Fibulin-1 is an extracellular matrix protein induced by estradiol in estrogen receptor (ER) positive ovarian cancer cell lines. Alternative splicing of ®bulin-1 mRNA results in four di erent variants named A, B, C and D that may have distinct biological functions. We studied the relative expression of ®bulin-1 mRNA variants and their estrogen regulation in human ovarian cancer cells. In ovarian tissues and cancer cell lines, ®bulin-1C and -1D are the predominant forms, whereas ®bulin-1A and -1B are weakly expressed. We developed a competitive PCR assay based on coampli®cation of ®bulin-1C and -1D to study the relative expression of these ®bulin-1 variants in human ovarian samples. In ovarian cancer cell lines and ovarian cancer samples, there was a marked increase in the ®bulin-1C:1D and ®bulin-1C:HPRT mRNA ratios as compared to normal ovaries. In the BG1 estrogen receptor positive ovarian cancer cell line, ®bulin-1C mRNA was induced by estradiol in a dose-and time-dependent manner. Since others and we have previously shown an increased expression of ERa as compared to ERb in ovarian cancer cells, we investigated whether ERa or ERb is involved in this induction. For this aim, MDA-MB-231 breast cancer cell line, which expresses both low basal levels of ERs and ®bulin-1, was infected with recombinant ERa or ERb encoding adenovirus and treated with estradiol. Fibulin-1C was induced by estradiol in ERa-but not ERb-infected cells, suggesting that ®bulin-1C induction is mediated through ERa. In ovarian tumors, a trend towards a correlation between ®bulin-1C and ERa expression levels was noted. In conclusion, this study showed an increased ®bulin-1C: -1D mRNA ratio in ovarian cancer cells as compared to normal ovaries. This ®nding suggests that the C variant may be involved in ovarian carcinogenesis. Fibulin-1C overexpression may thus be a clue for the understanding of a putative role of estrogens in ERa promoted ovarian tumor progression.

The nuclear peroxisome proliferator-activated receptors (PPARs) alpha, beta, and gamma activate the transcription of multiple genes involved in lipid metabolism. Several natural and synthetic ligands have been identified for each PPAR isotype but little is known about the phosphorylation state of these receptors. We show here that activators of protein kinase A (PKA) can enhance mouse PPAR activity in the absence and the presence of exogenous ligands in transient transfection experiments. Activation function 1 (AF-1) of PPARs was dispensable for transcriptional enhancement, whereas activation function 2 (AF-2) was required for this effect. We also show that several domains of PPAR can be phosphorylated by PKA in vitro. Moreover, gel retardation experiments suggest that PKA stabilizes binding of the liganded PPAR to DNA. PKA inhibitors decreased not only the kinase-dependent induction of PPARs but also their ligand-dependent induction, suggesting an interaction between both pathways that leads to maximal transcriptional induction by PPARs. Moreover, comparing PPAR alpha knockout (KO) with PPAR alpha WT mice, we show that the expression of the acyl CoA oxidase (ACO) gene can be regulated by PKA-activated PPAR alpha in liver. These data demonstrate that the PKA pathway is an important modulator of PPAR activity, and we propose a model associating this pathway in the control of fatty acid beta-oxidation under conditions of fasting, stress, and exercise.

Knockout and expression studies suggest that estrogen receptor ␤ (ER␤) plays a prominent role in ovarian function and pathology. Moreover, ovarian cancers are characterized by high morbidity and low responsiveness to anti-estrogens. Here we demonstrate, using quantitative PCR to measure ER␣ and ER␤ levels in 58 ovarian cancer patients, that ER␤ expression decreased in cysts and ovarian carcinomas as compared with normal ovaries and that this decrease is attributable only to a selective loss in ER␤ expression during cancer progression. To address the question of a possible involvement of ER␤ in ovarian cancers, we restored ER␣ and ER␤ expression in two human ovarian cancer cell lines PEO14 (ER␣-negative) and BG1 (ER␣-positive) using adenoviral delivery. ER␣, but not ER␤, could induce progesterone receptor and fibulin-1C. Moreover, ER␣ and ER␤ had opposite actions on cyclin D1 gene regulation, because ER␤ down-regulated cyclin D1 gene expression, whereas ER␣ increased cyclin D1 levels. Interestingly, ER␤ expression strongly inhibited PEO14 and BG1 cell proliferation and cell motility in a ligandindependent manner, whereas ER␣ had no marked effect. Induction of apoptosis by ER␤ also contributed to the decreased proliferation of ovarian cancer cells, as shown by Annexin V staining. This study shows that ER␤ is an important regulator of proliferation and motility of ovarian cancer and provides the first evidence for a proapoptotic role of ER␤. The loss of ER␤ expression may thus be an important event leading to the development of ovarian cancer.

The estrogen receptor (ER) belongs to a large family of nuclear receptors, many of whose members function as ligand-dependent transcriptional activators. The mechanism by which the receptor is converted from an inactive into an activated state is not yet completely understood. To investigate the kind of changes in receptor conformation and interactions that are involved in this activation, we have used the wild type ER and a set of constitutively active ER point mutants that show from 20% to nearly 100% activity in the absence of estrogen. These mutants are of particular interest as they could mimic, in the absence of ligand, the activated state of the wild type receptor. We have analyzed several transcriptional steps that could be involved in the activation: the ability of these receptors 1) to interact with several coactivators (steroid receptor coactivator-1, SRC-1; transcription intermediary factor-1, TIF-1; and estrogen receptor-associated protein 140, ERAP 140) and with members of the preinitiation complex [TATA box-binding protein (TBP), transcription factor IIB (TFIIB)]; 2) to exhibit conformational changes revealed by proteolytic digest patterns similar to those observed for the wild type hormone-occupied ER; and 3) to bend estrogen response element-containing DNA, which is thought to be one of the important phenomena triggering transcriptional activation. Our results demonstrate that the interaction of these mutant receptors with coactivators is likely to be one of the features of the activated step, as the mutant receptors interacted with some coactivators in a ligand-independent manner in proportion to their extent of constitutive activity. However, the different degrees of ligand-independent interaction of the mutant ERs with the three coactivators suggest that SRC-1, TIF-1, and ERAP 140 may play different roles in receptor activity. Limited proteolytic digest experiments reveal that the activated state of the receptor corresponds to a particular conformation of the receptor, which is fully observed with the mutant ER showing the highest activity in the absence of estrogen. Finally, it appears that in inactive or active states, the receptor exhibits distinctly different DNA-bending abilities. Addition of estradiol is able to modify the bending ability of only the wild type receptor, whereas estradiol has no influence on the constitutive receptors, which exhibited the same bending ability as that observed for the ligand-occupied wild type receptor. These data document that the ER undergoes major changes in its conformation and also in its functional properties when it is turned from an inactive into an active state and that mutational changes in the ER protein that result in constitutive, hormone-independent activation mimic many of the changes in ER properties that are normally under hormone regulation.