SEX HORMONE RECEPTORS: GENOMIC AND NON-GENOMIC ACTIONS IN LIVER CELLS MEDIATING HEPATOCARCINOGENESIS
Estrogen action and the role of ERs in carcinogenesis have been well documented in mammary carcinoma and the studies have revealed the involvement of estrogens in key cellular processes such as apoptosis, cell cycle, proliferation, oxidative stress and inflammation. The progress in understanding the role of estrogen in regulating various cellular events in liver carcinogenesis has been rather slow. However, the research conducted over recent years provides key insights in this direction.
The classical mode of estrogen action is the genomic mechanism in which ERs function as ligand-activated transcription factors. Activated ERs translocate to the nuclei and regulate the expression of specific target genes. These transcriptional regulations are achieved through interaction with estrogen responsive element (ERE) sequences located in the promoter region of the target gene. However, one third of the genes regulated by ERs in humans do not contain ERE-like sequences. ERs can also regulate the transcription of such genes without binding to DNA through protein-protein interactions with other transcription factors, such as AP-1 and Sp-1 in the nuclei. In addition, this transcriptional control at alternate response elements is also facilitated by non-genomic actions of estrogen. The non-genomic functions of estrogen are initiated by membrane-localized ERs and are associated with activation of various signaling pathways especially protein kinases. The functions of many transcription factors are regulated through protein kinase-mediated phosphorylation including CREB, NF-κB and AP-1 and these transcription factors may thus be targets for non-genomic actions of estrogens. This possible convergence of genomic and non-genomic actions at multiple response elements provides an extremely fine degree of control for the regulation of transcription for ERs (Figure 1)[105,110]. In the following section, we discuss the findings of recent in vitro studies highlighting the significance of ER in mediating genomic and non-genomic actions of estrogen in liver cells to modulate the expression of a number of genes involved in cellular processes central to carcinogenesis.
Telomerase activation has been implicated in hepatocarcinogenesis and the expression of human telomerase reverse transcriptase (hTERT) that encodes for the catalytic subunit of the multicomponent enzyme telomerase hTERT is the prerequisite for telomerase activation[111,112]. Several studies indicated that estrogen regulates transactivation of the hTERT gene by direct interaction of activated ER with an imperfect ERE sequence in the hTERT promoter. Estrogen treatment has been shown to up-regulate the expression of hTERT mRNA and protein in three normal human hepatic cell lines (hc-cells, hNheps and WRL-68) expressing ERα to varying degrees. Furthermore, estrogen exposure prevents shortening of telomeres and decreases the number of cells undergoing senescence, indicating that estradiol acts as a positive modulator of the hTERT gene in the liver. However, the mechanism of ER-mediated transactivation of hTERT in the liver is not well understood. In contrast, in HepG2 cells, estrogen modulation of telomerase activity has been found to be regulated post-transcriptionally via the IP3/PKC pathway[114,115]. IP3 production has been shown to be up-regulated by estrogens in HepG2 cells. Furthermore, estradiol-induced IP3/PKC-alpha production is dependent on either ERα or ERβ expression in both HepG2 and Hela cells. It is hypothesized that membrane ER-mediated IP3/PKC-alpha pathway represents an alternative signaling pathway utilized by cells when low ER levels are unable to activate classic ER-mediated genomic mechanisms as in HepG2 cells.
A similar regulatory mechanism has been observed in case of estrogen modulation of expression of cyclin D1 gene in hepatoma cells. Cyclin D1, important for progression of cells through G1 phase of cell cycle, is a well defined target for estrogen action in mammary carcinoma[116,117], although no detectable estrogen responsive element like sequence in the cyclin D1 gene promoter has been reported in these cells. The cyclin D1 mechanism identified in mammary carcinoma cells involves direct interaction of ERα and Sp1 or ERα and Ap-1. Interestingly, Marino et al demonstrated that in HepG2 cells, estrogen-induced activation of cyclin D1 transcription can occur independently of the transcriptional activity of ER. They further showed that the effect of 17-beta estradiol on HepG2 cells is mediated by activation of the MAPK/ERK pathway by membrane-localized ER that increases the expression of cyclin D1 gene through activation of AP-1 transcription factor, suggesting that non-genomic signaling pathways play an the pivotal role in estrogen-mediated regulation of gene expression at multiple response elements.
Besides, modulating the molecules involved in cell cycle control and cell proliferation, estrogen has also been shown to regulate the expression of genes crucial for apoptosis of hepatocytes and dysregulation of apoptosis in hepatic cells is reported to be a significant factor in accelerating hepatocarcinogenesis or tumor progression in HCC. The Bcl-2 family of proteins regulates one of the key steps in the conserved apoptotic pathway. Among the members of this family, Bcl-2 and Bcl-xL act as inhibitors of apoptosis where as Bax and Bak promote apoptosis[121,122]. Ethinyl estradiol is known to increase the levels of Bcl-2 protein in cultured female rat hepatocytes. Estradiol and idoxifene, two selective estrogen receptor modulators, are known to induce the expression of Bcl-2 protein in male rat liver tissues. Omoya et al and Inoue et al also demonstrated that estradiol is able to stimulate the expression of Bcl-2 and Bcl-xL and to suppress Bad expression in oxidative stress-induced early apoptotic rat hepatocytes. Similar findings have been recently documented in response to estradiol treatment of Huh-7 human hepatoma cells describing a dose dependent increase in expression of Bcl-2 and Bcl-xL and a reduction in Bad levels. No change was observed in expression of pro-apoptotic protein Bax. The regulation of Bcl-2 gene expression by estrogen in mammary carcinoma cells has been shown to be mediated indirectly through activation of Sp-1 transcription factor. However, the precise mechanism of Bcl-2 transactivation in hepatocytes has not been clearly understood.
One of the most interesting mechanisms of transcriptional regulation at alternate response elements by estrogen is through inhibition of transcription factor NF-κB. Studies demonstrating a mutually antagonistic cross-talk between these families of transcription factors have been recently reviewed. The ER has been shown to mediate opposition of NF-κB functions at various levels by inhibiting the activation of signaling pathways, preventing nuclear translocation, blocking DNA binding or inhibiting recruitment of co-activators for transcription. Estrogen has been shown to bring about its anti-inflammatory and anti-oxidant effects on liver cells by suppressing the NF-κB activity as evident from the following studies. It was reported that 17 beta-estradiol-bound ERα interferes with cytokine-induced activation of a NF-κB reporter in HepG2 cells, suggesting that estrogen exerts its anti-inflammatory and protective effects on human liver cells. Moreover, in an in vivo model, estrogen treatment has been shown to block the induction of hepatic expression of inflammatory vascular cell adhesion molecule-1 (VCAM-1), tumor necrosis factor-α (TNF-α), and regulate normal T-cell expression and secretion upon activation. In a mouse model of DEN-induced HCC, ERα was suggested to suppress IL-6 production, a pro-inflammatory molecule, through the involvement of the NF-κB pathway. Estrogen has also been reported to suppress oxidative stress-induced reactive oxygen species (ROS) generation, lipid peroxidation, activation of AP-1 and NF-κB as well as loss of Cu-Zn SOD activity in cultured rat hepatocytes[2,9].
In addition to genomic and non-genomic actions of estrogen mediated by nuclear and membrane ER, mitochondria have also recently been identified as important targets of estrogen and ERs. Early binding studies on sub-cellular fractions indicated that ER is present in rat liver mitochondria. Both ERα and ERβ have been reported to be present in the mitochondria of human HepG2 cells[130-132]. The mitochondrial genome has been shown to contain sequences that have partial homology to the estrogen responsive elements[132-134]. Both ERα and ERβ bind to mitochondrial DNA and the binding can be increased by estradiol using mobility shift assays and surface plasmon resonance. These results suggest that estradiol is directly involved in the regulation of mitochondrial DNA transcription (Figure 1). Regulation of apoptosis and oxidative metabolism by estrogens in mitochondria may be important in the normal liver and in the development of HCC.
Ethinyl estradiol treatment has been shown to elevate the expression levels of mitochondrial DNA-encoded cytochrome C oxidase subunit III (CO III) and ATP synthase 6 in vivo as well as in HepG2 cells. This increased expression of mitochondrial transcripts is accompanied by increased mitochondrial superoxide production and respiratory chain activity that require cytochrome P450-mediated biotransformation of ethinyl estradiol and 17-beta estradiol to catechol metabolites[136,137]. In addition to CO III, the levels of CO I and CO II encoded by mitochondrial DNA have also been found to be elevated in ethinyl estradiol treated female rat hepatocytes. This effect is accompanied by increased mitochondrial superoxide production, high ATP levels and increased Bcl2 production, and is suggested to play a role in ethinyl estradiol-mediated inhibition of apoptosis. In contrast, 17-beta estradiol and 17-beta estradiol like compounds, diethylstilbestrol (DES), tamoxifen and genistein, have been found to induce apoptotic effects in human hepatoma Hep3B cell line. These compounds cause the leaking of cytochrome C from mitochondria and activation of caspase-3 in an ER dependent manner. In another study, the two isoforms, ERα and ERβ, showed their opposing actions on apoptosis in a poorly differentiated HCC cell line HA22T. Over-expressed ERβ but not ERα induces the expression of caspase-8 and TNF-α in HA22T cells in response to estradiol treatment, indicating that the death receptor-mediated apoptotic pathway is activated.
Differential roles of ERα and ERβ have also been observed in non-genomic actions of estrogen in the liver[21,23]. There is indirect evidence that membrane ER may exist in human liver as the binding of gold tagged estrogen-BSA conjugate on the surface of clathrin-coated pits in HepG2 cells has been demonstrated by electron microscopic visualization. The non-genomic mechanism of action of sex steroids on the plasma membrane involves the activation of protein kinase cascades (Figure 1). Two major cascades, protein kinase C, and mitogen-activated protein (MAP) kinase are active and important in carcinogenic liver cells. Protein kinase C cascade and its second messenger IP3 are important in cell proliferation and have been discussed in this review in context of transcriptional regulation of hTERT expression by estrogen. The mitogen-activated protein (MAP) kinase cascade is another pathway that is regulated by the action of sex steroids at the plasma membrane. This complex signaling cascade involves three major pathways: ERK, p38, and JNK. In HepG2 cells, estradiol has been found to rapidly increase the phosphorylation of ERK[21,23]. Naringenin, an anti-estrogenic flavonone, induces the activation of p38 in ERα containing HepG2 cells or in ERβ containing human colon adenocarcinoma DLD-1 cells, suggesting that naringenin has an antiestrogenic effect only on the ERα expressing cells, whereas it mimicks the estradiol effects on ERβ expressing cells. The role of ERα and ERβ in the regulation of MAP kinase cascade has been further studied in cell lines expressing either ERα or ERβ. It was found that estrogen-bound ERα can rapidly activate the ERK and AKT signal transduction pathways leading to cell cycle progression and inhibition of apoptosis, whereas estrogen-complexed ERβ can induce rapid phosphorylation of p38 leading the cells to the apoptotic cycle and cell death. These studies further support the functional antagonism between ERα and ERβ with respect to estrogen-induced cell proliferation and emphasize the need to study the independent and interactive role of both isoforms in hepatocarcinogenesis.
In comparison with ER, there is limited information about genomic and non-genomic functions of AR in the liver. Like ER, AR has also been shown to regulate gene transcription by binding to androgen responsive sequences (ARE)[143,144]. Yoon et al demonstrated that androgen can directly regulate the expression of transformation growth factor-beta 1 (TGF-β1) through binding of AR to ARE in TGF-β1 promoter, suggesting that such activation might regulate the progression of HCC in both human and animal models. Furthermore, AR has been shown to interact with a newly identified transcription factor, paternally expressed gene 10 (PEG 10) in hepatoma cell line. PEG 10 has growth promoting properties and is implicated in hepatocarcinogenesis[147,148]. Dihydrotestosterone (DHT) promotes hepatoma formation in nude mice through PEG 10 activation. In addition, DHT treatment is shown to up-regulate hTERT expression in hepatoma cell lines in a PEG-10 dependent manner. These studies indicate that PEG-10-mediated transactivation of target genes by AR has an essential role in hepatocarcinogenesis.
To the best of our knowledge, AR has not been detected in the liver mitochondria. The information about the membrane localization of AR in human liver cells is also lacking. However, AR has been reported to occur in the plasma membranes of male rat liver. Androgens are also involved in the regulation of the MAP kinase signaling pathways as orchiectomy of H-ras 12V transgenic mice decreases phospho-MEK and phospho-ERK in liver tissues. In addition, orchiectomy reduces hepatotumorigenesis in male mice while ovariectomy increases phospho-MEK and phospho-ERK in liver tissue from female mice, but ovariectomy does not affect the incidence of tumorigenesis. Detailed investigations are urgently needed to confirm the existence of non-genomic signaling actions of androgens in liver and the role of AR in mediating these functions.