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The Myocardin-related transcription factor A [MRTFA, also known as Megakaryoblastic Leukemia 1 (MKL1))] is a major actor in the epithelial to mesenchymal transition (EMT). We have previously shown that activation and nuclear accumulation of MRTFA mediate endocrine resistance of estrogen receptor alpha (ERα) positive breast cancers by initiating a partial transition from luminal to basal-like phenotype and impairing ERα cistrome and transcriptome. In the present study, we deepen our understanding of the mechanism by monitoring functional changes in the receptor's activity. We demonstrate that MRTFA nuclear accumulation down-regulates the expression of the unliganded (Apo-)ERα and causes a redistribution of the protein localization from its normal nuclear place to the entire cell volume. This phenomenon is accompanied by a shift in Apo-ERα monomer/dimer ratio towards the monomeric state, leading to significant functional consequences on ERα activities. In particular, the association of Apo-ERα with chromatin is drastically decreased, and the remaining ERα binding sites are substantially less enriched in ERE motifs than in control conditions. Monitored by proximity Ligation Assay, ERα interactions with P160 family coactivators are partly impacted when MRTFA accumulates in the nucleus, and those with SMRT and NCOR1 corepressors are abolished. Finally, ERα interactions with kinases such as c-src and PI3K are increased, thereby enhancing MAP Kinase and AKT activities. In conclusion, the activation and nuclear accumulation of MRTFA in ERα positive breast cancer cells remodels both ERα location and functions by shifting its activity from nuclear genome regulation to extra-nuclear non-genomic signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The effects of some progestins on fish reproduction have been recently reported revealing the hazard of this class of steroidal pharmaceuticals. However, their effects at the central nervous system level have been poorly studied until now. Notwithstanding, progesterone, although still widely considered primarily a sex hormone, is an important agent affecting many central nervous system functions. Herein, we investigated the effects of a large set of synthetic ligands of the nuclear progesterone receptor on the glial-specific expression of the zebrafish brain aromatase (cyp19a1b) using zebrafish mechanism-based assays. Progesterone and 24 progestins were first screened on transgenic cyp19a1b-GFP zebrafish embryos. We showed that progesterone, dydrogesterone, drospirenone and all the progesterone-derived progestins had no effect on GFP expression. Conversely, all progestins derived from 19-nortesterone induced GFP in a concentration-dependent manner with EC50 ranging from the low nM range to hundreds nM. The 19-nortestosterone derived progestins levonorgestrel (LNG) and norethindrone (NET) were further tested in a radial glial cell context using U251-MG cells co-transfected with zebrafish ER subtypes (zfERα, zfERβ1 or zfERβ2) and cyp19a1b promoter linked to luciferase. Progesterone had no effect on luciferase activity while NET and LNG induced luciferase activity that was blocked by ICI 182,780. Zebrafish-ERs competition assays showed that NET and LNG were unable to bind to ERs, suggesting that the effects of these compounds on cyp19a1b require metabolic activation prior to elicit estrogenic activity. Overall, we demonstrate that 19-nortestosterone derived progestins elicit estrogenic activity by inducing cyp19a1b expression in radial glial cells. Given the crucial role of radial glial cells and neuro-estrogens in early development of brain, the consequences of exposure of fish to these compounds require further investigation., (Copyright © 2016. Published by Elsevier Inc.)

Unlabelled: The existence of a link between estrogen deprivation and osteoarthritis (OA) in postmenopausal women suggests that 17β-estradiol (17β-E2) may be a modulator of cartilage homeostasis. Here, we demonstrate that 17β-E2 stimulates, via its receptor human estrogen receptor α 66 (hERα66), type II collagen expression in differentiated and dedifferentiated (reflecting the OA phenotype) articular chondrocytes. Transactivation of type II collagen gene (COL2A1) by ligand-independent transactivation domain (AF-1) of hERα66 was mediated by "GC" binding sites of the -266/-63-bp promoter, through physical interactions between ERα, Sp1/Sp3, Sox9, and p300, as demonstrated in chromatin immunoprecipitation (ChIP) and Re-Chromatin Immuno-Precipitation (Re-ChIP) assays in primary and dedifferentiated cells. 17β-E2 and hERα66 increased the DNA-binding activities of Sp1/Sp3 and Sox-9 to both COL2A1 promoter and enhancer regions. Besides, Sp1, Sp3, and Sox-9 small interfering RNAs (siRNAs) prevented hERα66-induced transactivation of COL2A1, suggesting that these factors and their respective cis-regions are required for hERα66-mediated COL2A1 up-regulation. Our results highlight the genomic pathway by which 17β-E2 and hERα66 modulate Sp1/Sp3 heteromer binding activity and simultaneously participate in the recruitment of the essential factors Sox-9 and p300 involved respectively in the chondrocyte-differentiated status and COL2A1 transcriptional activation. These novel findings could therefore be attractive for tissue engineering of cartilage in OA, by the fact that 17β-E2 could promote chondrocyte redifferentiation., Key Messages: 17β-E2 up-regulates type II collagen gene expression in articular chondrocytes. An ERα66/Sp1/Sp3/Sox-9/p300 protein complex mediates this stimulatory effect. This heteromeric complex interacts and binds to Col2a1 promoter and enhancer in vivo. Our findings highlight a new regulatory mechanism for 17β-E2 action in chondrocytes. 17β-E2 might be an attractive candidate for cartilage engineering applications.

In addition to soluble factors, mechanical constraints and extracellular matrix stiffness are important regulators of cell fate that are mediated by cytoskeletal modifications. The EMT (epithelial-mesenchymal transition) that occurs during normal development and malignant progression is a typical example of the phenotypic switch associated with profound actin remodelling and changes in gene expression. For instance, actin dynamics control motile cell functions in EMT, in part, through regulating the subcellular localization of the myocardin-related transcription factor MKL1 (megakaryoblastic leukaemia translocation 1), a co-activator of SRF (serum-responsive factor). In the present paper, we show that MKL1 participates also to the control of the cellular switch between growth and quiescence. Experimental disconnection between MKL1 and G-actin (globular actin), by using an MKL1 mutant or enhancing the F (filamentous)-/G-actin ratio, generates a widely open chromatin state and a global increase in biosynthetic activity, classically associated with cell growth. Conversely, G-actin accumulation favours nuclear condensation and cell quiescence. These large-scale chromatin changes rely upon extensive histone modifications, exemplified by that of H3K9 (H3 Lys9) shifting from trimethylation, a heterochromatin mark, to acetylation, a mark of euchromatin. The present study provides the first evidence for a global reversible hetero/euchromatinization phenomenon triggered by the actin/MKL1 signalling pathway.

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.

The estrogen receptor alpha (ER alpha) is key in regulating normal breast development and function and is closely involved in the onset and progress of cancers. ER alpha transcriptional activity is mediated through two activation functions, AF1 and AF2, whose activity is tightly regulated in a cell-specific manner through yet unknown processes. Here, we demonstrate that cell-cell junctions generate cell permissiveness to AF1 through an up-regulation of the activity of an AF1 sub-region termed box 1. Moreover, the loss of E-cadherin expression is shown to silence the AF1 activity of ER alpha, allowing the receptor to mainly act through its AF2. This switch from an AF1 to an AF2 cell permissiveness also consequently results in the attenuation of ER alpha activity. Therefore, a loss of cell-cell junctions, a key process that occurs during the epithelial-mesenchymal transition, should have a broad impact on ER alpha transcriptional functions.

Purpose: Overexpression of PBR has been found in several tumor types including ovarian, colon, breast adenocarcinomas, esophageal cancer. There is evidence suggesting that PBR ligands regulate cell proliferation. However, their action is probably cell-type specific. We decided to evaluate mitotic activity of PBR ligands in some normal and neoplastic cell lines., Material and Methods: The cells were maintained according to standard procedures. Ligand binding assay was performed in cell extract using PK-11195 or Ro-54864 and [N-methyl-3H] Ro-54864 or [N-methyl-3H] PK-11195. Cell proliferation was evaluated using 5-[3H]-thymidine assay. Western Immunoblot assay was conducted using polyclonal anti-PBR antibody., Results: We have found that, macrophages evoked strong binding of both Ro-54864 and PK-11195. This phenomenon was accompanied by drastic decrease in the cell divisions. Similar effect was found only in the case of non-estrogen-dependent breast cancer cells MDA-MB 231. It suggest that PBR-ligand mediated inhibition of mitogenesis may represent a new anticancer strategy in non-estrogen-dependent breast cancer. In respect to macrophages inhibition of the cell division by both PBR ligands may have implication in modulation of inflammatory response. It has been postulated that PBR ligands may have anti-inflammatory activity in rheumatoid arthritis. The presence of peripheral benzodiazepine receptors in chondrocytes, T cells, macrophages and mesenchymal cells suggest that peripheral benzodiazepine receptor ligands may interfere with the cytokine network and thus modulate inflammatory response., Conclusions: The data suggest that PBR-ligand mediated inhibition of DNA synthesis in non-estrogen dependent breast cancer cells and in macrophages may represent a new therapeutic approach of breast anti-cancer and anti-inflammatory therapy.

The expression of two human estrogen receptor-alpha (hERalpha) isoforms has been characterized within estrogen receptor-alpha-positive breast cancer cell lines such as MCF7: the full-length hERalpha66 and the N terminally deleted hERalpha46, which is devoid of activation function (AF)-1. Although hERalpha66 is known to mediate the mitogenic effects that estrogens have on MCF7 cells, the exact function of hERalpha46 in these cells remains undefined. Here we show that, during MCF7 cell growth, hERalpha46 is mainly expressed in the nucleus at relatively low levels, whereas hERalpha66 accumulates in the nucleus. When cells reach confluence, the situation reverses, with hERalpha46 accumulating within the nucleus. Although hERalpha46 expression remains rather stable during an estrogen-induced cell cycle, its overexpression in proliferating MCF7 cells provokes a cell-cycle arrest in G(0)/G(1) phases. To gain further details on the influence of hERalpha46 on cell growth, we used PC12 estrogen receptor-alpha-negative cell line, in which stable transfection of hERalpha66 but not hERalpha46 allows estrogens to behave as mitogens. We next demonstrate that, in MCF7 cells, overexpression of hERalpha46 inhibits the hERalpha66-mediated estrogenic induction of all AF-1-sensitive reporters: c-fos and cyclin D1 as well as estrogen-responsive element-driven reporters. Our data indicate that this inhibition occurs likely through functional competitions between both isoforms. In summary, hERalpha46 antagonizes the proliferative action of hERalpha66 in MCF7 cells in part by inhibiting hERalpha66 AF-1 activity.

In this study, we hypothesized that many of the reported effects of phthalate esters and other peroxisome proliferators (PPs) in the testis are mediated by members of the PP- activated receptor (PPAR) family of transcription factors through alterations in proteins involved in steroidogenesis. Exposure of Leydig cells to PPs prevented cholesterol transport into the mitochondria after hormonal stimulation and inhibited steroid synthesis, without altering total cell protein synthesis or mitochondrial and DNA integrity. PPs also reduced the levels of the cholesterol-binding protein peripheral-type benzodiazepine receptor (PBR) because of a direct transcriptional inhibition of PBR gene expression in MA-10 Leydig cells. MA-10 cells contain mRNAs for PPARalpha and PPARbeta/delta, but not for PPARgamma. In vivo treatment of mice with PPs resulted in the reduction of both testis PBR mRNA and circulating testosterone levels, in agreement with the proposed role of PBR in steroidogenesis. By contrast, liver PBR mRNA levels were increased, in agreement with the proposed role of PBR in cell growth/tumor formation in nonsteroidogenic tissues. However, PPs did not inhibit testosterone production and testis PBR expression in PPARalpha-null mice. These results suggest that the antiandrogenic effect of PPs is mediated by a PPARalpha-dependent inhibition of Leydig cell PBR gene expression.

The peroxisome proliferator perfluordecanoic acid (PFDA) has been shown to exert an antiandrogenic effect in vivo by acting directly on the interstitial Leydig cells of the testis. The objective of this study was to examine the in vitro effects of PFDA and identify its site of action in steroidogenesis using as model systems the mouse tumor MA-10 and isolated rat Leydig cells. PFDA inhibited in a time- and dose-dependent manner the hCG-stimulated Leydig cell steroidogenesis. This effect was localized at the level of cholesterol transport into the mitochondria. PFDA did not affect either the total cell protein synthesis or the mitochondrial integrity. Moreover, it did not induce any DNA damage. Morphological studies indicated that PFDA induced lipid accumulation in the cells, probably due to the fact that cholesterol mobilized by hCG did not enter the mitochondria to be used for steroidogenesis. In search of the target of PFDA, we examined its effect on key regulatory mechanisms of steroidogenesis. PFDA did not affect the hCG-induced steroidogenic acute regulatory protein (StAR) levels. However, it was found to inhibit the mitochondrial peripheral-type benzodiazepine receptor (PBR) ligand binding capacity, 18-kDa protein, and messenger RNA (mRNA) levels. Further studies indicated that PFDA did not affect PBR transcription, but it rather accelerated PBR mRNA decay. Taken together, these data suggest that PFDA inhibits the Leydig cell steroidogenesis by affecting PBR mRNA stability, thus inhibiting PBR expression, cholesterol transport into the mitochondria, and the subsequent steroid formation. Moreover, this action of PFDA on PBR mRNA stability indicates a new mechanism of action of peroxisome proliferators distinct from the classic transcription-mediated regulation of target genes.

Background: Testicular Leydig cells use either exogenous or de novo synthesized cholesterol as the substrate for the production of testosterone with hormone stimulation. Although the long-term effect of trophic hormones on Leydig cell cholesterol uptake, storage, and deesterification has been well documented, the early effects of the human choriogonadotropin (hCG) on cell cholesterol/lipid distribution are not yet known., Methods: Sections of cells treated with hCG for 15 sec to 30 min were examined by electron microscopy (EM) for the surface density of lipid moieties in the cytoplasm. In addition, the time-dependent distribution of lipids within the cytoplasmic inclusions and the ultimate destination of this substrate were evaluated by EM. The results were analyzed with standard morphometric methods., Results and Conclusion: The surface density of cytoplasmic lipid pools increased significantly within the 15 sec following the exposure of cells to hCG, and it tapered off to the control level in the subsequent 30 min. Such a fluctuation in the amount of cytoplasmic lipids may be due to (1) the quantity of released substrate from the reticular compartment or (2) the rate of its transport across the outer mitochondrial membrane to the inner mitochondrial cytochrome P450 side-chain cleavage, where steroidogenesis begins with the conversion of cholesterol to pregnenolone. These two processes were not quantitatively coordinated in the stimulated cell during the initial 30 min, resulting in a surplus of cytoplasmic lipid pools. To compensate for such uneven metabolic balance, the cell apparently disposed of the excess substrate by a mechanism of molecular regrouping from a micellar configuration to a bilayer structure followed by exocytosis.

In order to clarify the role of germ cells in the regulation of Sertoli cell secretions, three experimental models of germ cell depletion were used: hypodactyl rat mutation (testis weight [TW]: 55% less than controls), in utero busulfan treatment (TW: 88% less than controls), and neonatal experimental cryptorchidism (TW: 72% less than controls). The aim of this work was to compare the numbers of Leydig and Sertoli cells and the production of germ cells in each experimental model to the in vitro secretions of Leydig and Sertoli cells in conditioned media and to the hormonal serum profiles of the same animal in vivo. In the three models, serum levels of hypophyseal and testosterone hormones were significantly increased and decreased, respectively. In the absence of germ cells, the total length of seminiferous tubules, the total numbers of Sertoli and Leydig cells, and the daily production of germ cells were significantly diminished. The addition of both control and damaged seminiferous tubule culture media (STM: media conditioned by 10 cm of seminiferous tubules) to 10(6) control or damaged Leydig cells led to a further increase of testosterone production after ovine LH stimulation. However, expressed per Sertoli cell, testosterone production by control Leydig cells was reduced by addition of damaged STM as compared to addition of control STM, and similarly, the addition of control STM to damaged Leydig cells enhanced testosterone production more than did the addition of damaged STM. Secretions of transferrin per Sertoli cell in STM were reduced as compared to controls by the absence of germ cells but to a lesser extent than was production of spermatocytes and of spermatids. In conclusion, secretions by Sertoli cells of the paracrine factor involved in the control of testosterone production by Leydig cells and of transferrin are modified by germ cells.

In order to elucidate the respective effects of depletion of germ cells and of increase in testicular temperature, rats of the same Wistar strain were rendered experimentally bicryptorchid or sterilized by a busulfan injection in utero and compared to control animals. In both models, germ cells were depleted but numeric evolution and functions of somatic cells differed. The aim of that work was to compare the numeric evolutions of testicular somatic and germ cells to their respective functions in each model before puberty and in adult rats of the same strain. Serum concentrations of FSH, LH and testosterone were compared at 20, 40 and 110 days of age. Histological analyses of Sertoli and germ cells in the seminiferous tubules and of Leydig cells in the intertubular tissue were performed before puberty and at adulthood. Testosterone serum concentrations were depleted in both models starting at 40 days of age and more in busulfan-treated rats. Both FSH and LH levels were increased from 20 days onwards in experimental rats. Additional cryptorchidism in busulfan-treated rats depressed the serum testosterone concentration. At 17 days of age, the cryptorchidism do not modify somatic or germ cell populations while busulfan treatment has induced a decrease of both these populations. Conversely, the cross sectional area of the somatic testicular cells was not affected whatever the treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadal steroidogenesis is regulated by pituitary gonadotropins and a locally produced, unidentified factor. A 70-kilodalton (kD) protein complex secreted from rat Sertoli cells was isolated. The complex, composed of 28- and 38-kD proteins, stimulated steroidogenesis by Leydig cells and ovarian granulosa cells in a dose-dependent and adenosine 3',5'-monophosphate-independent manner. The follicle-stimulating hormone-induced 28-kD protein appeared to be responsible for the bioactivity, but the 38-kD protein was indispensable for maximal activity. The 28- and 38-kD proteins were shown to be identical to the tissue inhibitor of metalloproteinase-1 (TIMP-1) and the proenzyme form of cathepsin L, respectively. Thus, a TIMP-1-procathepsin L complex is a potent activator of steroidogenesis and may regulate steroid concentrations and, thus, germ cell development in both males and females.

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.

Steroid biosynthesis activated by pituitary tropic hormones is known to be acutely regulated by cAMP acting via Protein kinase A. Because the mitochondrial benzodiazepine receptor (MBR) has been suggested to play a role in the activation of steroidogenesis, the present study investigates whether various protein kinases phosphorylate MBR. In rat and bovine adrenal mitochondrial preparations Protein kinase A, but not other purified protein kinases, was found to phosphorylate the 18 kDa MBR protein. In digitonin-permeabilized MA-10 Leydig tumor cells incubated with [gamma-32P]ATP, phosphorylation of MBR was detectable during treatment of the cells with dibutyryl cAMP. In conclusion, these data show that the MBR protein is an in vitro and in situ substrate of Protein kinase A, but the role of this phosphorylation in the regulation of steroidogenesis remains to be established.

In mature rat Leydig cells, the testosterone output (24 ng/10(6) Leydig cells/4hrs.) is increased 10 fold by LH; the addition of serum from either control or castrated or hypophysectomized rams inhibits (60%) the LH-stimulated testosterone production. Similarly, the incubation of immature rat Leydig cells with sera from hypophysectomized patients leads to a diminution (70 and 30% respectively) of both basal (0.98 ng) and LH stimulated (3.44 ng) testosterone biosynthesis. These data suggest the existence of an LH inhibitor (or inhibitors) in blood from ram and human; in addition, this substance is not only of testicular origin and is not an LH-related molecule.

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.

The existence of Sertoli cell factors which modulate the rat Leydig cell function prompted us to study the biological activity of selected proteins called CMB proteins and produced by immature rat Sertoli cells. Percoll purified Leydig cells (10(5)) from 20 days-old rats have been incubated 5 h at 32 degrees C in 1 ml Ham F12/DME medium with increasing concentrations of partially purified CMB proteins (0-1,000 ng/ml) either in presence or absence of oLH (25 ng/ml). Among the CMB proteins tested, only CMB-21 produces a dose related increase of testosterone production: from 2 to 500 pg/ml of CMB-21, testosterone output is unchanged (51 pg/10(5) cells) but 1 to 1,000 ng/ml of this protein produces a linear increase of testosterone productions (86 to 870 pg). In the presence of oLH which induces a 10-fold increase of testosterone production (499 pg), increasing doses of CMB-21 further stimulate testosterone output (775 to 2.272 pg/10(5) cells). Whatever the concentration of oLH used (0 to 50 ng/ml), CMB-21 (500 ng/ml) leads to a further 2 fold augmentation of testosterone synthesis; similarly, in the presence of dbcAMP (1 mM), CMB 21 increases the testosterone production but no effect is observed when Leydig cells are incubated in the presence of 22R-hydroxycholesterol (30 microM). The cAMP levels which are increased more than 4 fold by oLH, remain unchanged in the presence of CMB-21 either alone or with oLH, as observed when Sertoli cell culture medium is used.(ABSTRACT TRUNCATED AT 250 WORDS)