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Possible Genomic Consequence of Nongenomic Action of Glucocorticoids in Neural Cells

Yi-Zhang Chen^1,2,4 and Jian Qiu³

¹ Institute of Neuroscience,
² Department of Neurobiology, and
³ Department of Physiology, Second Military Medical University, Shanghai 200433; and
⁴ Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou 310031, China

	Abstract

 
The nongenomic, rapid effects of glucocorticoid activate multiple intracellular transduction pathways. This review proposes a possible genomic consequence of the nongenomic action of steroids. The genomic actions of hormonal steroids may be twofold: classic genomic and nongenomically induced genomic.

	Introduction

Top Introduction Nongenomic action mediated via... Multiple signal transduction... A new model of... Possible genomic sequel of... References

 
In the 1960s, the intracellular receptors for the steroid hormones were identified and the action of the steroid hormone was attributed to its binding with the receptor, which resulted in the activation of the genome in the cell nucleus as well as changes in the synthesis of new proteins. The genomic theory of steroid hormone action, which has been widely accepted for more than 30 years and is described in most discriminating textbooks as the sole mechanism of steroid hormone action, can satisfactorily account for most metabolic and developmental processes with slow onset and an extended time course. However, the theory met difficulties in explaining the rapid actions of steroid hormones in the body. As a complement to the classic genomic theory, a nongenomic mechanism has been proposed for the rapid action of hormonal steroids and has been widely documented (6, 13, 15, 18). In the present review, the pleiotropic pathways for the intracellular transduction pathways of the rapid action of glucocorticoid (GC) and the possible genomic sequel to the nongenomic action of GC are discussed. If the notion of a genomic sequel to the nongenomic action can be validated by substantial data, the genomic actions of hormonal steroids may be at least twofold: a "classic" genomic action and a nongenomically induced genomic sequel.

	Nongenomic action mediated via a putative membrane receptor as a plausible explanation for the rapid actions of the GC

Top Introduction Nongenomic action mediated via... Multiple signal transduction... A new model of... Possible genomic sequel of... References

 
Early observations indicated the existence of nongenomic actions of GCs. It has been observed that a rapid feedback action of GC on the pituitary occurs within 5 min after an increase of the plasma level of GC, that the rapid changes in firing activity of central neurons occurs after iontophoretic application of steroids, and that GC could rapidly inhibit the ascorbic acid uptake in the nerve terminals of the sectioned pituitary. These observations are hardly compatible with the traditional genomic theory of steroid action (5).

We revisited the issue and first proposed a hypothesis in 1987 on the basis of the facts that 1) GC could rapidly modulate neuronal excitability by hyperpolarizing the neuronal membrane and 2) it acted on the neuron from the external surface of the plasma membrane (3, 9) (Fig. 1). This notion envisages a new mechanism for the rapid action of GC on neurons that is mediated by the putative membrane receptor for the steroid hormone. Because the rapid action does not involve the genome, the term "nongenomic mechanism" was introduced.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. Membrane receptor-mediated effects of glucocorticoid (GC) on celiac ganglion neurons. A: BSA-conjugated cortisol (F-BSA) hyperpolarized a celiac ganglion cell in a dose-dependent manner. B and C: the action of F-BSA can be partially blocked by RU-38486. D: cortisol (F) apparently increased the membrane resistance during its hyperpolarizing effect, but when the membrane potential was manually clamped, it decreased the input resistance. The top and bottom tracings show membrane current and potential, respectively. E: in a spontaneously discharging neuron, F-BSA inhibited the discharge of the neuron while hyperpolarizing it. With the pen recorder, only excitatory postsynaptic potentials (upward deflections) and afterhyperpolarizations of the action potentials (downward deflections) were recorded, while the spikes of the action potential were too rapid to be followed. Figure reproduced from Ref. 4 with permission.

	Multiple signal transduction pathways are involved in the rapid, nongenomic effects of GC

Top Introduction Nongenomic action mediated via... Multiple signal transduction... A new model of... Possible genomic sequel of... References

 
Because the rapid, nongenomic effects of steroid hormones are well documented, their signal transduction pathways were explored. Recent experimental results indicate that they are remarkably pleiotropic.

G protein involvement.
Many observations indicated the possible involvement of G proteins in the rapid action of GC. For example, in normal cultured rat pituitary cells, GCs at a physiological concentration rapidly inhibit cAMP production and prolactin release induced by vasoactive intestinal peptide acting through specific GC receptors; GCs act rapidly in vitro to attenuate second-messenger responses to ACTH secretagogues in the rat. However, the precise mechanism underlying the process remains unknown (5).

Binding studies also supported the involvement of G proteins in GC's rapid actions. It has been found that, in Taricha, [³H]corticosterone ([³H]B) binding in neuronal membranes is negatively modulated by nonhydrolyzable guanine nucleotide analogs, especially GTPS, and that [³H]B-specific binding was enhanced in a concentration-dependent manner by adding Mg²⁺ to the assay buffer. The results are consistent with comparable studies for known G protein-coupled receptors and provide evidence that the putative membrane receptor of B is coupled to G protein (14, 15).

Protein kinase A pathway.
It has been shown that cortisol rapidly reduces prolactin release and cAMP and ⁴⁵Ca²⁺ accumulation in cichlid fish pituitary in vitro; that dexamethasone (100 µM) could potently suppress the forskolin-stimulated cAMP efflux, ACTH secretion, and proopiomelanocortin expression; and that a pertussis toxin-sensitive GTP-binding protein might, at least partly, participate in the effect (1). It was once suggested (5) that the protein kinase A (PKA) involvement is related to pituitary cells, but recent evidence seems to indicate that, in some cell lines, PKA is also involved in the rapid action of GC.

Protein kinase C pathway.
It has become clear that the G protein-PKA pathway may not be the sole route for signal transduction of GC's rapid action. The G protein-protein kinase C (PKC) pathway is also involved. Electrophysiological studies demonstrated that cortisol inhibited Ca²⁺ current in guinea pig hippocampal CA1 neurons via a G protein-coupled activation of protein kinase. In pertussis toxin-treated animals, cortisol inhibition was significantly diminished. Intracellular dialysis with GDPßS or with the pseudosubstrate of PKC (PKCI19-31) and PKC inhibitor bisindolylmaleide significantly diminished the cortisol inhibition of the Ca²⁺ current (7). The specific inhibitor of cAMP-dependent PKA, Rp-cAMPs, had no effect.

We have analyzed the inhibition by GC of intracellular Ca²⁺ concentration increment ([Ca²⁺]_i ) induced by various secretagogues in adrenal chromaffin cells as well as in PC12 cells. In the experiments, cells were loaded with fura 2-AM and the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in single cells was determined by the imaging system. When cultured PC12 cells were treated with acetylcholine, nicotine, bradykinin (BK), or high K⁺, an immediate and prompt increase in [Ca²⁺]_i ([Ca²⁺]_i) was observed. B as well as B-BSA can dose-dependently inhibit the [Ca²⁺]_i (5, 12, 16).

Nicotinic acetylcholine receptors are expressed in PC12 cells, and the inhibition by B of [Ca²⁺]_i induced by nicotine and high K⁺ can be blocked by verapamil and -conotoxin. Nicotine would be expected to raise [Ca²⁺]_i due to Ca²⁺ entry predominantly through voltage-dependent channels and additionally through nicotinic receptor-associated channels. B also significantly inhibited [Ca²⁺]_i induced by BK. The BK receptors can be categorized into two subtypes: BK₁ and BK₂. The BK receptor subtype in PC12 cells is exclusively BK₂, which is coupled to the G_q/11 family. In addition to intracellular Ca²⁺ release, BK can also induce extracellular Ca²⁺ influx through receptor-activated, non-voltage-gated Ca²⁺ channels. To dissociate the intracellular Ca²⁺ release and extracellular Ca²⁺ influx induced by BK, the Ca²⁺-free/Ca²⁺-reintroduction protocol was adopted in the experiment. The result showed that the Ca²⁺ influx induced by BK could be rapidly inhibited by B, but intracellular Ca²⁺ release was not affected (5).

Using [Ca²⁺]_i as an indicator, the involvement of the G protein-PKC pathway in the rapid, nongenomic action of GC in PC12 cells has been analyzed in detail. High K⁺ depolarizes the cell membrane, then opens the voltage-dependent Ca²⁺ channel, causing Ca²⁺ influx into cells. The inhibitory effects of B on the [Ca²⁺]_i induced by high K⁺ in PC12 cells is related to the time of B preincubation. The inhibitory effect started at 3 min of preincubation, reached maximum at 5 min, and was not obvious at 25 min (12). A G protein inhibitor, either pertussis toxin or GDPßS, significantly reduced the inhibitory effect of B and B-BSA. It is interesting to note that phorbol 12-myristate 13-acetate (PMA), a stimulator of PKC, could inhibit [Ca²⁺]_i induced by high K⁺ as well. On the other hand, inhibitors of PKC, chelerythrine chloride (CHE) and bisindolylmaleide, could significantly antagonize the inhibitory effect of GC on [Ca²⁺]_i. This inhibition of nicotine-induced [Ca²⁺]_i by B and B-BSA could also be mimicked by the PKC activator PMA and reversed by PKC inhibitors CHE and Gö-6976 (16). The results strongly suggested that the G protein-PKC system is involved in GC's rapid action in PC12 cells.

Although pharmacological experiments clearly demonstrated the involvement of the G protein-PKC pathway in GC's rapid action, direct measurement of the PKC activity in vitro was desirable. We determined PKC activity in the membrane fractions using the SignaTECT PKC assay system from Promega. The results showed that B could activate PKC activity and the PKC inhibitor CHE could completely block the activation of PKC. The time course showed that the PKC activity was kept at a high level from 5 to 15 min of B's action. The dose-response curve was bell-shaped, with a maximal activation at 10^–9 M at 37C but at 10^–5 M at 25C. This finding explained why the inhibitory effect was most efficient after 5 min preincubation with B before the addition of nicotine and high K⁺. It has also been shown that B-BSA can directly activate the PKC activity (Fig. 2).

View larger version (21K): [in this window] [in a new window]   FIGURE 2. Involvement of protein kinase C (PKC) in GC's inhibition of nicotine (Nic)-induced Ca2+ influx in PC12 cells. A: cells were preincubated with corticosterone (B) at various concentrations at 25C for 5 min and then were stimulated with Nic (100 µM). Intracellular Ca2+ concentration ([Ca2+]i) was determined by fura 2 fluorescence. Data are means ± SE (n = 20 cells/data point) of maximal [Ca2+]i over basal or resting values. The IC50 value for B to block the Nic-induced increase in [Ca2+]i was 0.61 ± 0.19 µM. B: effects of B, B-BSA, phorbol 12-myristate 13-acetate (PMA; a PKC activator), Gö-6976 and chelerythrine (PKC inhibitors), and verapamil and -conotoxin (Ca2+ channel blockers) on the Nic-induced increase in [Ca2+]i. **P < 0.01 vs. the Nic group. C: effect of PKC inhibitor chelerythrine on PKC activity stimulated by B and PMA. Cells were exposed to different drugs (B, 1 nM; PMA, 100 nM) at 37C for 5 min after preincubation with or without chelerythrine (100 µM) for 15 min. Then PKC activity in the membrane fractions was assayed with SignaTECT PKC assay system . Data are means ± SE of 3–5 experiments. **P < 0.01 vs. B group. D: time course of activation of PKC by B. E: dose-dependent activation of PKC by B and B-BSA. ** and ##, P < 0.01 vs. basal. Figure modified from Ref. 16 with permission.

In cerebral cortex synaptosomes and SK-N-SH cells, GDPßS significantly blocked the augmenting effect of B on uptake of glutamate. An activator of PKA, dibutyryl cAMP, had no effect on the uptake of glutamate in synaptosomes, SK-N-SH cells, or BT-325 cells. The results indicated that PKA could not be involved in the action of GC on the uptake of glutamate. On the other hand, the involvement of PKC was uncertain (18).

	A new model of GC's rapid nongenomic action

Top Introduction Nongenomic action mediated via... Multiple signal transduction... A new model of... Possible genomic sequel of... References

 
On the basis of previous studies of the rapid, nongenomic actions of GC and the protein kinase pathways, it is clear that multiple signal transduction pathways are involved in the rapid, nongenomic effects of GC in neural cells, and a new model of GC's rapid nongenomic action has been proposed (Fig. 4A). In the model, GC first acts on the putative membrane receptor and then via the PKA and pertussis toxin-sensitive G protein-PKC pathways to induce a variety of intracellular responses in neurons, glia, and pituitary cells. It should be emphasized that other pathways have been indicated (5) and new pathways might be disclosed. The pleiotropism in steroid's rapid action is still a big challenge for the future.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Models of pleiotropic signaling pathways in rapid, nongenomic action of GC. A: GC might act on putative membrane receptor and reduce [Ca2+]i via the PKC pathway and the protein kinase A (PKA) pathway in different types of cells. The steps that are not well defined are indicated by arrows with dotted lines. R, G protein-coupled GC receptor; G, G protein; PTX, pertussis toxin; VOC, voltage-operated channel; N-AChR, nicotinic ACh receptor; BK, bradykinin. B: a new model of the possible genomic consequence of the rapid, nongenomic action of B in PC12 cells. Arrows with dashed lines represent the steps that are not well defined. Reproduced from Refs. 5 and 10 with permission.

	Possible genomic sequel of nongenomic action of GCs

Top Introduction Nongenomic action mediated via... Multiple signal transduction... A new model of... Possible genomic sequel of... References

However, recent data showed that the activation of mitogen-activated protein kinases (MAPKs) is also involved in the rapid action of GC. Using Western immunoblot and protein kinase activity assays, for the first time we showed that B can induce a rapid activation of extracellular signal-related kinases (ERK)-1/2, p38, and c-Jun NH₂-terminal kinase (JNK) MAPKs in PC12 cells. The dose-response curve was bell shaped, with the maximal activation at 10^–9 M in 15 min (Fig. 3). Activation of ERK1/2, p38, and JNK by B was apparently not mediated by the classic cytosolic steroid receptors, because B-BSA can induce the phosphorylation of these MAPKs but the antagonist of GC nuclear receptor (RU-38486) cannot block the phosphorylation of ERK1/2, p38, or JNK induced by B. Phosphorylation of ERK1/2, p38, or JNK induced by B was not affected by a tyrosine kinase inhibitor (genistein), suggesting that the pathway does not involve the tyrosine kinase activity. On the other hand, PKC activator PMA can activate and PKC inhibitor Gö-6976 can block the activation of ERK1/2, p38, and JNK induced by B. Together, these data clearly demonstrated that B might act via putative membrane receptor and rapidly activate ERK1/2, p38, and JNK through PKC in PC12 cells, which might be mediated via cRaf-1 (10). The results from immunofluorescence staining also revealed that the activated ERK1/2 was translocated from cytoplasm to nucleus in PC12 cells in 15 min (17) (Fig. 3).

View larger version (34K): [in this window] [in a new window]   FIGURE 3. B-induced rapid phosphorylation of mitogen-activated protein kinases (MAPKs) in PC12 cells. A: concentration-dependent stimulation of p38 (top curve) and c-Jun NH2-terminal kinase (JNK; bottom curve) phosphorylation by B. Cells were stimulated with B (10–11–10–7 M), and the phosphorylation of p38 and JNK was determined by immunoblotting. Quantification of the p38 and JNK phosphorylation was determined by densitometric analysis of immunoblot. Data are means ± SE from 3 independent experiments. B: time course for the stimulation of p38 (top curve) and JNK (bottom curve) phosphorylation by B. Data are means ± SE from 3 independent experiments. C: effect of RU-38486 on B- and B-BSA-induced p38 and JNK phosphorylation. Cells were incubated with B (10–9 M), B-BSA (10–7 M), and BSA (10–7 M) for 15 min after pretreatment with GC nuclear receptor antagonist RU-38486 (10–6 M, 30 min) or no pretreatment. The phosphorylation of p38 (a, top) and JNK (b, top) was determined by immunoblot; a, bottom and b, bottom show total p38 and total JNK using phosphoindependent MAPK antibodies. D: effect of genistein on B-stimulated p38 and JNK phosphorylation. Cells were stimulated with B (1 nM) and nerve growth factor (NGF; 100 ng/ml) in the absence or presence of genistein (100 µM) for 15 min, and the phosphorylation of p38 (a, top) and JNK (b, top) was determined by immunoblot. E: effect of Gö-6976 on B-stimulated p38 and JNK phosphorylation. Cells were stimulated with B (10–9 M) in the absence and presence of Gö-6976 (10–7 M) for 15 min. Cells treated with PMA (10–6 M, 15 min) alone could also lead to the phosphorylation of p38 (a, top) and JNK (b, top). F: nuclear translocation of extracellular signal-related kinase (ERK)-1/2 MAPK in response to B, B-BSA, and NGF. Cells were either left untreated (control) or treated with BSA (5 nM), NGF (100 ng/ml), B (1 nM), or B-BSA (100 nM) for 15 min. Cells were fixed and permeabilized, then incubated with primary antibody anti-ERK1/2 (top left), anti-active ERK1/2 MAPK antibody (control and bottom), and then FITC-conjugated antibody. Images were captured using a confocal microscope. Results are representative of 3 independent experiments. A–E are reproduced from Ref. 10 with permission. F is reproduced from Ref. 17 with permission.

	Acknowledgments

 
We thank Dr. Xue-qi Wang and Ai-qun Qi for their technical assistance.

This work was supported by research grants from the National Basic Research Program of China (G 1999054000) and the Natural Science Foundation of China (39330100 and 39840019).

	References

Top Introduction Nongenomic action mediated via... Multiple signal transduction... A new model of... Possible genomic sequel of... References

 

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