Nuclear receptors outside the nucleus: extranuclear signalling by steroid receptors

Abstract

Steroid hormone receptors mediate numerous crucial biological processes and are classically thought to function as transcriptional regulators in the nucleus. However, it has been known for more than 50 years that steroids evoke rapid responses in many organs that cannot be explained by gene regulation. Mounting evidence indicates that most steroid receptors in fact exist in extranuclear cellular pools, including at the plasma membrane. This latter pool, when engaged by a steroid ligand, rapidly activates signals that affect various aspects of cellular biology. Research into the mechanisms of signalling instigated by extranuclear steroid receptor pools and how this extranuclear signalling is integrated with responses elicited by nuclear receptor pools provides novel understanding of steroid hormone signalling and its roles in health and disease.

Figure 1. Nuclear steroid signalling

a | Classic steroid sinalling pathway. Steroids enter cells through mechanisms that are still not understood. Some steroid receptors, such as glucocorticoid (GC) and androgen (A) receptors (GR and AR, respectively), are primarily in the cytoplasm as monomers bound to heat shock proteins (HSPs). Others, such as the oestrogen (E) receptor (ER), are located as monomers primarily in the nucleus, although a small percentage may also be bound to HSPs in the cytoplasm (not shown). In the case of GC and A, steroid binding to cytoplasmic receptors triggers release from the HSPs, receptor dimerization, alterations in receptor conformation and nuclear localization. In the case of E, the sex steroid binds to nuclear receptors to promote dimerization and changes in receptor conformation. In all cases, nuclear dimerized receptors then bind to specific steroid-response elements (SREs) and interact with various co-regulators to modulate gene transcription through either repression or activation. b | Tethered steroid signalling. Nuclear steroid receptors can also modulate gene expression without direct DNA binding. In this case, they bind to other transcription factors, such as AP1 or SP1, to either repress or activate transcription.

Figure 2. Events dictating trafficking of oestrogen receptor-α to and from the plasma membrane and its signal transduction

a | Monomeric oestrogen receptor-α (ER α_B) is sequentially bound by heat shock protein 27 (HSP27) molecules and palmitoyl acyltransferases (PATs) that attach palmitate (PA) to Cys447 of the E domain of this steroid receptor. Following palmitoylation, the steroid receptor associates with caveolin-1, which targets palmitoylated receptors to the caveolae of the plasma membrane, where they engage in signalling (see below in part c). Following de-palmitoylation, ERα undergoes internalization into endosomes, which can potentially result in alternative, endosomal signalling, lysosomal degradation of the receptor (and thus termination of signalling) or targeting of the internalized receptors to the Golgi for a subsequent round of palmitoylation. b | The palmitoylation motif is highly conserved in sex steroid receptors, and mutation of any of the residues indicated in pink severely restricts palmitoylation and subsequent trafficking of the receptors to, and their function at, the plasma membrane. c | Steroid receptors at the membrane are part of a large signalling complex, referred to as the signalsome, and mediate various signalling responses. For example, membrane-localized ERα can bind to the steroid hormone oestradiol (E2). Subsequent signal transduction from the membrane occurs through the physical interaction of membrane ERα with G proteins and kinases (such as AKT, Src and PI3K), which induce downstream signalling events (such as calcium flux, the generation of cyclic nucleotides and the activation of phospholipase C (PLC), leading to further signal propagation by various downstream kinases), as well as through the interaction of membrane ERα with linker proteins (including Pro-, Glu- and Leu-rich protein 1 (PELP1) and Src-homology 2 domain-containing protein (SHC)), which enhance signalling by mechanisms that are unclear. Steroid receptors at the membrane can also collaborate with growth factor receptors (GFRs) and have been implicated in GFR transactivation (directly by inducing their phosphorylation (P) or indirectly by promoting the expression and/or activation of metalloproteinases, resulting in the release of GFR ligands from the extracellular matrix; see also FIG. 3c) and thus in signalling from these receptors. In addition, ERα can associate with other membrane-localized sex steroid receptors (such as androgen receptor (AR) and progesterone receptor (PR)) and cooperate in signal transduction on ligand binding.

Figure 3. Membrane-localized steroid receptors can modulate gene expression in the nucleus

a | In adipocytes, following binding of the steroid hormone oestradiol (E2), oestrogen receptor-α (ERα) engages in phosphorylation (P) of Ser (S) and Thr (T) residues in subunits of carbohydrate-responsive element-binding protein (ChREBP) — a key transcription factor regulating the expression of genes involved in lipid biosynthesis. This phosphorylation (occurring through the activation of downstream kinases protein kinase A (PKA) and AMP-activated protein kinase (AMPK)) counteracts the dephosphorylation events (involving protein phosphatase 2 (PP2A)) stimulated by the binding of insulin to the insulin receptor and precludes ChREBP nuclear translocation. This decreases the expression of genes involved in lipogenesis, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC; also known as ACAC). b | When bound to the xenoestrogen diethyl-stilboestrol (DES) in myometrial uterine cells, ERα activates the PI3K-AKT signalling pathway, which results in phosphorylation of an epigenetic modifier, enhancer of Zeste homologue 2 (EZH2), which functions as a histone methyltransferase and introduces repressive trimethylation of histone H3 at Lys27 (H3K27me3). Phosphorylation of EZH2 decreases its histone methyltransferase activity and leads to derepression of genes, including ER-responsive genes. This altered expression of ER-responsive genes has been shown to disrupt uterine tissue morphology and function, increasing susceptibility to tumorigenic transformation. c | Extranuclear and intranuclear steroid signals can also cooperate in regulating gene expression. In prostate cancer cells, androgen (A) binds to membrane-localized androgen receptor (AR). This binding results in downstream signalling that activates metalloproteinases, which then act to release ligands of the membrane epidermal growth factor receptor (EGFR) from the extracellular matrix. This leads to transactivation of EGFR to trigger AKT (not shown) and ERK signalling. Activation of ERK signalling in part requires Src-mediated Tyr (Y) residue phosphorylation of paxillin. When ERK is activated, it regulates Ser phosphorylation of paxillin. Ser phosphorylated paxillin then enters the nucleus and assists in modulating both nuclear AR-mediated transcription and nuclear ERK-mediated transcription, promoting cell proliferation. Membrane AR signalling, in collaboration with nuclear signalling, therefore regulates prostate cancer epithelial cell growth.

Figure 4. Membrane steroid signalling in the regulation of metabolism, organ homeostasis and organogenesis

a | In breast cancer cells, membrane oestrogen receptor-α (ERα) and ERβ respond to the steroid hormone oestradiol (E2) to provide adaptation to glucose availability. When glucose is abundant, membrane ERs, through AKT activation, promote glucose uptake and use for glycolysis (the main source of energy for tumour growth), whereas when glucose levels are low, membrane ERs activate AMP-activated protein kinase (AMPK), which stimulates pyruvate dehydrogenase (PDH) to promote the metabolism of glucose through the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS), thereby enhancing cell viability under nutrient stress. b | In cardiomyocytes (left panel), membrane ERβ signalling upregulates modulatory calcineurin-interacting protein 1 (MCIP1), which binds to and inhibits calcineurin. Consequently, the transcription factor nuclear factor of activated T cells (NFAT) is not efficiently dephosphorylated and cannot translocate to the nucleus in response to pro-hypertrophic peptides angiotensin II (Ang II) and endothelin 1 (ET-1), meaning that it cannot engage in the transcription of hypertrophic genes. Ang II and ET-1 also activate calcium–calmodulin-dependent kinase II (CaMKII) and the protein kinase C (PKC)–PKD signalling axis, which both lead to the phosphorylation (P) of the anti-hypertrophic class II histone deacetylases (HDACs), leading to their nuclear exclusion. Membrane ERβ counteracts this phosphorylation. Similarly, membrane ERβ blocks Ang II- and ET-1-mediated activation of casein kinase 2 (CK2), which phosphorylates the pro-hypertrophic class I HDAC HDAC2, thereby promoting its nuclear localization. As a result, HDAC2 is retained in the cytoplasm and cannot deacetylate and activate pro-hypertrophic gene targets. In cardiac myofibroblasts (right panel), Ang II and ET-1 stimulate transforming growth factor-β (TGFβ) signalling, inducing the fibroblast to myofibroblast transition and the phosphorylation of the SMAD2 and SMAD3 transcription factors that is required for their nuclear translocation and subsequent stimulation of fibrosis-inducing genes. Membrane ERβ signalling inhibits this TGFβ axis, thereby preventing heart fibrosis. c | In endothelial cells, membrane ERα collaborates with membrane liver X receptor-β (LXRβ) and nuclear ERα signalling to stimulate the activity of endothelial cell nitric oxide synthase (eNOS), which leads to the generation of nitric oxide (NO) that then promotes artery relaxation and re-endothelialization of blood vessels, allowing endothelial repair after injury. 27-hydroxycholesterol (27HC) inhibits the actions of both the membrane and the nuclear ERα pools, acting as an endogenous selective ER modulator in this context. d | Androgens (A) enhance the effects of the pituitary hormone follicle-stimulating hormone (FSH) on the development of ovarian follicles. This requires both nuclear (genomic) and extranuclear (non-genomic) androgen receptor (AR) actions. Membrane AR transactivates epidermal growth factor receptor (EGFR), thereby activating ERK signalling and phosphorylation of paxillin (see also FIG. 3c). Through as-yet-unknown mechanisms, this then leads to increased translation of the FSH receptor. ERK activation and paxillin phosphorylation also lead to enhanced nuclear AR-mediated transcription of the microRNA miR-125b, which suppresses the expression of pro-apoptotic genes. This integrated response to androgens promotes follicle survival and growth. JNK, c-JUN N-terminal kinase (also known as MAPK); MEF2, myocyte-enhancing factor 2.