S TEROID HORMONE SIGNALING

Steroid hormones regulate many events during development, differentiation, reproduction and behavior. All steroid hormones are derived from cholesterol. In a first step cytochrome P-450 induces hydroxylation and cleavage of a side chain resulting in the formation of pregnolone (reviewed in (Yamamoto, 1985). This step is followed by a variety of different enzymatic steps that lead to the synthesis of all steroid hormones. Steroid hormones can be divided into five groups, the adrenal mineralocorticoids, responsible for salt balance and blood pressure regulation, the glucocorticoids, necessary for the regulation of carbohydrate metabolism, progesterones and estrogens, the female sex hormones important for reproduction and the establishment of secondary sex characteristics, and last, the androgens, the male sex hormones that are mainly produced in testis and regulate male secondary sex characteristics and are essential for fertility (summarized in (Heikkil, 2002;

Yamamoto, 1985)). All steroid hormones act via binding to specific intracellular receptors, which upon hormone binding are able to bind specific DNA sequences, so called hormone responsive elements (HREs), and regulate gene transcription (Evans, 1988).

Steroid hormones are produced by endocrine cells of gonads, the placenta and the adrenal cortex and distributed by the bloodstream throughout the whole body, where, due to their lipophilic character, they can enter cells mainly by diffusing through the plasma membrane. In the classical model of steroid hormone signaling, steroid hormone receptors are mainly localized in the cytoplasm and are bound to chaperone complexes in their inactive state. Upon hormone binding, the receptors are released and can enter the nucleus where they induce the transcription of target genes (Yamamoto, 1985). However, more recently it was shown that steroid hormone signaling is not restricted to the classical function and that their mechanism of action is somewhat more complicated. In addition to the long known classical signaling pathway (genomic pathway) of steroid hormone signaling, other more complex mechanisms, like membrane signaling (non-genomic pathway) and active transport mechanisms can be involved (Falkenstein et al., 2000; Galigniana et al., 2010).

Steroid hormone receptors are characterized by the presence of several domains, a DNA-binding domain with 2 conserved zinc fingers, which allows binding to specific HREs and a ligand binding domain in the C-terminus of the protein essential for hormone binding (Mangelsdorf et al., 1995). Due to its role in breast carcinogenesis

Introduction 18

and female reproduction, the estrogen receptor is one of the most extensively studied steroid hormone receptor.

1.6.1 Estrogen receptors

Estrogen receptors belong to the superfamily of ligand-inducible nuclear receptors.

Two different estrogen receptor variants are described until now, which are referred to as ERα and ERβ and are expressed from distinct genes. However, additional estrogen receptor variants, e.g. GPR-30 or ER_X, are still under debate, and it is speculated that these proteins reflect membrane associated and G-protein coupled estrogen receptor variants (Barnea and Gorski, 1970; Funakoshi et al., 2006; Kuiper et al., 1996; Toran-Allerand et al., 2002; Walter et al., 1985). Following translation, the estrogen receptors form a complex with proteins of the HSP family (Richter and Buchner, 2001). Chaperone interaction stabilizes the estrogen receptor, however this complex dissociates upon ligand binding, resulting in an active but unstable receptor (increased turnover-rate). Upon binding of estradiol, estrogen receptors undergo a conformational change and dimerize, which allows binding to estrogen-responsive elements (EREs), present in the promoters of target genes, and initiation of changes in gene expression. Classical EREs are inverse palindromic sequences with a length of 18bp (Levin, 2001).

Activated estrogen receptors can associate with a variety of different proteins (co-activators and co-repressors) that influence the activity of estrogen receptors, e.g., SRC1 and SRC3 (McKenna and O'Malley, 2000). It was also shown that activated estrogen receptors can interact with other transcription factors, like SP1 (specificity protein1), AP1 (activating protein1) or NFκB, thereby influencing the activity of these or vice versa (Biswas et al., 2005; Bjornstrom and Sjoberg, 2004, 2005). Additionally, the rapid non-genomic action of estrogen receptors, mediated by membrane-bound receptors is well established in the meantime. Estrogen receptors can be membrane associated via lipid anchors, e.g., by palmytoylation or by interaction with G-protein coupled receptors (Acconcia et al., 2005; Micevych and Dominguez, 2009). This membrane initiated estrogen signaling involves numerous protein kinase pathways.

Hence estradiol treatment results in the activation of PI3K, PLC, MAPK, PKC or PKA and the subsequent cellular consequences, including the activation of additional downstream transcription factors (Bjornstrom and Sjoberg, 2005; Levin, 2005).

Estradiol signaling is crucial for development and reproduction. Furthermore, deregulation of estrogen receptor signaling is associated with the progression of cancer. The most prominent hormone-related cancer type is breast cancer. However, additional cancer types have been associated with deregulated estradiol signaling,

Introduction 19

including prostate cancer, lung cancer or cervical cancer. Thus, treatments with so-called SERMs (Selective Estrogen Receptor Modulators), like Tamoxifen or Raloxifen that function as estradiol antagonists, are becoming increasingly important in cancer therapy (Thomas and Gustafsson, 2011).

The effects of estradiol on the brain to elicit reproductive behavior, like mating behavior, are known for decades. However, over the last decade an additional function for estrogen receptor signaling has been depicted in synaptogenesis and the formation of synaptic plasticity. Studies with ovariectomized rats implicated that estradiol influences the learning behavior, as these rats perform worse in learning tasks and show decreased density of dendritic spines in the CA1 stratum radiatum of the hippocampus, impaired recognition memory, and decreased NMDA (N-methyl-D-Aspartate) receptor expression. These impairments can be rescued by estradiol treatment (Cyr et al., 2001; Gould et al., 1990; Luine et al., 2003; Wallace et al., 2006). In addition, it was demonstrated that estradiol enhances long-term-potentiation (LTP) in the rat, one of the hallmarks of synaptic plasticity (Cordoba Montoya and Carrer, 1997). In accordance with these data, age-dependent, natural or surgical menopause results in cognitive impairments from rodents to man and can be at least partially reversed by estrogen replacement therapies. Thus it is suggested that estradiol signaling has neuroprotective effects (Sherwin, 2003). Although both estrogen receptor variants have been shown to participate in these functions, estrogen receptor knockout mouse models indicate that ERβ might be more important for the neurological functions of estradiol signaling than ERα. While ERα knockout mice display a severe reproduction phenotype, ERβ knockout mice are less impaired in reproduction, but show learning deficiency and poor synaptic plasticity (Couse and Korach, 1999; Rissman et al., 2002).

In conclusion, estrogen receptors control a vast number of crucial physiological processes, and deregulation of estrogen receptor signaling is involved in a number of diseases, ranging from cancer to neurological impairments.

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