Opposing effects of cortisol and dehydroepiandrosterone on the expression of the receptor for Activated C Kinase 1: Implications in immunosenescence

Abstract

Aging is associated to a decline in immune functions that are in part related to a defective protein kinase C dependent signal transduction machinery. RACK-1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. We have previously demonstrated, in the elderly, a defective PCKβII (Protein Kinase C βII) translocation related to a decrease in RACK-1 protein expression, which is correlated to the age-associated decline in DHEA (dehydroepiandrosterone) levels. As a consequence of this signal transduction impairment, a significant decrease in immune cells functionality was observed. Furthermore, we could demonstrate that in vivo and in vitro DHEA administration restored RACK-1 level and immune functions, indicating that this hormone behaved as a positive RACK-1 regulator.

We have most recently characterized the human GNB2L1 promoter region, coding for RACK-1 protein. Although no direct DHEA responsive elements were found, a glucocorticoid responsive element (GRE) was identified.

The purpose of this work was to investigate, in the human pro-myelocytic cell line THP-1, whether physiological cortisol concentrations were able to modulate GNB2L1 promoter activity, RACK-1 transcription as well as cytokine production. As DHEA is endowed of anti-glucocorticoid properties in several cellular systems, and as cortisol:DHEA ratio imbalance is relevant in aging, we also investigated their possible interaction at the RACK-1 expression level.

We could demonstrate that cortisol acted in a dose-related manner as a GNB2L1 promoter repressor, reducing RACK-1 mRNA expression and protein level. Probably by interfering with glucocorticoid receptor binding to GRE sequence, prolonged DHEA exposure counteracted cortisol effects, restoring RACK-1 levels and cytokine production, as assessed by LPS-induced TNF-α release.

Introduction

Aging is associated to a decline in immune functions that are in part related to a defective protein kinase C dependent signal transduction machinery, in particular related to reduced expression of the scaffold protein known as the Receptor for Activated C Kinase 1 (RACK-1) (Corsini et al., 1999). RACK-1 is a 36-kDa protein that contains seven WD-domain motifs and is related to G protein β subunits (McCahill et al., 2002). It is able to interact with different proteins in particular with PKCs (Mochly-Rosen et al., 1991), and preferentially with PKCε (Pass et al., 2001, Perry et al., 2004) and PKCβII (Ron et al., 1994), modulating their activity by stabilizing their active conformation (Ron et al., 1995) and promoting their translocation close to their specific substrates in order to activate defined pathways (Goodnight et al., 1995, Nishizuka, 1995). This capability to interact with various proteins suggests that RACK-1 integrates signaling pathways with different physiological functions. The age dependent reduction of RACK-1 expression (Corsini et al., 1999) reflects an impaired PKC signal transduction pathway, that specifically affects its ability to translocate to the physiological anchoring sites. Defective PKCβII translocation due to age-associated RACK-1 decline, was described in different immune cells (Fülöp, 1994, Delpedro et al., 1998), in rat brain (Pascale et al., 1996) and also in skin cells (Corsini et al., 2009). Furthermore we also demonstrated both in vitro and in vivo in rat, as well as in vitro in human leukocytes, that DHEA was able to restore the age-associated decline RACK-1 expression and immune functions (Battaini et al., 1997, Corsini et al., 2005, Corsin et al., 2002). Although a putative DHEA receptor in monocytes (McLachlan et al., 1995), T cells (Meikle et al., 1992) and endothelial cells (Liu and Dillon, 2002) has been described, we cannot establish whether DHEA or its metabolites increases directly RACK-1 transcription through interaction with the androgen receptor (Corsin et al., 2002) or by interacting with other downstream transcription factors. It is noteworthy that a non-genomic nature of the effect of DHEA has been postulated (Simoncini et al., 2003, Liu and Dillon, 2004), and that an indirect DHEA regulation, on the Bcl-2 promoter, was recently demonstrated (Liu et al., 2007).

In the last decade bioinformatics analysis have allowed the identification of several potential transcription factor binding sites, in the human, porcine and mouse GNB2L1 promoter region (Del Vecchio et al., 2009, Chou et al., 1999, Choi et al., 2003). Recently we have identified and characterized the human GNB2L1 promoter region, showing that it contains among others a binding consensus sequence for the glucocorticoid receptor (GRE sequence) (Del Vecchio et al., 2009). This finding is particularly relevant in the context of immunosenescence as one of the major hallmarks of aging is an increase in cortisol/DHEA ratio, mainly due to a decline in DHEA plasma levels (Corsini et al., 2006). Moreover, DHEA has been shown to exert anti-glucocorticoid effects in a number of systems, particularly within the immune system (Blauer et al., 1991, Daynes et al., 1995), with particular impact on immune functions in age related ailments (Butcher et al., 2005).

The purpose of this work was to study the effect of cortisol on RACK-1 expression and to assess in vitro the anti-glucocorticoid effect of DHEA in order to better define RACK-1 regulation. Finally, we turned our attention to measure TNF-α release after cortisol and cortisol/DHEA treatments to explore an example of the functional consequences of this interaction.