ß2-Adrenergic Receptors Potentiate Glucocorticoid Receptor Transactivation via G Protein ß{gamma}-Subunits and the Phosphoinositide 3-Kinase Pathway

Peer Schmidt, Florian Holsboer and Dietmar Spengler

Max Planck Institute of Psychiatry D-80804 Munich, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid hormones influence manifold neuronal processes including learning, memory, and emotion via the glucocorticoid receptor (GR). Catecholamines further modulate these functions, although the underlying molecular mechanisms are poorly understood. Here, we show that epinephrine and norepinephrine potentiate ligand-dependent GR transactivation in a hippocampal cell line (HT22) via ß2-adrenergic receptors. This enhancement was strongest at low concentrations of glucocorticoids and was accompanied by increased GR binding to a glucocorticoid-responsive element (GRE). ß2-Adrenergic receptor-mediated GR enhancement was relayed via G protein ß{gamma}-subunits, insensitive to pertussis toxin and independent of protein kinase A (PKA). In contrast, the catecholamine-evoked GR enhancement was strongly reduced by wortmannin, suggesting a critical role for phosphoinositide 3-kinase (PI3-K). In agreement, epinephrine directly activated PI3-K in vivo. Similarly, stimulation of tyrosine kinase receptors coupled to PI3-K activation, e.g. receptors for insulin-like growth factor I (IGF-I) or fibroblast growth factor (FGF), increased GR transactivation. Further analysis indicated that G protein-coupled receptor (GPCR) and tyrosine kinase receptor signals converge on PI3-K through separate mechanisms. Blockade of GR enhancement by wortmannin was partially overcome by expression of the downstream-acting protein kinase B (PKB/Akt). Collectively, our findings demonstrate that GPCRs can regulate GR transactivation by stimulating PI3-K. This novel cross-talk may provide new insights into the molecular processes of learning and memory and the treatment of stress-related disorders.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The glucocorticoid receptor (GR), NR3C1, is widely expressed in mammalian tissues and mediates the effects of glucocorticoids released from the adrenal cortex upon stress. This release is regulated by the hypothalamic-pituitary-adrenocortical (HPA) system, and the GR critically controls HPA activity by maintaining negative feedback control at several levels (1). High concentrations of GR are expressed in the hippocampus, an important region of the limbic system modulating HPA function, mood, learning, and memory (2).

The GR is a member of the steroid receptor family and is closely related to the mineralocorticoid, progesterone, androgen, and estrogen receptors. After glucocorticoid binding, the activated GR is released from its cytoplasmatic protein complex and translocates into the nucleus. There, it binds to glucocorticoid-responsive elements (GREs) located in the regulatory region of target genes and modulates their activity by interacting with the transcriptional machinery (3). Depending on the nature and location of the GREs and, in addition, on the presence of coregulators, the GR displays transactivation or transrepression. Furthermore, cross-talk between the GR and other transcription factors has been observed (3, 4).

Ligand-independent modulation of steroid receptor function in a cell type-specific manner is well known for the estrogen, progesterone, and androgen receptors with regard to growth factors, cyclins, cAMP, or other signals. In contrast, a huge number of studies agreed that the GR can be modulated by such stimuli only after ligand activation (for review see Refs. 5, 6). However, the mechanisms by which these signal transduction pathways modify GR function are less well understood. An advanced insight into these processes appears mandatory to improve our understanding of various physiological and pathophysiological conditions related to GR function and dysfunction.

A number of studies indicate that adrenergic and glucocorticoid signaling is intimately interwoven in the brain (2, 7). In particular, memory consolidation requires activation of both glucocorticoid- and norepinephrine-dependent pathways, and both kinds of stress hormones have been implicated in psychiatric disorders (8, 9). Despite this increased understanding at the systemic level, no evidence for a direct interaction of the two stress hormone pathways within neuronal cells has been reported so far. Hence, we set out to examine whether, at the cellular level, neuronal GR activity is modulated by catecholamines, and if so, to identify the relevant pathway.

The catecholamines epinephrine and norepinephrine bind to G protein-coupled receptors (GPCRs) in the cell membrane, leading to dissociation of the G protein {alpha} and ß{gamma} subunits. In addition to the well known signaling of GPCRs via G{alpha}, the interest in biological effects mediated by Gß{gamma} has increased continuously during recent years. In particular, ß{gamma}-subunits from inhibitory Gi proteins have been demonstrated to interact with specific isoforms of phosphoinositide-3 kinase (PI3-K) and to stimulate the mitogen-activated protein kinase (MAPK) pathway (10, 11).

In this study, we report that (nor)epinephrine, acting via ß2-adrenergic receptors, potentiates GR transactivation in hippocampal cells. Our data reveal a novel cross-talk between endogenous adrenergic receptors and GRs in neuronal cells via a pathway depending on PI3-K.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Catecholamines Potentiate Neuronal GR Activity via ß2-AR
The hippocampus regulates HPA axis activity and contains both glucocorticoid and adrenergic receptors (2, 12). Therefore, a murine hippocampus-derived cell line (HT22) was used as a cellular model to investigate the interaction between glucocorticoid and catecholamine signaling (13). HT22 cells were transiently transfected with a reporter plasmid containing the glucocorticoid-responsive mouse mammary tumor virus promoter upstream of the luciferase gene (MMTV-Luc). Treatment of transfected hippocampal cells with increasing concentrations of dexamethasone stimulated reporter gene expression in a dose-dependent manner, indicating the presence of functional GRs (Fig. 1AGo, open circles). Interestingly, when transfected cells were costimulated with dexamethasone and epinephrine, a marked increase in glucocorticoid-induced reporter activity was observed while the catecholamine alone was ineffective. A similar enhancement was obtained with the natural ligand corticosterone, which displays lower affinity for the GR (Fig. 1AGo).



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Figure 1. Epinephrine Potentiates Glucocorticoid Signaling through a GRE

A, Epinephrine enhances dexamethasone and corticosterone signaling. HT22 cells were transiently transfected with MMTV-Luc, stimulated with increasing concentrations of dexamethasone (dex, circles) or corticosterone (cort, diamonds), and cotreated with vehicle (open symbols) or epinephrine (30 µM, filled symbols). **, P < 0.01 compared with glucocorticoid treatment alone. B, Epinephrine reduces the ED50 of glucocorticoids. The results from panel A of treatment with dexamethasone are plotted as per cent of maximal induction of each condition. The strongest relative enhancement of GR transactivation is obtained at the lowest glucocorticoid concentrations (inset). C, Catecholaminergic potentiation of GR transactivation is mediated by ß2-adrenergic receptors. HT22 cells were transiently transfected with MMTV-Luc and stimulated with dexamethasone and epinephrine or norepinephrine. Where indicated, the antagonists RU 38486 (GR-specific, 1 µM), propranolol (ß-AR-specific, 30 µM), or ICI 118551 (ß2-AR-specific, 100 µM) were included. Results are shown as per cent induction of treatment with dexamethasone alone (dashed line). D, GR enhancement by catecholamines occurs through GREs. HT22 cells were transiently transfected with TK-Luc containing either no (-) or two (+) GREs and stimulated with dexamethasone (10 nM) and epinephrine (30 µM) as indicated. E, Epinephrine costimulation does not increase GR mRNA or protein levels. Cells were treated as indicated for 6 h before analysis of 20 µg RNA by Northern blot (top) or 50 µg cell lysate by Western blot (bottom). F, GR DNA binding is significantly increased after treatment with epinephrine. A representative image of an EMSA is shown in the left panel. Cellular extracts from HT22 cells pretreated with vehicle (lanes 1, 3, 4) or epinephrine (lane 2) were incubated with dexamethasone (10 nM). Specific GR-DNA complexes (arrow) disappeared in the presence of a 150-fold excess of unlabeled competitor (cold GRE, lane 3) but not mutated GRE (cold mtGRE, lane 4). In the right panel, EMSA results representing the average signal intensity + SEM of the specific GR-DNA complexes (n = 4, P < 0.05, Student’s t test) are expressed as per cent of dexamethasone treatment alone. Results in panels A and D are shown as fold induction. Data from transient transfections represent the mean + SEM of at least three independent experiments performed in duplicate.

 
The increase in GR transactivation is illustrated in Fig. 1BGo by presenting the data as per cent of maximal induction in the presence or absence of epinephrine. The left-shift caused by epinephrine reflects an enhanced effectiveness of available glucocorticoids, reducing the ED50 from 26 nM to 12 nM. Remarkably, catecholamine regulation was strongest at low doses of glucocorticoid, with a 5-fold increase of the induction by 3 nM dexamethasone alone (Fig. 1BGo, inset). The extent of potentiation gradually diminished at higher dexamethasone concentrations, but epinephrine still doubled GR transactivation at 100 nM of glucocorticoids and beyond. Hence, in neuronal cells epinephrine strengthens GR transactivation potency predominantly at low doses of GR agonists.

Epinephrine concentrations as low as 30 nM were sufficient for GR enhancement (data not shown). Moreover, treatment of HT22 cells with glucocorticoids and norepinephrine instead of epinephrine caused a similar enhancement of GR transactivation (Fig. 1CGo). Potentiation could be blocked by the GR antagonist RU 38486, demonstrating specificity of the transcriptional response (Fig. 1CGo). Furthermore, enhancement could be prevented by addition of the ß-adrenergic antagonist propranolol and also by ICI 118551, suggesting that GR enhancement was mediated by ß2-adrenergic receptors (AR) (Fig. 1CGo).

To examine whether enhanced GR transactivation occurred through the GREs or, alternatively, through unrelated regulatory elements in the MMTV promoter, we used a minimal thymidine kinase (TK) promoter as control. Neither stimulation with dexamethasone nor epinephrine increased reporter gene activity in HT22 cells. However, after insertion of two GREs adjacent to the regulatory region, this promoter displayed both glucocorticoid induction and potentiation by epinephrine (Fig. 1DGo). We conclude that the effect of epinephrine on enhanced GR transactivation requires a GRE and is not confined to the MMTV promoter context.

Enhanced GR function might be due to an increase in receptor concentration under catecholamine treatment. However, we found no significant change in GR transcript or protein levels after 6 h or 24 h of epinephrine treatment (Fig. 1EGo). Likewise, the number of ligand binding sites and the affinity constant were unaltered after incubation of HT22 cells with epinephrine for 0, 4, 8, or 24 h (not shown). Therefore, higher transactivation occurred despite unaltered GR binding parameters.

Binding to the GRE is an important step before induction of glucocorticoid-responsive genes. In nontransfected HT22 cells, electrophoretic mobility shift assay (EMSA) revealed that specific DNA binding of the GR is significantly stronger in extracts of cells stimulated with epinephrine than in those of untreated cells (Fig. 1FGo, lanes 1 and 2; quantification depicted in right panel). Specific GR-DNA complexes disappeared in the presence of an excess of cold competitor GRE oligonucleotides (Fig. 1FGo, lane 3). As a negative control, the same excess of cold mutated GRE oligonucleotides did not compete with complex formation (lane 4), demonstrating specificity of the complexes. These results suggest that adrenergic receptor activation potentiates GR function, at least in part, by improving DNA binding.

Enhanced GR Transactivation by Epinephrine Is Mediated via G Protein ß{gamma}-Subunits
ß2-ARs are G{alpha}s protein-coupled receptors leading to activation of protein kinase A (PKA) (14). To investigate the role of PKA in enhanced GR transactivation, we treated transfected HT22 cells with both dexamethasone, epinephrine, and specific inhibitors of PKA. Cotransfection of an expression vector encoding a PKA inhibitor (PKI) peptide or treatment with the inactive cAMP-analog Rp-cAMP did not reduce enhanced GR transactivation, suggesting that GR potentiation is not mediated by PKA in HT22 cells (Fig. 2AGo). Of note, pretreatment with H-89 further increased GR function (Fig. 2AGo), possibly due to effects of H-89 unrelated to PKA (15).



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Figure 2.{gamma} Subunits Confer GR Enhancement

A, Epinephrine-dependent GR enhancement does not require a PKA-dependent switch from Gs to Gi coupling. HT22 cells were transiently transfected with MMTV-Luc and PKI expression plasmid or empty vector (10 ng each) and stimulated with dexamethasone, epinephrine, and the PKIs Rp-cAMP (30 µM) or H-89 (1 µM) as indicated. Inset, Transfected cells were prestimulated for 90 min with epinephrine or vehicle. Where indicated, blockade of Gi proteins with Ptx (100 ng/ml) was performed before costimulation with dexamethasone. B, G protein ß{gamma}-subunits can replace epinephrine stimulation. HT22 cells were cotransfected with MMTV-Luc, Gß (30 ng) and G{gamma} (left bar), or inactive G{gamma}- (right bar) (100 ng each) and stimulated with dexamethasone. The long dashed line indicates GR transactivation after costimulation with epinephrine. C, GR enhancement by G{alpha}s-coupled receptors. Ectopic expression of the cDNAs of GPCRs for dopamine (D1-R, 1 µg), pituitary adenylate cyclase-activating peptide (PAC1-R, 100 ng), or LH (LH-R, 100 ng) enabled potentiation of GR function by the cognate ligands comparable to epinephrine via endogenous ß2-AR. Cells were stimulated with dexamethasone and dopamine (6 µM), PACAP-27 (20 nM), or LH (100 pM). Data are shown as per cent of dexamethasone induction (short dashed line) and represent the mean + SEM of at least three independent experiments performed in duplicate.

 
The preferential coupling of ß2-AR to G{alpha}s has been reported to switch to G{alpha}i as a consequence of receptor phosphorylation by PKA (16). In light of the above results, such a PKA-mediated switch from Gs to Gi proteins appeared unlikely to contribute to enhanced GR transactivation. To further exclude the involvement of Gi proteins, we investigated the effect of pertussis toxin (Ptx) on GR enhancement. HT22 cells preincubated with Ptx showed no reduction of GR enhancement after costimulation with dexamethasone and epinephrine (Fig. 2AGo, inset, right bar). As an additional control, cells prestimulated with epinephrine for up to 90 min were treated with vehicle or Ptx before the addition of dexamethasone. Blockade of Gi signaling by Ptx after prestimulation with epinephrine did not reduce the GR response either (Fig. 2AGo, inset). Finally, Ptx alone did not modulate reporter gene induction (not shown). Together, these findings demonstrate that 1) blockade of the cAMP-PKA pathway does not prevent enhanced GR transactivation by ß2-adrenergic receptors; and 2) differential coupling of ß2-AR to Gi proteins is not required for potentiation of GR transactivation.

Within recent years, increasing attention has been paid to the cellular actions of Gß{gamma}-subunits upon GPCR activation (17). Whereas the biological functions of ß{gamma}-subunits released from inhibitory G{alpha}i have been described in some detail, e.g. activation of PI3-K and MAPK (for review see Ref. 18), much less is known about the role of ß{gamma} signaling in the case of receptors coupled to stimulatory G{alpha}s. In view of our experiments, we reasoned that the stimulatory effect of epinephrine on the GR may be relayed by ß{gamma}-subunits in response to stimulation by ß2-ARs. To further test this hypothesis, we asked whether catecholamine treatment could be replaced by cotransfection of free Gß{gamma}-subunits. In fact, expression of Gß and G{gamma} cDNAs could substitute for epinephrine stimulation (Fig. 2BGo). In contrast, coexpression of Gß in conjunction with a mutated G{gamma}- that fails to associate with the membrane (10) did not increase GR transactivation, indicating that Gß{gamma}-subunits are critical for enhanced GR transactivation by epinephrine (Fig. 2BGo). Collectively, these data strongly suggest that ß2-adrenergic signaling to the GR is transduced via Gß{gamma}-subunits released from G{alpha}s.

To examine whether GR enhancement was restricted to ß2-AR, we ectopically expressed other GPCRs in HT22 cells. Costimulation with dexamethasone and dopamine, pituitary adenylate cyclase- activating peptide (PACAP), or LH did not influence GR transactivation in HT22 cells (not shown). In contrast, these ligands strongly enhanced GR activity when their cognate receptors were cotransfected, indicating that the observed effect on the GR is not specific to ß2-ARs but also extends to other GPCRs coupled to G{alpha}s (Fig. 2CGo).

PI3-K Relays Signaling to the GR
Several recent studies reported a cross-talk between Gß{gamma}-subunits released from G{alpha}i and the PI3-K cascade (11, 19). We asked whether PI3-K might also play a role in the G{alpha}s-mediated effect of epinephrine on GR transactivation. To test this hypothesis, we treated cotransfected HT22 cells with wortmannin, a potent and specific inhibitor of PI3-K (20). In fact, wortmannin decreased the enhancement of GR transactivation by epinephrine in a dose-dependent manner (Fig. 3AGo) while it was ineffective alone. Importantly, this reduction was also obtained with LY 294002, a structurally unrelated inhibitor of PI3-K, demonstrating specificity of the blockade (Fig. 3AGo). Inhibition by wortmannin was maximal at 100 nM, preventing approximately 80% of the epinephrine-mediated GR potentiation. This suggests a major role of PI3-K in GR enhancement.



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Figure 3. PI3-K Is Necessary and Sufficient for Enhanced GR Transactivation by Epinephrine and Growth Factors

A, Catecholamine-mediated GR enhancement depends on PI3-K. Transfected cells were treated with dexamethasone, epinephrine, and increasing concentrations of wortmannin or LY 294002 (10 µM), respectively. Data are shown as per cent of dexamethasone induction. *, P < 0.05; ***, P < 0.001. B, Activation of the PI3-K pathway is sufficient to enhance GR transactivation. Transfected cells were plated in serum-free medium and treated with 30 ng/ml IGF-I or bFGF and other substances as indicated. Note that the effects of epinephrine and either growth factor on steroid receptor function are additive. Furthermore, cells transfected with the constitutively active catalytic subunit p110* (1 µg) were plated in serum-free medium and stimulated as indicated. Data are shown as per cent of dexamethasone induction (short dashed line). The long dashed line indicates GR transactivation after costimulation with epinephrine. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with dexamethasone alone; {ddagger}, P < 0.05 compared with IGF-I + dexamethasone. C, Epinephrine does not require phosphotyrosine binding of p85. Cells were cotransfected with wild-type p85, the dominant negative form {Delta}p85, or empty vector (30 ng each) and costimulated with dexamethasone and IGF-I or epinephrine under serum-free conditions. Results are shown as per cent induction compared with costimulation in the presence of empty vector. Data represent the mean + SEM of at least three independent experiments performed in duplicate.

 
Since PI3-K acts downstream of ß2-ARs in HT22 cells, we reasoned that physiological stimuli of the phosphoinositide cascade, e.g. growth factors, might similarly modulate GR function. To this end, we treated transfected, serum-deprived HT22 cells with insulin-like growth factor 1 (IGF-I) or with basic fibroblast growth factor (bFGF). Similar to epinephrine, IGF-I and bFGF alone showed no induction of the GRE-containing promoter. In contrast, administration of either polypeptide significantly increased GR transactivation by dexamethasone, which could be blocked by wortmannin (Fig. 3BGo). Remarkably, cotreatment with both the catecholamine and either growth factor further enhanced the GR response in an additive manner, indicating that both signals were transduced through separate pathways (Fig. 3BGo).

The family of PI3-Ks comprises various isoforms, and those belonging to class I are composed of a heterodimer consisting of a regulatory and a catalytic subunit. Different isoforms of each subunit have been isolated, and their patterns of expression and modes of activation show some variation (21). The crucial role of PI3-K was further underlined by results obtained from cotransfecting a constitutively active catalytic subunit (p110*) of PI3-K into HT22 cells. In agreement with above data, expression of this kinase enhanced ligand-dependent GR transactivation by 2-fold in the absence of both IGF-I and bFGF (Fig. 3BGo). Consequently, PI3-K activation is necessary and sufficient for mediating growth factor-dependent GR potentiation and, additionally, represents a major pathway for catecholamine-mediated enhancement of GR transactivation in HT22 cells.

One of the PI3-K-regulatory subunits, p85, binds to phosphotyrosine residues, e.g. on receptor tyrosine kinases (RTK) upon growth factor stimulation, and recruits the catalytic subunit p110 to the membrane (21). To investigate whether phosphotyrosine binding of p85 was also required for ß{gamma}-induced signaling, we cotransfected HT22 cells with a dominant negative form of p85 ({Delta}p85) lacking the binding site for p110. {Delta}p85 can still bind to phosphotyrosine residues, but it can no longer recruit p110. In agreement with this prediction, the IGF-I-induced GR potentiation could be nearly completely blocked by cotransfection of {Delta}p85 but not wild-type p85 (Fig. 3CGo). In contrast, neither {Delta}p85 nor wild-type p85 significantly affected the enhancement conferred by epinephrine (Fig. 3CGo). We conclude that epinephrine-induced ß{gamma} signaling does not depend on phosphotyrosine binding of the PI3-K-regulatory subunit p85 but appears to lead to direct activation of p110. This confirms the notion that growth factor and ß{gamma} signaling occur via distinct mechanisms leading to stimulation of PI3-K.

Epinephrine Activates the PI3-K Cascade
To further confirm that epinephrine leads to activation of PI3-K in vivo, we treated nontransfected HT22 cells with vehicle or epinephrine under serum-free conditions and measured the activity of PI3-K immunoprecipitated by anti-p85-Ab. As shown in Fig. 4AGo, incubation with epinephrine stimulated the catalytic activity of PI3-K by 2.1-fold. As a control, PI3-K activity was also enhanced by 2-fold after incubation with IGF-I (not shown). These results corroborate our transfection studies and clearly demonstrate a cross-talk between adrenergic receptors and PI3-K in neuronal cells.



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Figure 4. Epinephrine Activates PI3-K and PKB Confers GR Enhancement

A, Epinephrine activates PI3-K. HT22 cells were stimulated with vehicle or epinephrine in vivo and PI3-K activity was measured in vitro. Products were separated by TLC and autoradiographed. Enhanced PI3-K activity is evidenced by, on average, 2.1-fold increased formation of radioactive phosphatidylinositol-3-phosphate (PtdIns-3-P; n = 3). A representative autoradiograph is shown. B, PKB enhances GR transactivation. Cells cotransfected with empty vector or a constitutively active form of PKB (PKB*) (10 ng each) were plated in serum-free medium and stimulated with dexamethasone. C, PKB can partly restore GR enhancement under wortmannin treatment. HT22 cells cotransfected with PKB*, SGK, p70S6K, or empty vector (10 ng each) were stimulated as indicated in serum-free medium. **, P < 0.01 compared with induction after wortmannin blockade. Data in panels B and C are shown as per cent of dexamethasone induction and represent the mean + SEM of at least three independent experiments performed in duplicate.

 
PI3-K may stimulate several downstream kinases including MAPK, protein kinase C (PKC), serum and glucocorticoid-inducible kinase (SGK), p70S6K, and protein kinase B (PKB/Akt) (22). To identify the PI3-K-dependent effectors responsible for enhanced GR transactivation, we examined each of these pathways individually. Activation of PKC by phorbol esters did not increase GR transactivation. Similarly, inhibition of MAPK kinase or cotransfection of dominant negative forms of SGK or p70S6K failed to decrease epinephrine-induced GR potentiation, arguing against a role of these kinases in GR enhancement (data not shown). In contrast, cotransfection of a constitutively active form of PKB (PKB*) strongly increased dexamethasone-induced GR transactivation under serum-free conditions (Fig. 4BGo). Next, we tested whether expression of PKB* could overcome the impaired epinephrine-mediated GR potentiation under wortmannin treatment. In fact, cotransfection of PKB* reversed nearly half of the wortmannin-mediated inhibition of GR enhancement, while SGK and p70S6K were ineffective (Fig. 4CGo). These findings strongly suggest a role for PKB in epinephrine signaling to the GR.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study, we employed a neuronal, hippocampus-derived cell line that expresses adrenergic receptors and GRs to examine the interaction of both classes of stress hormones at the cellular level and to elucidate the underlying signaling pathways. We observed that epinephrine enhances ligand-dependent GR transactivation and raises the efficiency of available glucocorticoid hormone levels. This increase in GR activity correlated with improved DNA binding, while receptor levels and ligand binding affinity remained constant. There was no indication of a switch in coupling from Gs to Gi proteins by ß2-ARs under prolonged epinephrine treatment. Moreover, ß2-AR activation of the GR was independent of PKA but, instead, was relayed by Gß{gamma}-subunits.

Interestingly, we further identified the phosphoinositide-3-phosphate signal transduction cascade as a major determinant downstream of ß2-ARs in HT22 cells. Our findings demonstrate that activation of PI3-K is an important step in GR enhancement. First, wortmannin, as well as the structurally unrelated PI3-K inhibitor LY 294002, blocked increased GR transactivation by epinephrine. Second, expression of PI3-K’s constitutively active catalytic subunit p110* augmented GR activity. Third, epinephrine directly stimulated PI3-K activity in neuronal cells. Our data point to a new mode of cross-talk between G{alpha}s-coupled receptors and PI3-K via Gß{gamma}-subunits in a neuronal cell line. In this view, our findings further exemplify the complexity of cross-talk between GPCRs and downstream kinases (18). Inhibition of GR potentiation by wortmannin could be partially overcome by PKB, which acts downstream of PI3-K. Hence, ß2-adrenergic stimulation of GR in HT22 cells is predominantly transferred by a pathway under the control of PI3-K (Fig. 5Go). In agreement with these findings, growth factor-mediated GR potentiation in HT22 cells could be completely blocked by wortmannin, again establishing the crucial role of PI3-K.



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Figure 5. Model of a New Cross-Talk between ß2-AR and the GR via PI3-K

Glucocorticoids induce a conformational change of the GR and its translocation to the nucleus. There, GR dimers can bind to GREs and stimulate gene transcription. (Nor)epinephrine, in turn, activates G protein-coupled ß2-adrenergic receptors. Subsequently, catecholamines enhance GR transactivation by Gß{gamma} signaling to PI3-K and its downstream target PKB. ß2-AR, ß2-adrenergic receptor; G{alpha}s, ß, {gamma}, GTP-binding protein subunits; GR, glucocorticoid receptor; GRE, glucocorticoid responsive element; PI3-K, phosphoinositide 3-kinase; PKB, protein kinase B (Akt).

 
ß2-AR-dependent signals are transduced mainly, although not exclusively, through PI3-K. Approximately 20% of the epinephrine-mediated enhancement was resistant to wortmannin, and this is mediated by a mechanism distinct from Gß{gamma} and PI3-K (P. Schmidt, F. Holsboer, and D. Spengler, unpublished observation). Elucidation of this additional pathway for GR enhancement will require further study.

Activation by G protein ß{gamma}-subunits has been shown for PI3-K{gamma} in vivo and for PI3-Kß in vitro (11, 23). However, only PI3-Kß is widely expressed in various tissues (21). Our results on PI3-K activation by epinephrine in vivo strongly suggest a role for PI3-Kß in HT22 cells, since PI3-K{gamma} does not immunoprecipitate with the regulatory subunit p85 (21). The additional increase in GR transactivation after costimulation with growth factors may be mediated by another PI3-K isoform recruited to tyrosine-phosphorylated residues, i.e. {alpha} or {delta}. If PI3-Kß mediated both signals, a synergistic rather than additive effect would be expected (23, 24). In agreement with the observation that the regulatory subunit p85 is required for growth factor signaling but does not interfere with Gß{gamma} signaling (24), our results using a dominant negative form of p85 support the possibility of two different mechanisms leading to PI3-K activation. In particular, they demonstrate that epinephrine signaling does not proceed via tyrosine phosphorylation of growth factor receptors in HT22 cells (25).

PI3-K can activate several downstream targets including PKB, PKC, MAPK, SGK, and p70S6K (22). We found that, with respect to these PI3-K targets, only PKB stimulated GR function in HT22 cells. Furthermore, PKB can, in part, restore the wortmannin-inhibited effect of catecholamines on GR function, suggesting an involvement of this kinase in GR enhancement. PKB has been reported to increase GR function in PC12 cells by antagonizing the inhibitory action of glycogen synthase kinase-3 (GSK-3) (26). In HT22 cells, however, coexpression of GSK-3 did not inhibit enhanced GR transactivation by epinephrine. Likewise, expression of a dominant negative form of GSK-3 did not improve GR function (P. Schmidt, F. Holsboer, and D. Spengler, unpublished observations). Therefore, our results gave no evidence for an involvement of GSK-3 in GR transactivation in HT22 cells, but they strongly support a critical role of the PI3-K target PKB in relaying adrenergic signaling to the GR.

Consistent with a number of previous studies, epinephrine was unable to activate the GR in the absence of glucocorticoids. In this respect, the GR seems to behave differently from most other steroid hormone receptors (5, 6). The strict ligand dependency of the epinephrine-induced GR potentiation in our model system distinguishes this regulation from a recent report on ligand-independent activation of the GR by ß2-adrenergic agonists in fibroblasts (27).

Interestingly, the highest enhancement by epinephrine was achieved at low concentrations of glucocorticoids and led to a 5-fold increase of GR transactivation compared with dexamethasone alone. Hence, the strongest potentiation of GR function occurred at glucocorticoid concentrations comparable to in vivo baseline conditions. Furthermore, we detected an equivalent enhancement of GR transactivation by epinephrine in the presence of the natural ligand corticosterone. The fact that the ED50 values observed were somewhat higher than expected from studies based on receptor overexpression may be due to the limited, but physiological, levels of GR expression in HT22 cells. Moreover, in this cell line the effect of a given concentration of glucocorticoids could be nearly doubled by blocking the membrane P-glycoprotein, which extrudes several lipophilic molecules from the cytoplasm, with verapamil (28). This was the case for both cortisol and dexamethasone (28), and we could reproduce this finding for corticosterone (data not shown).

The observed cross-talk between catecholamines and steroids might play a role under various physiological conditions. For instance, enhanced recruitment of the GR under low glucocorticoid levels may facilitate glucocorticoid-dependent memory storage (8). Furthermore, our findings might represent a molecular mechanism for fine tuning the neuronal stress response by (nor)epinephrine (2). Notably, epinephrine reduced the dose of glucocorticoids necessary for half-maximal GR activity by more than 50%, which might also be of relevance for stress-related disorders. Long-term treatment with antidepressants or lithium has been shown to influence GPCR and PI3-K signaling at various levels and might help to restore negative feedback regulation by glucocorticoids in depressed patients (9, 29).

The fact that both catecholamines and polypeptide growth factors enhance GR transactivation seems to indicate redundancy in this process. However, the complexity of these signaling systems suggests that distinct biological responses are achieved based on context-dependent activity profiles and cell type-specific expression patterns. In addition, epinephrine and IGF-I probably act via different isoforms of PI3-K, which may ensure selectivity. Thus, despite overlapping effects on GR transactivation, either stimulus may modulate glucocorticoid signaling in the nervous system in a specific and context-dependent manner. The interaction between growth factors and glucocorticoids may, for instance, play a role in neurodevelopment and neuronal plasticity of the hippocampus (30). Moreover, additional physiological functions in tissues other than the hippocampus might be suggested by our finding of GR enhancement by different G{alpha}s- coupled receptors.

In summary, this study reveals that adrenergic stress hormones enhance glucocorticoid activity in hippocampus-derived cells via the phosphoinositide pathway. Our findings demonstrate a novel cross-talk between GPCRs, PI3-K, and the GR and suggest a critical role for PKB in regulating steroid receptor function. These results imply a model of GR activity in which constant levels of steroid receptor and ligand may bring forth a broad range of activity in neuronal cells. This notion of GPCR-dependent regulation of GR function appears significant in the light of both physiological and pathophysiological processes.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids
cDNAs for p85 and {Delta}p85 (31) have been subcloned as BamHI-EcoRI fragments into the vector pRK7 harboring a cytomegalovirus (CMV) promoter (32). Construction of PKI (33), Gß{gamma} (34), G{gamma}- (10), constitutively active p110* (35), SGK (36, 37), p70S6K (38), PKB (39), constitutively active PKB* (40), and GSK-3 constructs (41) has been described.

Cell Culture and Transient Transfections
HT22 cells were grown in DMEM supplemented with 10% FCS (Life Technologies, Inc., Karlsruhe, Germany). Cells (3 x 106) were transfected with reporter plasmid [1 µg MMTV-Luc, TK-Luc, or TK-GRE2-Luc (42)] and, where indicated, with expression plasmids using an electroporation system (BTX, Inc., San Diego, CA). The ß-galactosidase expression vector pRK7-Gal [0.1 µg (32)] and carrier DNA (pGEM4, Promega Corp., Madison, WI) up to a final amount of 6 µg were included in each experiment. Electroporated cells were replated in DMEM supplemented with 10% charcoal-stripped, steroid-free FCS, except where indicated, and incubated with substances as described in the figure legends. Unless indicated otherwise, concentrations of 10 nM dexamethasone and 30 µM epinephrine were used. After 24 h, cell extracts were assayed for luciferase activity, and results were normalized by ß-galactosidase activity as described previously (42). ICI 118551 and PD 98059 were purchased from Tocris (Ballwin, MO) and IGF-I and bFGF from R&D Systems (Wiesbaden, Germany). Wortmannin, LH, and PACAP-27 were obtained from Calbiochem (Schwalbach, Germany). All other substances were from Sigma (Deisenhofen, Germany). Statistical evaluation was performed using Student’s t test.

Northern Blot Analysis
Cells were treated with dexamethasone (300 nM), epinephrine (30 µM), or both and total RNA was harvested using Trizol Reagent (Life Technologies, Inc.). Blotting was performed with 20 µg RNA by capillary transfer. As a probe, a fragment of the hGR cDNA was used (42). Signals were quantified using a digital image analysis system (BAS reader, Fuji Photo Film Co., Ltd.).

Western Blot Analysis
Total cellular lysates (50 µg) from cells pretreated with vehicle or dexamethasone (10 nM) and epinephrine (30 µM) were subjected to SDS-PAGE and blotted as described (43). Detection of GR was performed with polyclonal rabbit GR primary antibody (PA1–512, Affinity BioReagents, Inc. Golden, CO) and a secondary antibody coupled to horseradish peroxidase (Amersham Pharmacia Biotech, Freiburg, Germany) using enhanced chemiluminescence (Roche Molecular Biochemicals, Mannheim, Germany).

Electrophoretic Mobility Shift Assay
Annealed GRE-oligonucleotides (5'-GGAGCTTAGAACACAGTGTTCTCTAGAG-3' and 5'-GGAGTCCTCTAGAGAACACTGTGTTCTA-3') were labeled with 32P-{alpha}dCTP (Amersham Pharmacia Biotech) using Klenow fragment (New England Biolabs, Inc., Schwalbach, Germany). HT22 cells were homogenized in ice-cold buffer containing 20 mM Tris-HCl pH 7.5, 600 mM KCl, 20% glycerol, and 2 mM dithiothreitol (DTT) and centrifuged for 45 min (100,000x g, 4 C). Samples (20 µl) contained 10 mM HEPES, pH 7.9, 4% Ficoll, 1 mM DTT, 1 µg poly[dIdC], and 1 µl cell extract (10 µg protein). A 150-fold excess of unlabeled competitor GRE or mutated oligonucleotide was included in parallel samples to confirm specificity of the GR-DNA complex. After 10 min at 0 C, 0.1 ng 32P-labeled GRE was added and incubation was continued for 15 min at 25 C. DNA-protein complexes were resolved on a 4% polyacrylamide gel in 0.5x Tris-borate-EDTA (TBE), and autoradiographs were quantified with a digital image analysis system.

PI3-K Assay
HT22 cells grown to 70% confluence were treated for 1 min with epinephrine or vehicle before lysis in the presence of sodium orthovanadate. Precleared lysates were immunoprecipitated with anti-PI3K-p85 (No. 06–195, Upstate Biotechnology, Inc., Lake Placid, NY) according to the manufacturer’s instructions. Immunoprecipitates were resuspended three times in washing buffer (500 mM LiCl, 100 mM Tris-HCl, pH 7.4, 2 mM sodium orthovanadate) and twice in equilibration buffer (100 mM NaCl, 10 mM Tris-HCl, pH 7.4, 1 mM sodium orthovanadate). Equal concentrations of PI3-K as determined by immunoblotting were used for PI3-K assays. Reactions were started by adding 12.5 mM MgCl2 and 5 µM ATP in the presence of 10 µg phosphatidylinositol and 830 nM 32P-{gamma}dATP (3,000 Ci/mmol, Amersham Pharmacia Biotech). Incubation was stopped after 20 min with 1 M HCl and samples were extracted using chloroform-methanol (1:1). Products were separated by TLC (silica gel 60, Merck Eurolab, Ismaning, Germany) in chloroform-methanol-H2O- ammonium hydroxide (43:38:7:5) and dried plates were autoradiographed.


    ACKNOWLEDGMENTS
 
The authors are grateful to M. Greenberg, S. Gutkind, B. Hemmings, M. Kasuga, P. Klein, A. Klippel, F. Lang, M. Lohse, R. Maurer, R. Roth, and G. Thomas for the gift of expression plasmids and to C. Behl for the HT22 cell line. We thank S. Heck, T. Rein, J. Reul, and T. Trapp for valuable discussions and A. Hoffmann for excellent technical assistance. We are indebted to J. Reul and A. Gesing for the receptor binding data.


    FOOTNOTES
 
Address requests for reprints to: Florian Holsboer, Ph.D., M.D., Max Planck Institute of Psychiatry, Kraepelinstrasse 10, D-80804 Munich, Germany. E-mail: holsboer{at}mpipsykl mpg.de.

Received for publication August 14, 2000. Revision received December 1, 2000. Accepted for publication December 5, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. de Kloet ER, Vreugdenhil E, Oitzl MS, Joëls M 1998 Brain corticosteroid receptor balance in health and disease. Endocr Rev 19:269–301[Abstract/Free Full Text]
  2. de Kloet ER 1991 Brain corticosteroid receptor balance and homeostatic control. Front Neuroendocrinol 12:95–164
  3. Beato M, Herrlich P, Schütz G 1995 Steroid hormone receptors: many actors in search of a plot. Cell 83:851–857[Medline]
  4. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]
  5. Weigel NL, Zhang Y 1998 Ligand-independent activation of steroid hormone receptors. J Mol Med 76:469–479[CrossRef][Medline]
  6. Cenni B, Picard D 1999 Ligand-independent activation of steroid receptors: new roles for old players. Trends Endocrinol Metabol 10:41–46[CrossRef][Medline]
  7. McEwen BS, Sapolsky RM 1995 Stress and cognitive function. Curr Opin Neurobiol 5:205–216[CrossRef][Medline]
  8. Roozendaal B, Nguyen BT, Power AE, McGaugh JL 1999 Basolateral amygdala noradrenergic influence enables enhancement of memory consolidation induced by hippocampal glucocorticoid receptor activation. Proc Natl Acad Sci USA 96:11642–11647[Abstract/Free Full Text]
  9. Holsboer F, Barden N 1996 Antidepressants and hypothalamic-pituitary-adrenocortical regulation. Endocr Rev 17:187–205[Medline]
  10. Crespo P, Xu N, Simonds WF, Gutkind JS 1994 Ras-dependent activation of MAP kinase pathway mediated by G-protein ß{gamma} subunits. Nature 369:418–420[CrossRef][Medline]
  11. Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, Wetzker R 1997 Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI3-Kinase {gamma}. Science 275:394–397[Abstract/Free Full Text]
  12. Asanuma M, Ogawa N, Mizukawa K, Haba K, Hirata H, Mori A 1991 Distribution of the ß2-adrenergic receptor messenger RNA in the rat brain by in situ hybridization histochemistry: effects of chronic reserpine treatment. Neurochem Res 16:1253–1256[Medline]
  13. Davis JB, Maher P 1994 Protein kinase C activation inhibits glutamate-induced cytotoxicity in a neuronal cell line. Brain Res 652:169–173[CrossRef][Medline]
  14. Lefkowitz RJ, Caron MG 1988 Adrenergic receptors - models for the study of receptors coupled to guanine-nucleotide regulatory proteins. J Biol Chem 263:4993–4996[Free Full Text]
  15. Shoshan MC, Ljungdahl S, Linder S 1996 H-89 inhibits collagenase induction by phorbol ester through a mechanism that does not involve protein kinase A. Cell Signal 8:191–195[CrossRef][Medline]
  16. Daaka Y, Luttrell LM, Lefkowitz RJ 1997 Switching of the coupling of the ß2-adrenergic receptor to different G proteins by protein kinase A. Nature 390:88–91[CrossRef][Medline]
  17. Clapham DE, Neer EJ 1997 G protein ß{gamma} subunits. Annu Rev Pharmacol Toxicol 37:167–203[CrossRef][Medline]
  18. Luttrell LM, Daaka Y, Lefkowitz RJ 1999 Regulation of tyrosine kinase cascades by G-protein-coupled receptors. Curr Opin Cell Biol 11:177–183[CrossRef][Medline]
  19. Murga C, Laguinge L, Wetzker R, Cuadrado A, Gutkind JS 1998 Activation of Akt/protein kinase B by G protein-coupled receptors. A role for {alpha} and ß{gamma} subunits of heterotrimeric G proteins acting through phosphatidylinositol-3-OH kinase {gamma}. J Biol Chem 273:19080–19085[Abstract/Free Full Text]
  20. Ui M, Okada T, Hazeki K, Hazeki O 1995 Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem Sci 20:303–307[CrossRef][Medline]
  21. Vanhaesebroeck B, Waterfield MD 1999 Signaling by distinct classes of phosphoinositide 3-kinases. Exp Cell Res 253:239–254[CrossRef][Medline]
  22. Chan TO, Rittenhouse SE, Tsichlis PN 1999 AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 68:965–1014[CrossRef][Medline]
  23. Kurosu H, Maehama T, Okada T, Yamamoto T, Hoshino S, Fukui Y, Ui M, Hazeki O, Katada T 1997 Heterodimeric phosphoinositide 3-kinase consisting of p85 and p110ß is synergistically activated by the ß{gamma} subunits of G proteins and phosphotyrosyl peptide. J Biol Chem 272:24252–24256[Abstract/Free Full Text]
  24. Maier U, Babich A, Nürnberg B 1999 Roles of non- catalytic subunits in Gß{gamma}-induced activation of class I phosphoinositide 3-kinase isoforms ß and {gamma}. J Biol Chem 274:29311–29317[Abstract/Free Full Text]
  25. Maudsley S, Pierce KL, Zamah AM, Miller WE, Ahn S, Daaka Y, Lefkowitz RJ, Luttrell LM 2000 The ß2-adrenergic receptor mediates extracellular signal-regulated kinase activation via assembly of a multi-receptor complex with the epidermal growth factor receptor. J Biol Chem 275:9572–9580[Abstract/Free Full Text]
  26. Rogatsky I, Waase CLM, Garabedian MJ 1998 Phosphorylation and inhibition of rat glucocorticoid receptor transcriptional activation by glycogen synthase kinase-3 (GSK-3). Species-specific differences between human and rat glucocorticoid receptor signaling as revealed through GSK-3 phosphorylation. J Biol Chem 273:14315–14321[Abstract/Free Full Text]
  27. Eickelberg O, Roth M, Lörx R, Bruce V, Rüdiger J, Johnson M, Block L-H 1999 Ligand-independent activation of the glucocorticoid receptor by ß2-adrenergic receptor agonists in primary human lung fibroblasts and vascular smooth muscle cells. J Biol Chem 274:1005–1010[Abstract/Free Full Text]
  28. Herr AS, Wochnik GM, Rosenhagen MC, Holsboer F, Rein T 2000 Rifampicin is not an activator of glucocorticoid receptor. Mol Pharmacol 57:732–737[Abstract/Free Full Text]
  29. Chalecka-Franaszek E, Chuang D-M 1999 Lithium activates the serine/threonine kinase Akt-1 and suppresses glutamate-induced inhibition of Akt-1 activity in neurons. Proc Natl Acad Sci USA 96:8745–8750[Abstract/Free Full Text]
  30. MacLennan KM, Smith PF, Darlington CL 1998 Adrenalectomy-induced neuronal degeneration. Prog Neurobiol 54:481–498[CrossRef][Medline]
  31. Kotani K, Yonezawa K, Hara K, Ueda H, Kitamura Y, Sakaue H, Ando A, Chavanieu A, Calas B, Grigorescu F, Nishiyama M, Waterfield MD, Kasuga M 1994 Involvement of phosphoinositide 3-kinase in insulin-induced or IGF-1-induced membrane ruffling. EMBO J 13:2313–2321[Abstract]
  32. Spengler D, Waeber C, Pantaloni C, Holsboer F, Bockaert J, Seeburg PH, Journot L 1993 Differential signal transduction by five splice variants of the PACAP receptor. Nature 365:170–175[CrossRef][Medline]
  33. Day RN, Walder JA, Maurer RA 1989 A protein kinase inhibitor gene reduces both basal and multihormone-stimulated prolactin gene transcription. J Biol Chem 264:431–436[Abstract/Free Full Text]
  34. Quitterer U, Lohse MJ 1999 Crosstalk between G{alpha}i- and G{alpha}q-coupled receptors is mediated by Gß{gamma} exchange. Proc Natl Acad Sci USA 96:10626–10631[Abstract/Free Full Text]
  35. Klippel A, Escobedo M-A, Wachowicz MS, Apell G, Brown TW, Giedlin MA, Kavanaugh WM, Williams LT 1998 Activation of phosphatidylinositol 3-kinase is sufficient for cell cycle entry and promotes cellular changes characteristic of oncogenic transformation. Mol Cell Biol 18:5699–5711[Abstract/Free Full Text]
  36. Park J, Leong MLL, Buse P, Maiyar AC, Firestone GL, Hemmings BA 1999 Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI3-kinase-stimulated signaling pathway. EMBO J 18:3024–3033[Abstract/Free Full Text]
  37. Waldegger S, Barth P, Raber G, Lang F 1997 Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume. Proc Natl Acad Sci USA 94:4440–4445[Abstract/Free Full Text]
  38. Jefferies HBJ, Fumagalli S, Dennis PB, Reinhard C, Pearson RB, Thomas G 1997 Rapamycin suppresses 5'TOP mRNA translation through inhibition of p70S6K. EMBO J 16:3693–3704[Abstract/Free Full Text]
  39. Franke TF, Yang S-I, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN 1995 The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell 81:727–736[Medline]
  40. Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a constitutively active Akt Ser/Thr kinase in 3T3–L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:31372–31378[Abstract/Free Full Text]
  41. Pierce SB, Kimelman D 1995 Regulation of Speman organizer formation by the intracellular kinase XGSK-3. Development 121:755–765[Abstract/Free Full Text]
  42. Rupprecht R, Arriza JL, Spengler D, Reul JMHM, Evans RM, Holsboer F, Damm K 1993 Transactivation and synergistic properties of the mineralocorticoid receptor: relationship to the glucocorticoid receptor. Mol Endocrinol 7:597–603[Abstract]
  43. Spengler D, Villalba M, Hoffmann A, Pantaloni C, Houssami S, Bockaert J, Journot L 1997 Regulation of apopotosis and cell cycle arrest by Zac1, a novel zinc finger protein expressed in the pituitary gland and the brain. EMBO J 16:2814–2825[Abstract/Free Full Text]