A growing body of evidence concerning estrogen effects cannot be explained by the classic model of hormone action, which involves the binding to estrogen receptors (ERs) ␣ and ER␤ and the interaction of the steroid-receptor complex with specific DNA sequences associated with target genes. Using c-fos proto-oncogene expression as an early molecular sensor of estrogen action in ER␣-positive MCF7 and ER-negative SKBR3 breast cancer cells, we have discovered that 17␤-estradiol (E2), and the two major phytoestrogens, genistein and quercetin, stimulate c-fos expression through ER␣ as well as through an ER-independent manner via the G protein-coupled receptor homologue GPR30. The c-fos response is repressed in GPR30-expressing SKBR3 cells transfected with an antisense oligonucleotide against GPR30 and reconstituted in GPR30-deficient MDA-MB 231 and BT-20 breast cancer cells transfected with a GPR30 expression vector. GPR30-dependent activation of ERK1/2 by E2 and phytoestrogens occurs via a G␤␥-associated pertussis toxin-sensitive pathway that requires both Src-related and EGF receptor tyrosine kinase activities. The ability of E2 and phytoestrogens to regulate the expression of growth-related genes such as c-fos even in the absence of ER has interesting implications for understanding breast cancer progression. Estradiol (E2)1 and natural estrogen-like compounds, such as genistein and quercetin, bind to and activate estrogen receptors (ER) ␣ and ␤, which in turn regulate the expression of target genes directly and/or indirectly via protein-protein interactions with other transcription factors (1-7). Although it is often, but not always, straightforward to link the physiological effects of estrogens to the genomic model of the ligand-receptor complex activity, considerable controversy still exists on the ability of E2 to elicit transcriptional responses independently of ER␣ and ER␤.

High consumption of soybean products, such as phytoestrogens, has been hypothesized to contribute to a reduced incidence of prostate cancer in Southeast Asian people, although there have been inconsistent results among studies. Human LNCaP cells, extensively used as a model for androgen-dependent prostate tumor, express the androgen receptor (AR) mutant T877A promiscuously transactivated by estrogens and other ligands, which may further facilitate cancer progression. Here, for the first time to our knowledge, we demonstrate that genistein and quercetin, two phytoestrogens abundantly present in soybeans, activate either the AR mutant T877A in LNCaP or in transfected Chinese hamster ovary cells. This observation is supported by their capability to induce AR accumulation in the nuclear compartment of LNCaP together with mRNA down-regulation of the androgen target genes AR and PAP, and PSA up-regulation. Of interest, at concentrations eliciting transcriptional activity, both genistein and quercetin stimulate LNCaP cell growth, whereas at high levels, they become cytotoxic independently of AR expression, as ascertained in steroid receptor-negative Hela cells. The results of our study provide evidence that phytoestrogens may regulate several signaling processes in LNCaP cells; however, further studies are needed to assess their potential capability to restrain prostate tumor progression.

Xenoestrogens are chemically distinct industrial products potentially able to disrupt the endocrine system by mimicking the action of endogenous steroid hormones. Among such compounds, the ubiquitous environmental contaminants bisphenol A (BPA) and 4-nonylphenol (NPH) may promote adverse effects in humans triggering estrogenic signals in target tissues. Following a research program on human exposure to endocrine disruptors, we found contamination of fresh food by BPA and NPH. More important, these contaminants were found to display estrogen-like activity using as a model system the estrogen-dependent MCF7 breast cancer cells (MCF7wt); its variant named MCF7SH, which is hormone-independent but still ERalpha-positive, and the steroid receptor-negative human cervical carcinoma HeLa cells. In transfection experiments BPA and NPH activated in a direct manner the endogenous ERalpha in MCF7wt and MCF7SH cells, as the antiestrogen hydroxytamoxifen was able to reverse both responses. Moreover, only the hormone-binding domains of ERalpha and ERbeta expressed by chimeric proteins in HeLa cells were sufficient to elicit the transcriptional activity upon BPA and NPH treatments. Transfecting the same cell line with ERalpha mutants, both contaminants triggered an estrogen-like response. These transactivation properties were interestingly supported in MCF7wt cells by the autoregulation of ERalpha which was assessed by RT-PCR for the mRNA evaluation and by immunoblotting and immunocytochemistry for the determination of protein levels. The ability of BPA and NPH to modulate gene expression was further confirmed by the upregulation of an estrogen target gene like pS2. As a biological counterpart, concentrations of xenoestrogens eliciting transcriptional activity were able to stimulate the proliferation of MCF7wt and MCFSH cells. Only NPH at a dose likely too high to be of any physiological relevance induced a severe cytotoxicity in an ERalpha-independent manner as ascertained in HeLa cells. The estrogenic effects of such industrial agents together with an increasing widespread human exposure should be taken into account for the potential influence also on hormone-dependent breast cancer disease.

CREB3 proteins comprise a set of ER-localised bZip transcription factors defined by the presence of a transmembrane domain. They are regulated by inter-compartmental transport, Golgi cleavage and nuclear transport where they promote appropriate transcriptional responses. Although CREB3 proteins play key roles in differentiation, inflammation and metabolism, a general framework relating their defining features to these diverse activities is lacking. We identify unique features of CREB3 organisation including the ATB domain, which we show is essential for transcriptional activity. This domain is absent in all other human bZip factors, but conserved in Drosophila CREBA, which controls secretory pathway genes (SPGs). Furthermore, each of the five human CREB3 factors was capable of activating SPGs in Drosophila, dependent upon the ATB domain. Expression of the CREB3 protein, CREB-H, in 293 cells, upregulated genes involved in secretory capacity, extracellular matrix formation and lipid metabolism and increased secretion of specific cargos. In liver cells, which normally express CREB-H, the active form specifically induced secretion of apolipoproteins, including ApoA-IV, ApoAI, consistent with data implicating CREB-H in metabolic homeostasis. Based on these data and other recent studies, we propose of a general role for the CREB3 family in regulating secretory capacity, with particular relevance to specialised cargos.

MAPK phosphorylation of various substrates is medi-