17β-estradiol stimulates substance P receptor gene expression

Abstract

The actions of substance P (SP), a widely distributed tachykinin neuropeptide, are mediated by the NK1 receptor, a seven trans-membrane spanning domain cell surface receptor coupled to heterotrimeric G-proteins. SP regulates cellular processes in the CNS, placenta and vasculature including permeability, inflammation, mitogenesis and transformation. Examples of sexual dimorphism in tissue distribution and expression of SP and the SP receptor (SPR) in various organ systems (breast, uterus, brain) suggest the SPR may be under hormonal control. Using Northern blot analysis of SPR mRNA levels, we studied the effects of 17β-estradiol (E₂) on SPR gene expression in AR42J (rat pancreatic acinar) cells which constitutively express high levels of SPR. E₂ (100 nM) led to a 2.5-fold increase in SPR mRNA levels (4.7 kb band) which was time- and concentration-dependent. The increase was inhibited by the RNA polymerase inhibitor actinomycin D (5 μg/ml) but not by the translational inhibitor cycloheximide (10 μg/ml). In addition, the antiestrogen tamoxifen (1 μM) blocked the stimulatory effect of E₂ on SPR mRNA. Increased SPR mRNA levels in response to E₂ were linearly related to increased [³H]SP binding to the SPR. This study has implications for understanding molecular mechanisms of hormonal control of receptor gene expression.

Introduction

Tachykinins constitute a group of widely distributed neuropeptides with a wide range of biological activities (Pernow, 1983). The mammalian tachykinins include substance P (SP), substance K (SK) and neuromedin K (NMK). These peptides exert their effects by binding to cell surface receptors which belong to the superfamily of seven trans-membrane spanning domain receptors which couple to heterotrimeric G proteins (Hanley, 1989, Nakanishi et al., 1990, Nakanishi, 1991). Intracellular signalling occurs through hydrolysis of inositol phospholipids, releasing diacylglycerol and inositol trisphosphate. Although crossreactivity exists in receptor binding among the three mammalian tachykinins (SP, SK and NMK) and their respective receptors (NK1, NK2 and NK3), SP binds preferentially to its NK1 receptor, the substance P receptor (SPR). The SPR has been cloned and sequenced in various species (Masu et al., 1987, Yokota et al., 1989, Hershey and Krause, 1990, Takahashi et al., 1992). Binding of SP to its receptor has been associated with a number of functionally diverse sequelae including mediation of cellular growth, wound healing, permeability and inflammation. Although much is known about the structure and function of the SPR (Krause et al., 1992), relatively little is known about regulation of gene expression for this receptor.

Tachykinins and their receptors are differentially expressed in the nervous system and peripheral tissues. Gender differences in tissue distribution, localization and regulation of SP have also been recently identified (Brown et al., 1990, Brown et al., 1991a, Brown et al., 1991b, Rance, 1992, Akesson, 1994). These have been largely limited to the brain and reproductive tissues. In these systems, SP has been shown to be differentially regulated by gonadal steroids (Kerdelhué et al., 1985, O'Halloran et al., 1990, Kerdelhué et al., 1993, Sarajari et al., 1995). Similarly, sex differences in pre-protachykinin (SP-encoding precursor) gene expression have been demonstrated to be hormonaly regulated (Jonassen and Leeman, 1991). These studies are consistent with sexual dimorphism in SP expression and may predict parallel sexual dimorphism, arising from direct regulation of the SPR. However, regulation of SPR gene expression by sex steroids has not been reported.

Sex steroids may be important regulators of gene expression. Indeed, receptor regulation by estrogen has been described previously for nuclear hormone receptors, such as the progesterone receptor (Horwitz and McGuire, 1978, Olster and Blaustein, 1992) and cell surface receptors, such as the prostaglandin (Rage et al., 1995) and oxytocin receptors (Ostrowski et al., 1995). This study was undertaken to study the possible regulation of gene expression of the SPR by 17β-estradiol (E₂), the most abundant and physiologically important estrogen. We report here that E₂ rapidly stimulated increased expression of the SPR gene in the rat pancreatic acinar cell line (AR42J), leading to increased specific binding of SP.