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International audience; Although involved in processes leading to the emergence and development of hormone-dependent breast cancers, the estrogen receptor alpha (ERalpha) also prevents transformed cells from progressing toward a more aggressive phenotype. The transcriptional activity of ERalpha is mediated through two transactivation functions, called activation function 1 and 2, whose respective involvement varies in a cell-specific manner. Here, we identify the Rho/megakaryoblastic leukemia 1 (MKL1) signaling pathway as a main actor in controlling the cell-specific activity of both transactivation functions of ERalpha. Notably, we show that, when the coregulator MKL1 is sequestered in an inactive form by unpolymerized actin, the transcriptional activity of ERalpha mainly relies on the activation function 1. The activation of MKL1, which results from its dissociation from unpolymerized actin, promoted by the ability of Rho to support polymeric actin accumulation, silences the activation function 1 of ERalpha and allows the receptor to mainly act through its activation function 2. Importantly, this switch in the respective contribution exerted by both transactivation functions is correlated with an impaired ability of ERalpha to efficiently transactivate estrogen-regulated reporter genes. MKL1 is further shown to be present on estrogen-responsive genes in vivo. Interestingly, the Rho/MKL1 signaling pathway is activated during the epithelial-mesenchymal transition. A reduced transactivation efficiency of ERalpha, resulting from the activation of this pathway, may therefore suppress the protective role exerted by ERalpha toward tumor progression and invasiveness.

In nuclear physics experiments, a decision maker named as “trigger” gives a bit pattern which allows the fired detectors identification. As the data acquisition dead time is greater than the time between physical events, timing information is essential. We add to the trigger function a Time to Digital Converter (TDC) in order to make a separation between events. The paper describes the architecture chosen for the TDC and illustrates the contribution of each element to the TDC performance. An eight-bit counter is used for the dynamic range of the TDC (in microsecond) associated to a delay line improving the resolution (in nanosecond). The study shows that exploiting the two system clock states (high and low) allows to reduce the number of delay line cells. The Differential Nonlinearity Measurements are given for different resolutions (1, 2 and 5 ns) and illustrate the clock period, the clock duty cycle and the delay line contributions to the TDC performances.

Calcitonin is a peptide which acts in the brain to modulate behavior and hormone release, possibly through an interaction with serotonergic systems. We investigated the effects of chronic systemic injections of salmon calcitonin on the [3H]-8-OHDPAT binding to 5-HT1A receptors in the frontal cortex and hippocampus in adrenalectomized and intact (non adrenalectomized) rats. The results show that salmon calcitonin increases the maximal density of 5-HT1A binding sites in both structures in adrenalectomized animals (and decreases the affinity in the frontal cortex only). Calcitonin does not alter this binding in intact rats. These results demonstrate the existence of interactions between calcitonin, serotonin and glucocorticoids, and raise the hypothesis of a neurotrophic effect of calcitonin on serotonergic neurons.

Steroidogenesis begins with the metabolism of cholesterol to pregnenolone by the inner mitochondrial membrane cytochrome P450 side-chain cleavage (P450scc) enzyme. The rate of steroid formation, however, depends on the rate of cholesterol transport from intracellular stores to the inner mitochondrial membrane and loading of P450scc with cholesterol. In previous in vitro studies, we demonstrated that a key element in the regulation of cholesterol transport is the mitochondrial peripheral-type benzodiazepine receptor (PBR). We also showed that the polypeptide diazepam binding inhibitor (DBI), an endogenous PBR ligand, stimulates cholesterol transport and promotes loading of cholesterol to P450scc in vitro, and that its presence is vital for hCG-induced steroidogenesis by Leydig cells. Based on these data and the observations that i) the mitochondrial PBR binding and topography are regulated by hormones; ii) the 18-kDa PBR protein is functionally coupled to the mitochondrial contact site voltage-dependent anion channel protein; iii) the 18-kDa PBR protein is a channel for cholesterol, as shown by molecular modeling and in vitro reconstitution studies; iv) targeted disruption of the PBR gene in steroidogenic cells dramatically reduces the ability of the cells to transport cholesterol in the mitochondria and produce steroids; v) endocrine disruptors, with known anisteroidogenic effect, inhibit PBR ligand binding; and vi) in vivo reduction of adrenal PBR expression results in reduced circulating glucocorticoid levels, we conclude that PBR is an indispensable element of the steroidogenic machinery.

Native MA-10 mouse Leydig tumor cell mitochondrial preparations were examined by transmission electron (TEM) and atomic force (AFM) microscopic procedures in order to investigate the topography and organization of the peripheral-type benzodiazepine receptor (PBR). Mitochondria were immunolabeled with an anti-PBR antiserum coupled to gold-labeled secondary antibodies. Results obtained indicate that the 18,000 MW PBR protein is organized in clusters of 4-6 molecules. Moreover, on many occasions, the interrelationship among the PBR molecules was found to favor the formation of a single pore. Taking into account recent observations that the 18,000 MW PBR protein is functionally associated with the pore forming 34,000 MW voltage-dependent anion channel (VDAC) these results suggest that (i) the mitochondrial PBR complex could function as a pore, thus allowing the translocation of cholesterol and other molecules to the inner mitochondrial membrane, and (ii) the native receptor is a multimeric complex of an approximate 140,000 MW composed on an average of five 18,000 PBR subunits, one 34,000 VDAC subunit, and associated lipids.

Purpose: Type II collagen, encoded by COL2A1 gene, is a phenotypic marker of articular cartilage whose expression is strongly decreased during osteoarthritis (OA) process. Specific proteins (Sp) and Sox proteins have been previously characterized to be major regulators of this gene. Besides, the existence of a link between estrogen deprivation and osteoarthritis in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a critical modulator of cartilaginous matrix homeostasis. The aim of present study was to investigate the molecular mechanisms regulating type II collagen gene expression under the effect of 17β-E2, and to characterize the genomic pathway via estrogen receptors (ER). Methods: Articular chondrocytes were isolated from 3-week old rabbits and incubated for 24 hours with increasing concentrations of 17β-E2 (0 to 10 nM). Nuclear proteins were extracted and submitted to gel retardation assays in order to determine Sp and Sox proteins binding activities on COL2A1 gene. In some experiments, relative expression of Sp1/Sp3 or Sox-9 was inhibited by siRNAs strategies. Moreover, chromatin immunoprecipitation was used to study protein-protein interactions on COL2A1 promoter and enhancer sequences. Results: We have previously shown that 17β-E2 could stimulate type II collagen neosynthesis, protein level and corresponding steady-state levels of COL2A1 mRNAs in primary and in vitro dedifferentiated rabbit articular chondrocytes. 17β-E2 action was mediated, in part, by an ERα/Sp transactivating effect on the -266/-63 bp region of COL2A1 proximal promoter. Our new data showed that 17β-E2 also increases the DNA-binding activities of Sp1/Sp3 and Soxs proteins to the first intron region including COL2A1 specific enhancer. In addition, Sp1, Sp3 and Sox-9 siRNAs prevented hERα66-induced transactivation of COL2A1, suggesting that these factors are required for COL2A1 up-regulation. Moreover, chromatin immunoprecipitation assays indicated that ERα physically interacts in vivo with Sp1, Sp3, Sox-9 and p300 transcription factors, on both COL2A1 promoter and enhancer sequences. Thus, considering that all these transcription factors are known to act in concert, we proposed that 17β-E2 in vivo effect could be mediated by a bridging complex, composed of ERα, Sp1, Sp3, Sox-9 and p300 transcription factors, which could establish a physical link between the two main cisregulatory regions of the gene, and consequently, may facilitate COL2A1 transcription process. Besides, this transcriptional activation of COL2A1 gene requires the ERα transactivation domains, AF-1 and AF-2, as demonstrated by using ERα mutants lacking these functional domains. Conclusions: Understanding the molecular basis for 17β-E2/ERα induction of COL2A1 transcription provides new insights into molecular mechanisms of OA and could be therefore attractive for some applications in the tissue engineering of cartilage.

A single i.p. injection of either cisplatin or bleomycin affects the testosterone production by the Leydig cells in mature rats. It is noteworthy that on day 30 after treatment, a complete recovery of Leydig cell function is found in bleomycin-treated rats whereas in cisplatin-injected animals, 25-48% decreases of testosterone synthesis are still observed, respectively, under basal and LH-stimulated conditions. Together with the germ cell destruction, the low testosterone levels probably contribute to the sterility in cisplatin-treated rats.