Af1

DNA-binding domain Dimerization

Ligand-binding domain AF-2 Dimerization

Activation of the promoter of the target gene

Growth factor receptoi

Activation of the promoter of the target gene

Growth factor receptoi

Activation of the promoter of the target gene

(D) 17-b-Estradiol

: Estrogen receptors.

(D) 17-b-Estradiol

Activation of the promoter of the target gene

Figure 14.1A: The Structure of the Estrogen Receptor. The estrogen receptor is composed of several domains, including A/B domain (located near the amino terminus), C domain (the DNA binding domain, D (the hinge region), and E/F domain (the ligand-binding domain near the COOH terminus. Activation Factors are depicted as AF-1 and AF-2. Figure 14.1B: The ligand-dependent

Continues

: Estrogen receptors.

Activation of the promoter of the target gene

Figure 14.1A: The Structure of the Estrogen Receptor. The estrogen receptor is composed of several domains, including A/B domain (located near the amino terminus), C domain (the DNA binding domain, D (the hinge region), and E/F domain (the ligand-binding domain near the COOH terminus. Activation Factors are depicted as AF-1 and AF-2. Figure 14.1B: The ligand-dependent

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Lymphoid and nonlymphoid cells in both mice and humans possess ERs (Ansar Ahmed, 2000; Weihua et al., 2003). ERa is expressed on nearly all lymphocyte subsets from the thymus and thymic stromal cells. The double-positive CD4+CD8+ thymocytes and thymic stromal cells express ERp (Mor et al., 2001). Studies in many laboratories are under way to identify the type of ERs in distinct cells of the immune system. ERa is obligatory for functional development of the thymus, since ERa-deficient mice have hypoplasia of the thymus and T cell developmental impediments such as a decrease in numbers of mature CD4+CD8-thymocytes (Erlandsson et al., 2003). The expression of ERp is essential for estrogen-mediated thymic cortex atrophy. In SLE patients, monocytes, T cells, and B cells express transcripts of ER (Suenaga et al., 2001). Reverse transcription and polymerase chain amplification indicate that ERa and ERp mRNA are expressed in human T cells (Rider and Abdou, 2001).

ERp and ERa are essential for regulating B cell responses, splenic size, and cytokine responses (Erlandsson et al., 2003). Much of the data supporting this are derived from mice lacking ERa and/or ERp genes. ERa-deficient mice have decreased splenic size, while ERp-deficient mice exhibit splenomegaly, thus suggesting that ERs are important in maintaining normal spleen size (Erlandsson et al., 2003). Complete B lymphopoesis in the bone marrow and spleen was only observed in wild-type mice with intact ERa and ERp, when compared to ERa and ERp-deficient mice given estrogen. The ERa-deficient mice given estrogen had fewer B cell subpopulations in the bone marrow, whereas wild-type mice given estrogen had decreased early hematopoietic B cell progenitors and a shift toward a mature B cell subpopulation. ERa is mainly responsible for regulating estrogen-induced B cell changes and immunoglobulin secretion. The loss of ERp resulted in hyperplasia in the bone marrow, increased B cell numbers in the blood, and lymphadenopathy (Shim et al., 2003). The expression of ERs is also important for T helper cell activities. ERa, but not ERp, is essential for enhanced estrogen-induced Th1 cell responses such as increased IFNy secretion (Maret et al., 2003).

Recent investigations have shown that estrogen-responsive genes can be activated by a variety of mechanisms (Nilsson and Gustafsson, 2002), which are depicted in Figure 14.1B4-2. First, in the classical pathway (Figure 14.1), the binding of the ER to estrogen results in a conformational change involving dimerization

Figure 14.1. Continued Classical Estrogen Receptor Pathway. The binding of estrogen receptor to estrogen results in a conformational change in the receptor. This estrogen-estrogen receptor complex is then translocated to the nucleus. This complex will specifically bind to the estrogen response elements (ERE) in the promoter of target genes and affect their transcription. Figure 14.1C: Ligand-Independent Pathway. Estrogen receptors can be activated even in the absence of their usual ligand, estrogen. For example, estrogen receptors can be phosphorylated and activated following signaling mediated by growth factors, Epidermal Growth Factor (EGF) and other molecules. The activated estrogen receptor can bind to ERE to affect the transcription of target genes. Figure 14.1D: Ligand-dependent, but Estrogen Receptor Element (ERE)-independent Pathway. Estrogen receptors can "cross-talk" with other transcriptional molecules. For example, estrogen-estrogen receptor complex can bind to Jun and Fos, that are in turn bound to AP-1 site. The coactivators recruited by Jun/Fos bind to the estrogen receptors and affect the transcription of genes.

and release of the ER from heat shock proteins. This conformational change of the ER involves exposing a hydrophobic surface in the AF-2 region of the ERs, allowing the coactivator proteins to bind. Transcription of the target gene is mediated by binding of the DNA-binding domain of the ER to an estrogen response element (ERE) that is present in the promoter of estrogen-responsive genes. Once bound to an ERE, the ER dimer interacts with coactivators to promote chromatin remodeling and recruits other transcription factors. This results in the upregula-tion or downregulation of target gene expression. Interestingly, both ERa and ERp can exist intracellularly as homodimers or heterodimers (Levin, 2002). The different transcriptional activities of homodimers or heterodimers formed by ERa and ERp may explain the selective actions of estrogen on different cell types and genes (Katzenellenbogen, 1996). Although these ERs are both expressed in tissues and form functional heterodimers, when coexpressed, ERp inhibits the tran-scriptional activity of ERa at saturating hormone levels (Matthews and Gustafsson, 2003). Thus, overall estrogen responsiveness may be determined by the ERa versus ERp ratio in cells where both receptors are expressed.

Second, in a ligand-independent pathway, ERs can be activated in the absence of their usual ligand, estrogen (Figure 14.1C). ERs can be activated by many nonestrogenic physiological molecules, such as growth factors (EGF, IGF-1), cell cycle proteins, and protein kinases. EGF-mediated signaling involving the MAP kinase pathway can activate ERs by phosphorylation. The activated ER can then bind to EREs to affect gene transcription (Bunone et al, 1996). ERs can also be phosphorylated by cyclins (Rogatsky et al., 1999), general regulators such as protein kinase C (PKC) (Lahooti et al., 1998), or protein kinase A (PKA) (Bunone et al., 1996).

Third, in the ERE-independent pathway, ERs can "cross-talk" with other transcription molecules (Figure 14.1D). For example, estrogen-activated ER complexes can physically interact with key transcription molecules including fos-jun complexes, NF-kB, and GC box-bound SP-1 to modulate the transcription of target genes (Kushner et al., 2000). Both ERa and ERp when complexed with estrogen bind to Jun and Fos located on the AP-1 site. The coactivator (p160) recruited by Jun/Fos binds to the ERs and triggers increased transcription of the target gene. In addition, the interaction between ERa and c-Rel prevents NF-kB from binding to the IL-6 promoter and inhibits protein expression of IL-6 (Ray et al., 1997). ERa also interacts with the transcription factor Sp-1, regardless of the presence or absence of estrogen (Porter et al., 1997).

Fourth, in the nongenomic pathway, also called the cell surface ER signaling pathway, estrogen can induce rapid signaling by binding to ERs on the cell surface that reside in cell membrane domains named caveolae (Levin, 2002). Estrogen treatment stimulates the protein synthesis of caveolae structural coat protein, caveolin-1, which facilitates ER translocation to the cell membrane (Razandi et al., 2002). The estrogen-ER complex in the caveolae may participate in signaling by activating G proteins. The activation of G proteins leads to rapid and specific signaling through activation of phosphoinositol 3-kinase/Akt (PI3K) and MAPK pathways resulting in rapid gene transcription and biological effects implying that this pathway does not involve ER-dependent genomic alterations

(Pedram et al., 2002). Estrogen-ER signaling can also occur via other signaling molecules such as p21ras (Migliaccio et al, 1996), B-Raf (Singh et al., 1999), and Src (Migliaccio et al., 2000). The existence of this pathway in cells of the immune system has also been demonstrated (Guo et al., 2002).

3.2. Estrogen Alterations of B cells

One important mechanism by which estrogen promotes lupus-like features in mice may be via inducing alterations in B cell differentiation and maturation. We have shown that estrogen treatment of non-autoimmune C57BL/6 mice enhanced B cell differentiation into plasma cells that secreted autoantibodies against dsDNA and phospholipids (Ansar Ahmed et al., 1999; Ansar Ahmed, 2000). Further, in a lupus model (BALB/c mice transgenic for the heavy-chain of a pathogenic anti-DNA antibody), estrogen decreased immature transitional B cells and increased mature anti-dsDNA autoantibody-secreting marginal zone (MZ) B cells, implicating this B cell subset in autoimmunity (Grimaldi et al., 2001).

Defective apoptosis is well known to be involved in autoimmunity. In BALB/c mice transgenic for the heavy-chain of a pathogenic anti-DNA antibody, the expression of the anti-apoptotic protein, Bcl-2, in splenic B cells of estrogen-treated mice was increased (Bynoe et al., 2000). Further, estrogen treatment protected isolated primary B cells from B cell receptor-mediated apoptosis. Estrogen upregulated several genes that are involved in B cell activation and survival, including cd22, shp-1, bcl-2, and vcam-1. These effects of estrogen are mediated through the ERs (Grimaldi et al., 2002). In our model of estrogen treatment of non-autoimmune mice, we also noticed that splenic B cells demonstrated a remarkable ability to survive in culture. This survival of splenic B cells from estrogen-treated mice is even more pronounced with anti-CD40 antibody treatment with concomitant upregulation of antiapoptotic proteins, Bcl-2 and Bcl-xL. Thus, estrogen can alter B cell development, leading to the survival, expansion, and activation of a population of autoreactive B cells (Ansar Ahmed et al., 1999; Grimaldi et al., 2001). In humans, direct exposure of PBMC from SLE patients to 17P-estradiol resulted in decreased apoptosis of blood monocytes and decreased secretion of TNFa, which may allow survival of autoreactive cells in SLE patients (Evans et al., 1997).

It is possible that estrogens can regulate the levels of B cell activation factor (BAFF), an aspect that is currently being examined in our laboratory. This factor is crucial for the survival of MZ and transitional-2 B cells and is potentially implicated in a number of autoimmune diseases (Mackay and Kalled, 2002).

3.3. Estrogen Effects on Cytokines

The immune system uses cytokines as molecular messengers to coordinate the functioning of diverse cells of lymphoid and nonlymphoid organs. It is therefore not surprising that in an immune-dysregulated state such as autoimmune disease, significant alterations in the profile and levels of cytokines or response to these cytokines are evident. One mechanism by which estrogens influence autoimmunity is by regulating the secretion of cytokines and signaling responses to these cytokines. 17P-estradiol affects several cytokines and chemokines including IFNy, IL-1a, IL-4, IL-6, TNFa, IL-10, and IL-12 (Deshpande et al, 1997; Carruba et al., 2003). It is beyond the scope of this chapter to discuss at length the various cytokines that sex hormones regulate. Instead, we focus here on IFNy, a proinflammatory cytokine that plays an important role in autoimmune diseases, including lupus, and is highly responsive to estrogens.

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