 cells. Scatchard analysis of [~H]DEX b i n d i n g in a whole cell p r e p a r a t i o n foll o w i n g an A23187 i n d u c e d increase in i n t r a c e l l u l a r c a l c i u m d e m o n s t r a t e d a decrease in [ 3 H ] D E X b i n d i n g

Calcium/PKC and GR Regulation with no apparent change in the affinity of D E X for the receptor. T w o other possible explanations for the A23187 induced decrease in G R binding is a decrease in receptor n u m b e r or conversion of the receptor to a non-binding form. A23187 has previously been d e m o n strated to decrease protein synthesis and overall protein degradation in other cells [49, 50]. However, following 3 h of A23187 treatment A t T - 2 0 total cellular protein did not significantly decrease, and acutely inhibiting protein synthesis with cycloheximide for 3 h did not mimic the A23187 induced decrease in G R binding. T h u s , these data indicate that the A23187 induced decrease in G R binding is probably not via the inhibition of G R synthesis, nor via a non-specific decrease in all cellular proteins. T h e discrepancy in [3H]DEX binding between intact cells and cytosol extract following A23187 treatment is strong evidence that in whole cells A23187 converts the receptor to a non-binding form. T h e fact that binding can be recovered when cells are lysed and binding measured in cytosol extract indicates that this is not a p e r m a n e n t change in G R , and also that the cellular milieu plays an important role in maintaining the receptor in a non-binding form. T o further characterize the specificity of the calcium effect on G R expression we treated cells with a specific L - t y p e calcium channel agonist BAY K8644. Corticotropes exhibit spontaneous calcium-dependent action potentials [51, 52] which are mediated through two inward calcium currents, a low threshold fast inactivating current ( T - t y p e ) and a higher threshold slow inactivating current ( L - t y p e ) [ 5 3 - 5 5 ] , the latter being sustained open by BAY K8644 [55]. A t T - 2 0 cells treated with BAY K8644 at a concentration (1.0/~M) that induced a three fold increase in fl-endorphin secretion, and which has previously been shown to induce a maximal release of fl-endorphin from these cells [46], decreased G R binding, though the effect was not as dramatic as that observed for A23187. T h e difference between A23187 and BAY K8644 treatment on G R expression may be explained by the mixture of L - t y p e calcium channel agonist and antagonist enantiomers of BAY K8644. Even though the antagonistic properties of BAY K8644 on A t T - 2 0 cells are not p r e d o m i n a n t until concentrations greater than 1.0 ~tM are used [46], the antagonistic enantiomer m a y decrease the effectiveness of the agonist at lower concentrations. Another possible explanation is that A23187 increases intracellular calcium through both extracellular influx and release of calcium from intracellular stores [56, 57], whereas BAY K8644 only increases intracellular calcium by extracellular influx and only through L type calcium channels. T h u s the rate of calcium influx and/or the concentration of intracellular free calcium m a y also be important in the potency of a c o m p o u n d to decrease G R binding. T h e G R is a phosphoprotein and the phosphorylation state of the receptor has been implicated in terms of its ability to bind steroid and translocate to the nucleus [58-60]. Activation of P K C has also been SB 4 8 / ~ 3

343

proposed to play a role in the phosphorylation state of the G R [61], and recently was demonstrated to downregulate both estrogen and progesterone receptors in M C F - 7 cells [62]. In addition, activation of P K C in corticotropes increases influx of calcium through L - t y p e calcium channels [47, 63]. In contrast to the rapid effects of A23187 and BAY K8644, activation of P K C with the phorbol ester P M A had very little effect on G R binding, though the increase in fl-endorphin release observed indicates that P M A did activate P K C in these cells. T h e inability of P M A to alter G R binding indicates that if phosphorylation of the G R by protein kinase C is occurring, it does not influence the ability of steroid to bind to the receptor. Given that we have measured nuclear localized binding, it is also clear that phosphorylation has not decreased the ability of the steroid/receptor complex to translocate to the nucleus. T h e fact that P M A has a biphasic effect on calcium influx, in that it initially increases intracellular free calcium (which lasts several minutes) followed by inhibition [47], may explain the inability o f a P M A - i n d u c e d calcium influx to acutely regulate G R binding, and in addition, suggests that a prolonged elevation of calcium may be required. T h e fact that 16 h of P M A treatment was required to slightly decrease [3H]DEX binding suggests that this decrease is probably not due to the initial transient increase in intracellular free calcium. We have previously demonstrated that chronic C R F treatment or activation of adenylate cyclase decreases G R binding in A t T - 2 0 cells, and that this effect is mediated, at least in part, through a decrease in steady state m R N A levels [24]. T h e present study d e m o n strates that influx of extracellular calcium induces a more rapid and potent decrease in G R binding (A23187 effect reached m a x i m u m within 3 h, whereas the C R F effect took 16-24h). F u r t h e r m o r e , the mechanism involved differs from that induced by C R F , in that there is no initial decrease in G R m R N A levels, and the receptor appears to be present but unable to bind ligand. In addition, the present data have demonstrated that activation of P K C has no apparent effect on G R expression nor the nuclear translocation process in A t T - 2 0 cells. Although the physiological relevance of these results remains to be determined, the association of calcium influx with a rapid decrease in G R binding potentially has very important implications in the ability of G R to regulate m a n y physiological pathways. In terms of the stress response, one can speculate that stress paradigms that lead to chronic release of C R F and/or other factors that induce prolonged elevations of intracellular calcium in corticotropes would lead to desensitization of the negative feedback actions of glucocorticoids at the level of the pituitary. In contrast, those stressors that promote corticotrope A C T H release via activation of P K C (such as AVP) may remain sensitive to glucocorticoid feedback. REFERENCES

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