Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.

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␤.

Estrogens play a crucial role in the development of ovarian tumors; however, the signal transduction pathways involved in hormone action are still poorly defined. The orphan G protein-coupled receptor 30 (GPR30) mediates the nongenomic signaling of 17B-estradiol (E2) in a variety of estrogen-sensitive cancer cells through activation of the epidermal growth factor receptor (EGFR) pathway. Whether estrogen receptor A (ERA) also contributes to GPR30/ EGFR signaling is less understood. Here, we show that, in ERA-positive BG-1 ovarian cancer cells, both E2 and the GPR30-selective ligand G-1 induced c-fos expression and estrogen-responsive element (ERE)-independent activity of a c-fos reporter gene, whereas only E2 stimulated an EREresponsive reporter gene, indicating that GPR30 signaling does not activate ERA-mediated transcription. Similarly, both ligands up-regulated cyclin D1, cyclin E, and cyclin A, whereas only E2 enhanced progesterone receptor expression. Moreover, both GPR30 and ERA expression are required for c-fos stimulation and extracellular signal-regulated kinase (ERK) activation in response to either E2 or G-1. Inhibition of the EGFR transduction pathway inhibited c-fos stimulation and ERK activation by either ligand, suggesting that in ovarian cancer cells GPR30/EGFR signaling relays on ERA expression. Interestingly, we show that both GPR30 and ERA expression along with active EGFR signaling are required for E2-stimulated and G-1-stimulated proliferation of ovarian cancer cells. Because G-1 was able to induce both c-fos expression and proliferation in the ERA-negative/GPR30-positive SKBR3 breast cancer cells, the requirement for ERA expression in GPR30/EGFR signaling may depend on the specific cellular context of different tumor types.

The proto-oncogene-encoded transcription factor c-Jun activates genes in response to a number of inducers that act through mitogen-activated protein kinase (MAPK) signal transduction pathways. The activation of c-Jun after phosphorylation by MAPK is accompanied by a reduction in c-Jun ubiquitination and consequent stabilization of the protein. These results illustrate the relevance of regulated protein degradation in the signal-dependent control of gene expression.

The growth of both normal and transformed epithelial cells of the female reproductive system is stimulated by estrogens, mainly through the activation of estrogen receptor alpha (ERalpha), which is a ligand-regulated transcription factor. The selective ER modulator tamoxifen (TAM) has been widely used as an ER antagonist in breast tumor; however, long-term treatment is associated with an increased risk of endometrial cancer. To provide new insights into the potential mechanisms involved in the agonistic activity exerted by TAM in the uterus, we evaluated the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, to transactivate wild-type ERalpha and its splice variant expressed in Ishikawa and HEC1A endometrial tumor cells, respectively. OHT was able to antagonize only the activation of ERalpha by 17beta-estradiol (E2) in Ishikawa cells, whereas it up-regulated c-fos expression in a rapid manner similar to E2 and independently of ERalpha in both cell lines. This stimulation occurred through the G protein-coupled receptor named GPR30 and required Src-related and epidermal growth factor receptor tyrosine kinase activities, along with the activation of both ERK1/2 and phosphatidylinositol 3-kinase/AKT pathways. Most importantly, OHT, like E2, stimulated the proliferation of Ishikawa as well as HEC1A cells. Transfecting a GPR30 antisense expression vector in both endometrial cancer cell lines, OHT was no longer able to induce growth effects, whereas the proliferative response to E2 was completely abrogated only in HEC1A cells. Furthermore, in the presence of the inhibitors of MAPK and phosphatidylinositol 3-kinase pathways, PD 98059 and wortmannin, respectively, E2 and OHT did not elicit growth stimulation. Our data demonstrate a new mode of action of E2 and OHT in endometrial cancer cells, contributing to a better understanding of the molecular mechanisms involved in their uterine agonistic activity.

The higher incidence of thyroid carcinoma (TC) in women during reproductive years compared with men and the increased risk associated with the therapeutic use of estrogens have suggested a pathogenetic role exerted by these steroids in the development of TC. In the present study, we evaluated the potential of 17␤-estradiol (E2), genistein (G), and 4-hydroxytamoxifen (OHT) to regulate the expression of diverse estrogen target genes and the proliferation of human WRO, FRO, and ARO thyroid carcinoma cells, which were used as a model system. We have ascertained that ARO cells are devoid of estrogen receptors (ERs), whereas both WRO and FRO cells express a single variant of ER␣ that was neither transactivated, modulated, nor translocated into the nucleus upon treatment with ligands. However, E2, G, and OHT were able either to induce the transcriptional activity of c-fos promoter constructs, including those lacking the estrogen-responsive elements, or to increase c-fos, cyclin A, and D1 expression. It is noteworthy that we have demonstrated that the G protein-coupled receptor 30 (GPR30) and the mitogen-activated protein kinase (MAPK) pathway mediate both the up-regulation of c-fos and the growth response to E2, G, and OHT in TC cells studied, because these stimulatory effects were prevented by silencing GPR30 and using the MEK inhibitor 2Ј-amino-3Ј-methoxyflavone (PD 98059). Our findings provide new insight into the molecular mechanisms through which estrogens may induce the progression of TC.

Tandem affinity purification (TAP) and mass spectrometric peptide sequencing showed that the DEAD-box RNA helicase RHII/Gu is a functional interaction partner of c-Jun in human cells. The N-terminal transcription activation region of, c-Jun interacts with a C-terminal domain of RHII/Gu. This interaction is stimulated by anisomycin treatment in a manner that is concurrent with, but independent of, c-Jun phosphorylation. A possible explanation for this effect is provided by the observation that RHII/Gu translocates from nucleolus to nucleoplasm upon anisomycin or UV treatment or when JNK signaling is activated by overexpression of a constitutively active form of MEKK1 kinase. Several experiments show that the RNA helicase activity of RHII/Gu supports c-Jun-mediated target gene activation: dominant-negative forms of RHII/Gu, as well as a neutralizing antibody against the enzyme, significantly interfered with c-Jun target gene activity but not with transcription in general. These findings clarify the mechanism of c-Jun-mediated transcriptional regulation, and provide evidence for an involvement of RHII/Gu in stress response and in RNA polymerase II-catalyzed transcription in mammalian cells.

The Notch signaling pathway acts in both physiological and pathological conditions, including embryonic development and tumorigenesis. In cancer progression, diverse mechanisms are involved in Notch-mediated biological responses, including angiogenesis and epithelial-mesenchymal-transition (EMT). During EMT, the activation of cellular programs facilitated by transcriptional repressors results in epithelial cells losing their differentiated features, like cell–cell adhesion and apical–basal polarity, whereas they gain motility. As it concerns cancer epithelial cells, EMT may be consequent to the evolution of genetic/epigenetic instability, or triggered by factors that can act within the tumor microenvironment. Following a description of the Notch signaling pathway and its major regulatory nodes, we focus on studies that have given insights into the functional interaction between Notch signaling and either hypoxia or estrogen in breast cancer cells, with a particular focus on EMT. Furthermore, we describe the role of hypoxia signaling in breast cancer cells and discuss recent evidence regarding a functional interaction between HIF-1α and GPER in both breast cancer cells and cancer-associated fibroblasts (CAFs). On the basis of these studies, we propose that a functional network between HIF-1α, GPER and Notch may integrate tumor microenvironmental cues to induce robust EMT in cancer cells. Further investigations are required in order to better understand how hypoxia and estrogen signaling may converge on Notch-mediated EMT within the context of the stroma and tumor cells interaction. However, the data discussed here may anticipate the potential benefits of further pharmacological strategies targeting breast cancer progression.