In vitro modulation of the glucocorticoid receptor by antidepressants

Abstract

Clinical studies have demonstrated an impairment of glucocorticoid receptor (GR)-mediated negative feedback on the hypothalamus–pituitary–adrenal (HPA) axis in patients with major depression (GR resistance), and its resolution by antidepressant treatment. Accordingly, reduced GR function has also been demonstrated in vitro, in peripheral tissues of depressed patients, as shown by reduced sensitivity to the effects of glucocorticoids on immune and metabolic functions. We and others have shown that antidepressants in vitro are able to modulate GR mRNA expression, GR protein level and GR function. This paper reviews the in vitro studies that have examined the effect of antidepressants on GR expression, number and function in human and animal cell lines, and the possible molecular mechanisms underlying these effects. Antidepressants are shown to both increase and decrease GR function in vitro, based on different experimental conditions. Specifically, increased GR function is likely to be mediated by an increased intracellular concentration of glucocorticoids, while decreased GR function seems to be the consequence of GR downregulation. We suggest that the study of the effects of antidepressants on glucocorticoid function might help clarify the therapeutic action of these drugs.

• Corticosterone
• cortisol
• dexamethasone
• major depression
• mineralocorticoid receptor
• multidrug resistance P-glycoprotein

The HPA axis in major depression

The hypothalamus–pituitary–adrenal (HPA) axis is the main hormonal system involved in major depression. The HPA axis is coordinated by neuropeptides produced by neurones in the paraventricular nucleus of the hypothalamus, including the corticotropin releasing hormone (CRH) and vasopressin (Antoni 1993). CRH then stimulates the production of adrenocorticotropin (ACTH) from the anterior pituitary corticotrophs, which in turn stimulates the production of glucocorticoids by the adrenal gland. Glucocorticoids—cortisol in humans and corticosterone in rodents—then interact with their receptors in multiple target tissues. These include the hypothalamus and anterior pituitary, where they are responsible for the negative feedback regulation of the HPA axis through actions on paraventricular nucleus CRH/vasopressin neurones and corticotrophs, respectively. Glucocorticoids also exert their negative feedback actions at other levels in the brain, including the hippocampus, which is an upstream regulator of the HPA axis (Tasker et al. 2006; de Kloet et al. 2007). Glucocorticoids have notable actions on the neural pathways projecting to the CRH neurons from distant limbic-midbrain and cortical brain regions, like the amygdala and prefrontal anterior cingulate cortex (de Kloet et al. 2007). Although glucocorticoids regulate the function of almost every tissue in the body, major physiological effects of these hormones are in the regulation of energy metabolism, through increased gluconeogenesis, increased lipolysis, and increased protein degradation, and in the modulation of immune cell function (Pariante 2004).

The effects of glucocorticoids are mediated by nuclear intracellular receptors, namely the glucocorticoid receptor (GR) and the mineralocorticoid receptor (MR) (de Kloet et al. 1998). These intracellular receptors initiate transcriptional activation or repression by translocating, after ligand-binding, to the nucleus, and exert their effect either by interacting to a glucocorticoid response element (GRE) sequence in the promoter region of different glucocorticoid-regulated genes, or by interacting with other transcription factors (Mangelsdorf et al. 1995; de Kloet et al. 1998; Falkenstein et al. 2000). Changes in receptor number or function can be expected to alter the homeostatic function of the HPA axis.

The GR is expressed in almost all tissues in the body, although tissue and cell cycle-specific regulation of GR levels have been reported (Cidlowski et al. 1990; Oakley et al. 1996; Lu and Cidlowski 2005). In the brain, the GR is widely expressed, and is abundant in hypothalamic CRH neurons; it is also expressed in the anterior pituitary (de Kloet et al. 1998). Apart from binding glucocorticoids, the MR also binds aldosterone and is involved in the regulation of salt appetite and autonomic outflow (beyond the scope of this review). The MR is found in some hypothalamic sites, although the highest MR expression is found outside the hypothalamus, in the hippocampus, where it binds glucocorticoids at low, basal levels (de Kloet et al. 1998).

The MR and GR mediate different actions of glucocorticoids. The MR has a high affinity for endogenous glucocorticoid and for aldosterone, and is considered to be proactive and to regulate basal HPA activity (de Kloet et al. 1998; Juruena et al. 2004). In contrast to the MR, the GR has a high affinity for dexamethasone but a low affinity for endogenous corticosteroids. The GR is therefore considered to be more important in the regulation of the response to stress when endogenous levels of glucocorticoids are high (McEwen et al. 1997; de Kloet et al. 1998).

Classically, the MR and the GR mediate the slow, genomic actions of glucocorticoids; rapid action responses have also been described, and these could possibly include the classical intracellular receptor acting at the membrane through non-genomic signaling mechanisms (Tasker et al. 2006). Indeed, Karst et al. (2005) reported a fast glucocorticoid action critically dependent on the MR, and suggested that a membrane location for the MR could mediate faster glucocorticoid actions when the levels of this hormone are high.

The HPA axis has been found to be hyperactive in many depressed patients and this disturbance is analogous to a sustained stress response in the absence of a stressor. The hyperactivity of the HPA axis in major depression is one of the most consistent findings in psychiatry, occurring in up to 80% of the patients when severely depressed (reviewed in Heuser et al. 1994; Nemeroff 1996; Holsboer 2000; McQuade and Young 2000; Pariante and Miller 2001a, 2003). In brief, the hyperactivity of the HPA axis is characterized by high cortisol levels in the plasma, urine and cerebrospinal fluid (Gold et al. 1988), impairment in the negative feedback regulation of the HPA axis (Carroll et al. 1981; Nemeroff 1996), and hyperplasia of the adrenal and pituitary glands (Axelson et al. 1992; Rubin et al. 2001). Consistent with these findings, increased volume of the pituitary in patients with affective and non-affective psychosis was shown by our group using brain magnetic resonance imaging (Pariante et al. 2004c, 2005). Other HPA axis abnormalities have been found in victims of suicide—many of whom were presumably depressed—including a downregulation of CRH receptors in the frontal cortex (Merali et al. 2004) and increased weight of the adrenal glands (Dumser et al. 1998).

Such disturbance of the HPA axis has been also verified indirectly, using tests designed to measure the integrity of the GR-mediated negative feedback mechanism, and these studies support the notion that GR-mediated negative feedback is impaired in major depression. There is a multitude of studies demonstrating non-suppression of cortisol secretion following administration of the synthetic glucocorticoid, dexamethasone (dexamethasone suppression test, DST), and more recent studies have shown a lack of inhibition of ACTH responses to CRH following dexamethasone pre-treatment (dexamethasone/CRH test) (Nemeroff 1996; Holsboer 2000). While non-suppression by dexamethasone in the DST and the dexamethasone/CRH test likely represent impaired feedback inhibition at the level of the pituitary (de Kloet et al. 1998), impaired responsiveness to hydrocortisone challenge in depressed patients suggests these feedback alterations occur in the brain (Young et al. 1991), although this latter finding has not been always replicated (Cooney and Dinan 1996).

The relevance of DST and the dexamethasone/CRH test comes from the finding that the responses to these tests are not only a biomarker of depression, but also a biomarker of treatment success. Indeed, efficacious antidepressant treatment is associated with resolution of the disturbance in the negative feedback in patients who are non-suppressors before treatment, with up to 75% of non-suppressor patients switching to suppressor status coincident with a treatment response (Linkowski et al. 1987; Heuser et al. 1996).

Resolution of the HPA disturbance is also seen after non-pharmacological antidepressant treatments like electroconvulsive therapy (Yuuki et al. 2005; Kunugi et al. 2006) and transcranial magnetic stimulation (Pridmore 1999; Reid and Pridmore 1999; Zwanzger et al. 2003). Moreover, persistence of non-suppression after antidepressant treatment is associated with high risk of early relapse and a poor outcome after discharge; specifically, in one study 90% of patients who were still non-suppressors after initial resolution of depressive symptoms had an early relapse (Zobel et al. 1999, 2001). Although normalization of the hyperactivity of the HPA axis occurs after successful antidepressant therapy, data in humans and animals (discussed below) suggest that the mechanism by which antidepressants normalize the HPA axis is at least partially independent of changes in monoaminergic neurotransmitter systems—the accepted mechanism of action of antidepressants.

In agreement with the studies mentioned above is the finding that first-degree relatives of depressed patients also show the same impaired GR-mediated negative feedback, which may therefore represent a genetic (trait) vulnerability to depressive disorders (Lauer et al. 1998; Modell et al. 1998). Moreover, a preliminary prospective study conducted in these first-degree relatives has shown that 26% of the subjects develop depression within 4 years (Ising et al. 2005). In addition, the importance of the impaired GR-mediated negative feedback in depression is underlined by genetic investigations which found that polymorphisms of the GR gene and of genes encoding for chaperones of the GR possibly contribute to the development of depressive symptoms and play a role in the variability of antidepressant response (Binder et al. 2004; van Rossum et al. 2006).

It is also of note that clinical studies show that manipulation of GR function has antidepressant effects. Thus, treatment with GR and MR agonists, like dexamethasone, prednisolone and cortisol, has shown their antidepressant action (Dinan et al. 1997; Bouwer et al. 2000; DeBattista et al. 2000). In contrast, GR antagonists, like RU486, have therapeutic effects in the treatment of psychotic depression (Belanoff et al. 2001) and bipolar disorder (Young et al. 2004). These findings could appear to contradict each other. However, the therapeutic effects of GR antagonists could also be explained by the action of these drugs in blocking negative feedback, thus causing a persistent elevation of cortisol levels. The increased cortisol levels, in turn, would increase the cortisol signal in the brain, similar to a treatment with an agonist. Consequently, during treatment with a GR antagonist, the increased cortisol levels could increase MR stimulation since this receptor is not blocked by RU486.

Interestingly, although typical major depression is associated with a hyperactive HPA system and hypersecretion of CRH, the same system may be underactive in atypical depression. It has been speculated that an underactive HPA axis might contribute to the profound fatigue and reversed neurovegetative symptoms that characterize this specific subtype of major depression (Gold and Chrousos 2002; Levitan et al. 2002; Stewart et al. 2005).

Why depressed patients have a hyperactive HPA axis is still a matter of debate. Some authors suggest that it is related to the abnormal GR-mediated feedback found in these patients, and thus to glucocorticoid resistance (Pariante and Miller 2001). This may be due to a reduced GR sensitivity, a reduced GR number, or to both, a view that is supported by reduced GR function in peripheral blood cells (Yehuda et al. 1993) and in the skin (Fitzgerald et al. 2006), and by the lack of cushingoid stigmata (Murphy 1991), in hypercortisolaemic depressed patients. This suggests that GR dysfunction generalizes to tissues outside the HPA axis, but the extent of this dysfunction is not known. Because of this uncertainty, in this review the GR dysfunction will be called partial glucocorticoid resistance.

Evaluation of glucocorticoid receptor function in major depression

Given the limited access to the brain GR in clinical populations, in vitro studies have been used to understand the molecular mechanisms underlying GR abnormalities in patients with major depression. Indeed, data have demonstrated similar regulation of GR in the brain and in the immune system of laboratory animals. For example, Lowy (1990) has shown that treatment of rats with reserpine, the amine depleting drug that is known to induce depressive symptoms in humans and to produce dexamethasone non-suppression in rats, decreases GR levels in the hippocampus, frontal cortex and pituitary as well as in lymphocytes and spleen. Similarly, Spencer et al. (1991) have found that, both in the brain and in the immune system, GR is up-regulated following adrenalectomy and down-regulated following chronic treatment with corticosterone. Therefore, many studies have relied on the measurement of peripheral GR number and function in blood cells, but it is still unknown whether these results reflect the actual values in key areas of the brain associated with clinical depression.

A detailed analysis of these studies has been presented (Pariante et al. 2001b; Pariante 2004, 2006). Briefly, depressed patients have shown some alterations of lymphocyte GR number (Gormley et al. 1985; Whalley et al. 1986). Although most studies have found no evidence of reduced GR number when measured in the whole cell, some studies found reduced GR levels in the cytoplasm, which is likely due to increased sequestration in the nucleus, as discussed previously (Pariante et al. 2001b; Pariante 2004). More consistently, depressed patients have shown reduced GR function when investigated in vitro in immune cells from the peripheral blood. The studies have focused on the well-known capacity of glucocorticoids to inhibit the ability of peripheral blood mononuclear cells to proliferate in response to polyclonal mitogens. Interestingly, in these studies immune cells from depressed patients showed partial glucocorticoid resistance, seen as a reduced dexamethasone-induced inhibition of this proliferative response in vitro (Lowy et al. 1984, 1988; Rupprecht et al. 1991; Wodarz et al. 1991, 1992). The partial glucocorticoid resistance found in lymphocytes correlates with the partial glucocorticoid resistance found with the DST mentioned above. When patients are non-suppressors in the DST, indicating in vivo partial glucocorticoid resistance, they also show reduced dexamethasone-induced inhibition of the lymphocyte proliferative response. In addition, when patients respond positively to treatment and overcome the suppression found on the DST, the sensitivity of lymphocytes to dexamethasone also returns to normal levels (Wodarz et al. 1992).

HPA abnormalities in depressed patients have been shown also by other techniques. A remarkable lack of translocation response of GR was found when measured by cytosolic binding following incubation with dexamethasone (Wassef et al. 1990; Yehuda et al. 2002); probing peripheral cutaneous GR function by the steroid vasoconstriction assay also revealed partial GR resistance (Fitzgerald et al. 2006). Consistently, in these experiments the lack of response is particularly evident in those who are non-suppressors in the DST (Calfa et al. 2003).

Although the glucocorticoid-mediated negative feedback action is mediated by both GR and MR, most of the studies have indeed focused on the GR instead of the MR. In order to evaluate the contributions of both receptors to the HPA disturbance in depression in vivo, we have proposed a suppressive test using prednisolone (PST), a synthetic glucocorticoid that, in contrast to dexamethasone, is more similar to the endogenous glucocorticoid cortisol in its pharmacodynamic and pharmacokinetic features (Pariante et al. 2002b, 2004a) and therefore should probe both receptors. Using the PST, we have shown that depressed patients who present partial glucocorticoid resistance of the GR, still have intact MR function (Juruena et al. 2006). In accordance, the only previous study that specifically examined MR function in depression using an MR antagonist—spironolactone—also found that this pathway is intact, and possibly oversensitive (Young et al. 2003).

The effect of antidepressants on the glucocorticoid receptor

The demonstrated efficacy of antidepressants in modifying the HPA axis when inducing the therapeutic effects suggests that the HPA axis could be a target of antidepressant action. Can antidepressants directly interact with the HPA axis? Can these drugs change the number or the function of the GR or of the MR, in order to overcome this partial glucocorticoid resistance? And if so, what are the molecular mechanisms that could be operative?

Following the initial study by Peiffer et al. (1991), considerable evidence from humans, animals and in vitro work has accumulated demonstrating that antidepressants do interact with the GR and increase GR-mediated negative feedback. Specifically, studies in depressed patients, animals and cellular models, have demonstrated that, depending on the tissue, antidepressants are able to increase GR expression, enhance GR function and promote GR nuclear translocation (Pariante et al. 2001b). This review is focused on the in vitro models as these allow examination of the effects of functional GR responsiveness without the confounding pharmacodynamic or pharmacokinetic effects that are present with either acute or long-term in vivo treatment studies. A summary of the studies discussed here on the effect of antidepressants on GR is shown in Tables I–III, respectively, in human, rat and mouse cells. Like the studies of primary HPA abnormalities in depression, most of the studies on the effects of antidepressants have investigated GR only. The only study that has measured MR in vitro has found no effect of antidepressants (Lai et al. 2003).

Table I.  In vitro effects of antidepressants on the GR and the MR in human cells.

Source	Treatment (concentration in μM)	Duration (days)	Cells	Expression assay	Results	Function assay	Results
Funato et al. (2006)	CMI (1 and 0.1) DMI (1 and 0.1) PAR (1 and 0.1)	Until translocation completed	SY5Y (neuroblastoma)	mRNA GR	→ ↑ ↑ ↑ ↑	Dex 2 TR/ICC TR/ICC TR/ICC	↑ ↑ →
Funato et al. (2006)	CMI (1 and 0.1) DMI (1 and 0.1) PAR (1 and 0.1)	2 and 7 2 and 7 2 and 7	Jurkat cells (lymphoma)	mRNA GR	→ ↑ ↑ ↑ ↑
Cai et al. (2005)	DMI (10) FLU (10) DMI (10) FLU (10)	3 3 3 3	Huh7 (hepatoblastoma) Jurkat cells	mRNA GR mRNA GR WB WB	↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
Heiske et al. (2003)	IMI (0.1) MAP (0.1) MIR (0.1) DMI (0.1)	2.5–48 h 1 1 1	Leukocytes monocytic U-937	mRNA GR	↑ – ↓ ^# → → ↓	TR/ICC	↑
Okuyama-Tamura et al. (2003)	DMI (0.1) FLU (0.1) MNP (0.1) CLR (0.1)	1 and 2 1 and 2 1 and 2 1 and 2	Lymphocytes	Reporter plasmid mRNA GR	↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓	TR/ICC	↑ ↑ ↑ ↑
Vedder et al. (1999)	AMI (0.1–10)	1	Whole blood	mRNA GR	↑ ↑ ↑

AMI indicates, amitriptyline; CIT, citalopram; DMI, desipramine; FLU, fluoxetine; IMI, imipramine; MAP, maprotiline; MIA, mianserin; MNP, minalcipran; MIR, mirtazapin; MOC, moclobemide; PAR, paroxetine; PARG, pargyline; PHE, phenelzine; TIA, tianeptine; TRA, trazodone; RBX, reboxetine; VEN, venlafaxine.

#Time dependent effect, being higher after 2.5 h and lower after 48 h.

Table II.  In vitro effects of antidepressants on the GR and the MR in rat cells.

Source	Treatment (concentration in μM)	Duration	Cells	Expression assay	Results	Function assay	Results
Lai et al. (2003)	AMI (1) FLU (1) AMI (1) FLU (1)	4 4 4 4	HC	GR mRNA GR mRNA WB WB	↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
Hery et al. (2000)	CIT (0.1) PARG (10)	4 4	HC RM	Cyt	↑ ↑ ↓
Okugawa et al. (1999)	DMI (0.1)	2		Cyt, mRNA GR	↑	TR/ICC	↑
		14		mRNA GR	↑ ↑ ↑		→
	AMI (0.1)	2 and 14	HC	mRNA GR	↑ ↑ ↑
	MIA (0.1)	14		mRNA GR	↑ ↑ ↑		↑
	PAR (0.1)	14		mRNA GR	↑ ↑		↑
Pariante et al. (2003a)	CMI (10)	1	Cortex	WB	↓ ↓ ↓
Pepin et al. (1989)	DMI (0.1) AMI (0.1) IMI (0.1) TRA (0.1) MAP (0.1)	2 2 2 2 2	HT, AM, Cortex HT, AM, Cortex HT, AM, Cortex HT, AM, Cortex HT, AM Cortex	mRNA GR	↑ ↑ ↑ ↑ → → ↑ ↑ ↓

AM indicates amygdala neurones; HC, hippocampal neurones; HT, hypothalamic neurones; RM, raphe magnus neurones.

Table III.  In vitro effects of antidepressants on the GR and the MR in mouse cells.

Source	Treatment (concentration in μM)	Duration (days)	Cells	Expression assay	Results	Function assay	Results
Augustyn et al. (2005)	IMI (3, 10 and 30) VEN (30) MNP(3, 10 and 30) RBX (10 and 30) CIT (30) MIR (30)	5 5 5 5 5 5	L929/LMCAT (fibroblasts)			RG^c	↓ ↓ ↓ → ↓ ↓ ↓
Barden (1996)	DMI (1)	1–4	LTK- (fibroblasts) Neuroblastoma	Cyt MRNA	↑ ↑	RG^c	↑
Budziszewska et al. (2000)	IMI (100) AMI (10–100) DMI (10–100) FLU (100) TIA (10–100) MIA (10–100) MOC (100)	5	L929/LMCAT (fibroblasts)			RG^c	↓ ↓ ↓ ↓ ↓ ↓ → ↓ ↓ ↓ → ↓ ↓ ↓ ↓
Miller et al. (2002)	DMI (10)	1	L929/LMCAT (fibroblasts)			RG^b	↑ ↑ ↑
Pariante et al. (1997)	DMI (1–10)	1–3	L929/LMCAT (fibroblasts)	Cyt	↓	TR/ICC TR/WB RG^a RG^b	↑ ↑ ↓ ↓ ↑ ↑ ↑
Pariante et al. (2001a)	AMI (10) FLU (10) PAR (10) CMI (10)	1	L929/LMCAT (fibroblasts)			RG^b	↑ ↑ ↑ ↓ ↑ ↑ ↑ ↑ ↑ ↑
Pariante et al. (2003a)	CMI (10)	1	L929/LMCAT	WB	↓ ↓
Pariante et al. (2003b)	FLU (10)		L929/LMCAT (fibroblasts)			RG^b	↑ ↑ ↑
Pepin et al. (1992a)	DMI (1)	1–2 3–4 1–4 1 2–3 4	LTK (fibroblasts) LTK (fibroblasts) Neuroblastoma Neuroblastoma Neuroblastoma Neuroblastoma	Cyt Cyt Cyt mRNA mRNA mRNA	→ ↑ ↑ ↑ ↑ ↑ → ↑ ↑ ↑	RG^a RG^a	↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ →

Cyt indicates, cytosolic binding; GRmRNA, glucocorticoid receptor messenger RNA; WB, Western blotting. RG, a reporter gene whose regulation is dependent on glucocorticoid responsive elements (see notes for experimental conditions); TR/ICC, translocation of the receptor assessed by immunocytochemistry; TR/WB, translocation of the receptor assessed using Western blotting in the cytosolic and nuclear fractions. Up arrow indicates a significant increase in antidepressant-treated cells vs. controls; down arrow, a significant decrease in antidepressant-treated cells vs. controls; right arrow, no difference between antidepressant-treated cells and controls. One arrow represents 0–30% difference from basal conditions; two arrows, 30–65% and three arrows, >65%.

^a Followed by corticosterone

^b Together with dexamethasone

^c Followed by dexamethasone.

In animals, tricyclic and SSRI antidepressants increase GR mRNA expression in many cells including neuronal cell cultures (Pepin et al. 1989; Okugawa et al. 1999; Hery et al. 2000; Lai et al. 2003) and fibroblasts (Pepin et al. 1992a). Interestingly, increased GR expression has also been confirmed in human peripheral blood mononuclear cells (Vedder et al. 1999; Heiske et al. 2003; Funato et al. 2006). It is of note that the effect of antidepressants on GR mRNA levels can actually be time-dependent as reported by Heiske et al. (2003). These authors found increased GR mRNA levels in human leukocytes after 2.5 h treatment with imipramine, which decreased after longer periods of incubation.

GR protein has also been quantified, either indirectly using cytosolic binding or directly using Western blot techniques. Interestingly, some of the studies that have analyzed GR mRNA expression and quantified GR protein in the same cells have detected a positive correlation of these two measurements (Pepin et al. 1992b; Barden 1996; Lai et al. 2003). Nevertheless, the results using GR protein have been more difficult to interpret, with studies finding both increased (Pepin et al. 1992a; Hery et al. 2000; Lai et al. 2003; Cai et al. 2005), decreased (Pariante et al. 1997; Hery et al. 2000; Pariante 2003a) or unaltered level (Pepin et al. 1992a; Hery et al. 2000). Most studies that have looked at the immediate in vitro effects, that is, within the first 24 h, have found that antidepressants reduce GR protein level (Pariante et al. 1997, 2003a; Heiske et al. 2003; Okuyama-Tamura et al. 2003). This is particularly important in the light of the studies (described below) that have evaluated GR function.

GR function has been investigated using basically two different approaches: translocation of the receptor to the nucleus, or GR-mediated gene transcription. GR translocation has been measured by immunocytochemistry and Western blot; whereas GR-mediated gene transcription has been measured using a mouse fibroblast cell line transiently transfected with the MMTV–chloramphenicol acetyltransferase (MMTV–CAT) reporter gene (LMCAT cells). Expression of the CAT reporter gene by these cells is under glucocorticoid control by virtue of several GRE residing within the MMTV promoter, which lies upstream of the CAT reporter gene (Sanchez et al. 1994).

Using the reporter gene technique above specified, Pepin in their pivotal paper showed that in rat fibroblasts, desipramine enhances GR function, measured as GR-mediated gene transcription after 24 h (while up-regulating GR protein after 72 h), suggesting that antidepressants directly increase GR function before (and in the absence) of GR upregulation (Pepin et al. 1992a). Subsequently, our group showed that 24 h treatment with desipramine in mouse fibroblasts also increases GR function: specifically, it increases GR translocation in the absence of any glucocorticoids (Pariante et al. 1997). Following our work, other groups confirmed that antidepressants increase GR translocation using this and other animal species, including human (Heiske et al. 2003; Okuyama-Tamura et al. 2003; Funato et al. 2006).

Moreover, we found that 24 h coincubation of antidepressants and dexamethasone or cortisol enhances GR-mediated gene transcription compared to cells treated with the glucocorticoids alone (Pariante et al. 1997, 2001a, 2003b). The increased GR-mediated gene transcription is evident in the absence of any GR upregulation, suggesting that the GR upregulation described by other authors after antidepressants is a consequence, rather than a cause, of increased receptor function (Pariante et al. 1997). This is consistent with the study discussed above (Pepin et al. 1992a), where the increased GR function was already present after treatment with desipramine for 1 day, while the increased GR number was only seen after 3 days of desipramine treatment.

It is important to mention that some studies, including from our own group, also found GR function to be reduced after antidepressant treatment (Pariante et al. 1997, 2001a). Mouse fibroblasts cells (LMCAT cells) show reduced GR-mediated gene transcription after antidepressant treatment when followed by (rather than coincubated with) dexamethasone (Pariante et al. 1997), or in the presence of another glucocorticoid hormone, corticosterone (both when coincubated with and when following the antidepressants). This finding has been replicated by other authors using various antidepressants (Augustyn et al. 2005; Budziszewska et al. 2000,2005). Also confirming our results, Miller et al. (2002) have found that incubation with desipramine alone induces a small decrease in unstimulated GR-mediated gene transcription. Therefore, under some experimental conditions, antidepressants decrease rather than increase GR function.

At first, the differential effect of antidepressants on GR function can seem confusing. However, it is of note that the results agree with each other when studies are separated according to the experimental condition. When cells are pre-treated with antidepressants, before incubation with any glucocorticoids, GR function is decreased (Barden 1996; Pariante et al. 1997; Budziszewska et al. 2000, 2005; Augustyn et al. 2005). When cells are coincubated with antidepressants and cortisol or dexamethasone, GR function is increased, while if cells are coincubated with antidepressants and corticosterone, GR function is decreased (Pariante et al. 1997; Miller et al. 2002). The only result that reports the opposite has used a different cell culture model—LTK fibroblasts and neuroblastoma cells (Pepin et al. 1992a).

Interesting, and possibly relevant for the differential effect of antidepressants on glucocorticoid function, is the finding that antidepressants directly inhibit membrane steroid transporters that expel glucocorticoids out of cells. By blocking these transporters, antidepressants increase the intracellular levels of glucocorticoids: an effect that is independent of any neurotransmitter action, and which we have described in both mice fibroblasts and rat cortical neurons (Pariante et al. 2003a,b, 2004b). The inhibiting effects of antidepressants on membrane steroid transporters has been shown, by us and others, for tricyclic antidepressants (Merry et al. 1991; Varga et al. 1996; Szabo et al. 1999; Pariante et al. 2003a) and newer antidepressants including fluoxetine, sertraline, paroxetine, fluvoxamine, reboxetine, citalopram and venlafaxine (Pariante et al. 2003b; Weiss et al. 2003). In support of a role for steroid transporters in the effects of antidepressants on GR function are the findings described above that coincubation of antidepressants and glucocorticoids leads to increased GR function if the glucocorticoids are substrate for the transporters (dexamethasone and cortisol), while it leads to decreased GR function if the glucocorticoid is not a substrate for the transporters (corticosterone) (Pariante et al. 2001a); moreover, we have also shown that if cells are treated with verapamil, an inhibitor of these steroid transporters, then antidepressants decrease GR function even when coincubated in the presence of dexamethasone or cortisol (Pariante et al. 2001a); and finally, antidepressants directly increase the intracellular concentrations of (radioactive) cortisol, but not of (radioactive) corticosterone (Pariante et al. 2003a,b). Moreover, Herr et al. (2003) have also shown that treatment of mouse hippocampal cells with verapamil reduces the effects of antidepressants on GR-mediated gene transcription.

Mechanisms for the effect of antidepressants on the GR: A possible model

Our group has hypothesized a possible explanation for the effect of antidepressants on GR function, as depicted in Figure 1. As discussed above, this model supports a role for the membrane steroid transporters, like the multidrug resistance P-glycoprotein (MDR PGP). Overall, treatment with antidepressants (for 24 h or less) inhibits steroid transporters, induces GR translocation, and reduces GR expression (Pariante et al. 1997, 2001b, 2003a,b). These three effects could be mediated by the same mechanism. For example, blocking of steroid transport can increase the intracellular levels of steroids taken up from the medium, thus leading to translocation of the GR. Data showing that inhibitors of the steroid transporter P-glycoprotein induce partial GR translocation in human ovarian cancer cells support this possibility (Prima et al. 2000); although a recent study found no effect of verapamil on GR translocation in human lymphocytes (Okuyama-Tamura et al. 2003). Translocation of GR, in turn, leads to the reduction in GR expression (Schaaf and Cidlowski 2002). Indeed, GR translocation by both GR agonists and GR antagonists has been associated with GR downregulation (Burnstein et al. 1994). This downregulation takes place over a few hours and is due to a reduction in the protein half-life and an inhibition of GR mRNA synthesis; it is temporary and can be followed by a subsequent upregulation (Schmidt and Meyer 1994). Therefore, it is possible that the inhibition of the transporters precedes (and causes, by inducing GR translocation) the GR downregulation.

Figure 1 Theoretical model of antidepressant differential effects on GR function. We hypothesise that the final in vitro effect of antidepressants on glucocorticoid receptor (GR) function depends on the experimental conditions, and especially on the ability of antidepressants to block membrane steroid transporters like P-glycoprotein (PGP) that expel glucocorticoids from the cells. In conditions that do elicit effect on the PGP (A), antidepressants lead to increased GR function by blocking steroid transporters and therefore increasing steroid level inside the cells; these effects compensate and overcome the effects of the GR downregulation. By contrast, in conditions that do not elicit effects on PGP, antidepressants decrease GR function because the effects on GR downregulation predominate (B).

If cells are treated in experimental conditions that do elicit the effects on the transporter (Figure 1A), as when cells are coincubated for 24 h with antidepressants and a glucocorticoid that is expelled by the transporter, such as dexamethasone or cortisol, enhanced GR-mediated gene transcription is evident (Pariante et al. 1997, 2003a,b, 2001b). This is because the increase in the intracellular levels of the glucocorticoid overcomes, and possibly precedes, the GR downregulation.

However, if cells are treated in experimental conditions that do not elicit the effects on the transporter (Figure 1B), the GR downregulation leads to a reduced GR-mediated gene transcription. This could explain why antidepressants give a reduction in GR-mediated gene transcription when cells are coincubated with corticosterone (Pariante et al. 2001a, 2003b), which is not a substrate of P-glycoprotein, or in the presence of the transporter inhibitor, verapamil (Pariante et al. 2001a, 2003b), or when cells are treated with antidepressants alone (Miller et al., 2002). Finally, pre-incubation of cells with antidepressants inhibits GR-mediated gene transcription induced by a subsequent short treatment (1.5–2 h) with dexamethasone or corticosterone (Pariante et al. 1997). In this case, even if the inhibition of the transporter increases the intracellular levels of the glucocorticoid, as confirmed by our in vitro experiments with radioactive cortisol (Pariante et al. 2003a), this is unable to compensate for the GR downregulation, possibly because of the short incubation, or possibly because the GR downregulation is present before the glucocorticoid is added (in contrast with the coincubation experiments).

Interestingly, this model is supported by our most recent study in mice showing that treatment with desipramine induces GR downregulation in P-glycoprotein knockout mice—and therefore in a condition that is similar to the in vitro experiments that do not elicit the effect on the transporter; on the other hand, desipramine induces GR upregulation in control mice—and therefore in a condition that is similar to the in vitro experiments which do elicit effects on the transporter (Yau et al. 2007). Of course, these animal data, taken together with the clinical data reviewed above, support the notion that the GR downregulation is a transient effect, possibly due to the initial GR activation, and remains evident only under artificial conditions such as in the P-glycoprotein knockout; while the long-term effect of antidepressants under normal conditions is that of increased GR expression and function.

Mechanisms for the effect of antidepressants on the GR: Other possible models

Undoubtedly, other molecular pathways are involved in the molecular mechanisms of the effects of antidepressants on GR, besides the effects on the membrane steroid transporters. There is now considerable evidence that phosphorylation of the GR by cAMP-dependent protein kinase has a relevant role in the regulation of GR function. For example, adenylate cyclase activators, PKA activators and phosphodiesterase type 4 inhibitors—all compounds that increase PKA activity—increase GR function in vitro (Rangarajan et al. 1992; Miller et al. 2002), and β-agonists have been shown to translocate the GR from cytoplasm to nucleus via the cAMP/protein kinase A pathway (Eickelberg et al. 1999). These findings are particularly intriguing in view of the finding that cultured fibroblasts from depressed patients exhibit reduced cAMP-dependent protein kinase activity (Manier et al. 2000). Moreover, recent work on the mechanism of action of antidepressants suggests that cAMP and protein kinase A play an important role as mediators of the psychotropic effects of these agents, possibly leading to increased neurogenesis (Rasenick et al. 1996; Duman et al. 2001). Indeed, the phosphodiesterase type 4 inhibitor rolipram has been shown not only to increase GR function alone but also to potentiate the antidepressant-induced increase of GR function (Miller et al. 2002). Therefore, it is possible that disruption in the cAMP/protein kinase A pathway described in major depression is linked to GR resistance in this disorder, and that antidepressants may overcome these receptor alterations via a direct effect on this pathway.

Antidepressants are also known to influence the activity of other intracellular protein kinases such as protein kinase C and the Ca²⁺ –calmodulin-dependent protein kinase (Silver et al. 1986; Nalepa and Vetulani 1991; Bouron and Chatton 1999; Budziszewska et al. 2000; Augustyn et al. 2005). These kinases seem to be involved in the antidepressant-induced decrease of GR-mediated gene transcription (and therefore possibly in the antidepressant-induced GR downregulation, although this has not been investigated yet). Some authors have shown that the effect of imipramine on the corticosterone mediated gene transcription depends partly on the PLC/PKC pathway (Budziszewska et al. 2000; Augustyn et al. 2005).

Finally, another possible pathway by which GR function is abnormal in depression could be the activation of mediators of the immune response. Pro-inflammatory cytokines, like interleukins 1 and 6, can induce partial glucocorticoid resistance and result in hyperactivity of the HPA axis (Miller et al. 1999; Pariante et al. 1999; Maddock and Pariante 2001). A number of studies have demonstrated that treatment with pro-inflammatory cytokines induces a decrease in GR function as evidenced by decreased sensitivity to the effects of glucocorticoids on functional endpoints and decreased GR affinity for ligand. Moreover, studies performed on peripheral cells and tissues of patients with inflammatory diseases such as asthma, ulcerative colitis, acquired immunodeficiency syndrome and rheumatoid arthritis, especially those showing resistance to the therapeutic effects of glucocorticoids, have also demonstrated reductions in GR function and affinity that are similar to those induced by cytokines (Miller et al. 1999; Pariante et al. 1999; Maddock and Pariante 2001). Indeed, major depression has also been associated with evidence of immune inflammation and increased levels of pro-inflammatory cytokines (Raison and Miller 2003). In turn, treatment with pro-inflammatory cytokines, or treatments that increase the production of pro-inflammatory cytokines like interferon-α for chronic viral hepatitis, can induce depressive symptoms (Pariante et al. 1999, 2002a; Raison and Miller 2003, Maddock et al. 2004, 2005). We have shown that the pro-inflammatory cytokine interleukin-1 directly blocks GR translocation and function in vitro (Pariante et al. 1999), an effect that is virtually opposite to that of antidepressants in the same experimental system (Pariante et al. 1997). Wang et al. (2004) have shown that these effects of interleukin-1 are mediated by stimulating the p38 mitogen-activated protein kinase signal transduction pathway.

It is of note that a few studies have examined the effect of antidepressants on GR function in populations of psychiatric patients. For example, Yehuda et al. (2004, 2006) have found increased GR function in leukocytes of patients with post-traumatic stress disorder, which is reduced by 3 days of in vitro treatment with sertraline. These findings are consistent with an extensive literature supporting the notion that the GR function in these patients is virtually opposite to that in depressed patients, that is, it is hypersensitive (Yehuda 2001; Yehuda et al. 2004, 2006). This study supports once again the effect of antidepressants on the GR and the fundamental ability of these in vitro tests to reproduce the function of GR in the HPA axis tissues.

Conclusions

In conclusion, in vitro use of antidepressants is able to extensively modulate GRs irrespective of their antidepressant structure and putative target molecule. Hence, the study of the in vitro effect of antidepressants and GR are of particular relevance to understand the molecular mechanisms underlying GR abnormalities in depressed patients and its regulation by antidepressant treatment. Moreover, this approach might prove to be successful in finding strategies to maximize therapeutic antidepressants effects.

Acknowledgements

Livia A. Carvalho is currently funded by a grant from the UK Medical Research Council (MRC). Carmine M. Pariante has been funded by the UK MRC since 1999, first as a Clinical Training Fellow, and currently as an MRC Clinician Scientist Fellow. His research is also funded by the NIHR South London and Maudsley NHS Foundation Trust & Institute of Psychiatry Specialist Biomedical Research Centre for Mental Health, the NARSAD, the APIRE, and the British Academy. The authors have no relevant financial interest to disclose.