Estrogen receptor α regulates the expression of syndecan-1 in human breast carcinoma cells.

Breast cancer (BC) is the primary cause of cancer-related mortality among women. Patients who express the estrogen receptor (ER), which mediates the tumorigenic effects of estrogens, respond to antihormonal therapy. Loss of ER expression or acquired resistance to E2 is associated with aggressive malignant phenotypes, which lead to relapse. These BC subtypes overexpress syndecan-1 (SDC1), a transmembrane heparan sulfate proteoglycan that mediates angiogenesis as well as the proliferation and invasiveness of cancer cells. We showed here that the activation of ER-alpha (ERα) by estrogens induces downregulation of SDC1 expression in ER(+) MCF7 cells but not in T47D cells. Loss of ERα expression, induced by RNA interference or a selective ER downregulator, led to subsequent SDC1 overexpression. E2-dependent downregulation of SDC1 expression required de novo protein synthesis and was antagonized by treatment with BAY 11-7085, an irreversible inhibitor of IκBα phosphorylation, which inhibits the activation of NFκB. Downregulation of SDC1 expression required ERα and activation of IKK, but was independent to downstream transcriptional regulators of NFκB. BAY 11-7085 prevented E2-mediated phosphorylation of ERα on Ser118, increasing its proteasomal degradation, suggesting that IKK stabilized E2-activated ERα, leading to subsequent downregulation of SDC1 expression. Our results showed that sustained ER signaling inhibits SDC1 expression. Such antagonism elucidates the inverse correlation between SDC1 and ER expression in ER(+) BC as well as the overexpression of SDC1 in hormone receptor-negative BC subtypes with the most aggressive phenotypes. These results identify SDC1 as an attractive therapeutic target for BC as well as for other endocrine-associated cancers.

Increase in hyaluronic acid degradation decreases the expression of estrogen receptor alpha in MCF7 breast cancer cell line.

The loss of estrogen receptor α (ERα) expression in breast cancer constitutes a major hallmark of tumor progression to metastasis and is generally correlated to a strong increase in Hyaluronic Acid (HA) turnover. The aim of our study was to search for a putative link between these two major events of breast cancer progression in the estrogen receptor-positive (ER+) MCF7 breast cancer cell line. The increase in HA turnover was performed by stable overexpression of the standard CD44 (CD44S) isoform and also by treatment with exogenous Hyaluronidase (Hyal) to allow an increase in HA catabolism. Stable overexpression of CD44S in MCF7 cells was correlated to a decrease in ESR1 gene expression, which did not lead to alteration of estrogen response. Moreover, our results showed that the exposure to exogenous Hyal stimulates the proliferation and strongly decreases the expression of ERα whatever the expression level of CD44 in the MCF7 cell line. The culture in the presence of Hyal led to the decrease in estrogens responsiveness and to hormonal therapy resistance. The effect on growth is correlated to the activation of MAPK/ERK and PI3K/Akt signaling pathways while the Hyal-induced down-regulation of ESR1 gene expression involves the activation of PI3K/Akt and NF-κB signaling pathways. Many of our data suggest that the effects of Hyal described here could be related to the activation of TLR signaling. Taken together, our results demonstrate that the increase in HA degradation could be involved in breast cancer progression and in resistance to hormonal therapy.

Syndecan 1 represses cell growth and FSH responsiveness in human granulosa cells.

Albeit devoid of intrinsic catalytic activity, the transmembrane heparan sulphate proteoglycan syndecan 1 plays critical roles in cellular processes such as extracellular matrix crosstalk, cytoskeletal organization, cell spreading, proliferation and differentiation. During the ovarian cycle, the expression of syndecan 1 in granulosa cells shows cyclic variation suggesting that it might fulfil specific roles in follicle development. To investigate its physiological roles on granulosa cells, syndecan 1 was overexpressed in human granulosa cell line KGN which retains features of granulosa cells from small antral follicle such as estradiol (E2) synthesis and low expression of functional FSH receptor (FSHR). We demonstrated that overexpression of syndecan 1 in immature granulosa cells (KGN-SDC1) induces a profound alteration in their intrinsic characteristics including enhanced spreading and attachment, both associated with a reduced growth rate. Flow cytometry analysis revealed that syndecan 1 overexpression increases the percentage of KGN cells in quiescent phase. This partial cell cycle exit is concordant with downregulated levels of CCND1 and CDK4 and upregulated expression of CDK inhibitor CDKN1A In parallel both unstimulated and FSH-induced E2 synthesis are reduced in KGN-SDC1 through both repression of CYP19A1 and FSHR mRNA associated with decreased levels of potential regulators NR5A1 and ESR2 Additionally, we provide evidence that transient cAMP accumulation reduction in cells overexpressing syndecan 1 is accompanied by an increase in cAMP-hydrolysing PDE activity. Our results demonstrated that syndecan 1 might regulate differentiation of granulosa cells and follicular development by means of various mechanisms involving morphological changes, control of signalling pathways and alterations in gene expressions.Free French abstract: A French translation of this abstract is freely available at http://www.reproduction-online.org/content/153/6/797/suppl/DC1.Reproduction.

Hyaluronate synthase-2 overexpression alters estrogen dependence and induces histone deacetylase inhibitor-like effects on ER-driven genes in MCF7 breast tumor cells.

In breast carcinoma cells, high levels of hyaluronan (HA) and its CD44 receptor are frequently associated with alteration in estrogen signaling. We demonstrate that stable hyaluronate synthase 2 (HAS2) overexpression in estrogen receptor α (ERα) -positive MCF7 cells oppositely altered estrogen dependence of cell growth and its sensitivity towards antiestrogens. Albeit without effect on ERα expression and estradiol binding properties, HAS2 overexpression increased ERα Ser118 phosphorylation as well as transcriptional activity of estrogen in an ERE-luciferase reporter gene assay. However, HAS2 overexpression induced partial silencing of E2 driven-genes without affecting the magnitude of regulation by estradiol. This effect was associated with half-reduction in the activity of nuclear histone deacetylases (HDACs) through a post-translational mechanism likely consecutive to the enhanced expression of the histone acetyl-transferase EP300. In conclusion, increase in HA/CD44 interactions may contribute, through an HDAC inhibitor-like and ER-independent mechanism, to the silencing of estrogen-driven genes in breast carcinoma.

Alteration of cell membrane proteoglycans impairs FSH receptor/Gs coupling and ERK activation through PP2A-dependent mechanisms in immature rat Sertoli cells.

BACKGROUND: During the pre-pubertal life, the cessation of Sertoli cell proliferation and the onset of differentiation are associated with a shift in the FSH-mediated signaling leading to inhibition of the ERK-mitogenic pathway and to a concomitant sensitization of cAMP/PKA pathway. METHODS: To highlight the role of cell proteoglycans (PGs) in the shift of FSH signaling, both FSH-induced cAMP production and ERK1/2 inactivation were studied in untreated and sodium chlorate PG-depleted cultured Sertoli cells from 20day-old rats. RESULTS: Depletion of cell membrane PGs by sodium chlorate reduced FSH-, but not cholera toxin-stimulated cAMP production as well as basal ERK phosphorylation through an okadaic acid (OA)-sensitive mechanism. Involvement of PP2A was further substantiated by a marked decrease in membrane- associated PP2A activity under SC conditions and by the OA-induced restoration of PKA-dependent ERK inactivation in SC-treated cells. CONCLUSIONS: In 20-day-old rat Sertoli cells, transmembrane cell PGs, through tethering/activation of PP2A activity exerts regulatory control on both FSH receptor/Gs coupling and ERK phosphorylation. GENERAL SIGNIFICANCE: Besides their antiproliferative roles, cell PGs such as syndecan-1, could be involved in the increase in cAMP response to FSH occurring in Sertoli cells at the time of transition between proliferative and differentiated states.

GnRH agonist and GnRH antagonist protocols in ovarian stimulation: differential regulation pathway of aromatase expression in human granulosa cells.

Gonadotrophin-releasing hormone (GnRH) agonists and antagonists have been widely used to prevent premature LH surge during ovarian stimulation. However, studies have shown a significantly lower serum oestradiol concentration on the day of human chorionic gonadotrophin administration for cycles using GnRH antagonist. This study compared aromatase gene expression in granulosa lutein cells from 50 women randomly assigned to receive either GnRH agonist (group 1, n=28) or GnRH antagonist (group 2, n=22). The cellular mechanism involved in the observed effects was also investigated. GnRH antagonist treatment significantly affected serum oestradiol concentration (1894+/-138 versus 1074+/-63 pg/ml; P < or = 0.001), follicular-fluid oestradiol concentration in large follicles (18,565+/-2467 versus 10,184+/-1993 pg/ml; P < or = 0.05), aromatase activity (9600+/-1179 versus 5376+/-997 fmol/10(6) cells/h; P < or = 0.05) and mRNA aromatase/mRNA glyceraldehyde 3-phosphate dehydrogenase (15+/-3 versus 6+/-1; P < 0.05). Protein kinase C (PKC) activity in granulosa lutein cells from the GnRH antagonist group was 2.5-fold higher than in the GnRH agonist group. In-vitro experiments showed that selective down-regulation of PKC was only observed in GnRH-desensitized granulosa lutein cells. This report suggests that, in granulosa lutein cells, the modulation of the FSH-induced protein kinase A pathway by PKC was different in agonist versus antagonist cycles.

FSH-induced phosphoprotein phosphatase 2A-mediated deactivation of particulate phosphodiesterase-4 activities is abolished after alteration in proteoglycan synthesis in immature rat Sertoli cells.

Cessation of rat testicular Sertoli cells proliferation around days 15-20 post partum is associated in vitro with the highest rise in rolipram-sensitive cAMP-catabolizing phosphodiesterase-4 activities (PDE4s) triggered by FSH during the early postnatal period. The transient nature of FSH-induced increase in PDE4s suggests concomitant changes in both PKA-mediated activation and subsequent deactivation of these activities. In this study, we demonstrated that the deactivation of FSH-stimulated particulate, but not soluble, PDE4s in cultured Sertoli cells from 20-day-old rats was inhibited by phosphoprotein phosphatase (PP) inhibitors, okadaïc acid, and calyculin A. Moreover, the deactivation of FSH-stimulated particulate PDE4s was timely related with the gonadotropin-induced increase in both particulate PP2A activity and particulate PP2A catalytic subunit immunoreactive expression independently of any transcriptional regulation of that subunit. Both the FSH-induced increase in recruitment/activation of particulate PP2A and the subsequent deactivation of particulate PDE4 were abolished when Sertoli cell proteoglycans (PGs) synthesis was altered by sodium chlorate. Sodium chlorate effect was developmentally regulated as evidenced by its ability to silence particulate PDE4 deactivation only in non-proliferating (from 20- to 30-day-old rats) but not in proliferating (from 10-day-old rats) Sertoli cells. All these data suggested that PGs could be involved in the FSH-induced recruitment/activation of PP2A. Particularly, developmentally regulated transmembrane syndecans, the most abundant PGs in Sertoli cells, by targeting PP2A at the membrane level could allow developmental control of activated particulate PDE4s and, potentially, other signaling phosphoproteins, including the FSH receptor, during the early postnatal period.

Expression of the cAMP-phosphodiesterase PDE4D isoforms and age-related changes in follicle-stimulating hormone-stimulated PDE4 activities in immature rat sertoli cells.

Major changes in the cAMP-dependent signal transduction pathway triggered by FSH take place during transition of rat Sertoli cells from proliferative to the quiescent/terminally differentiated state. Using Sertoli cell cultures isolated from 10-, 20-, and 30-day-old rats, we recorded a specific increase in PDE4 activity in both the soluble and particulate subcellular fractions of 20-day-old Sertoli cells, which also displayed the highest cAMP response to FSH and the highest FSH-induced increase in PDE4 activity in both subcellular compartments. RT-PCR and immunoblotting experiments showed that almost all the PDE4D isoforms, known as the main cAMP-regulated rolipram-sensitive PDE in Sertoli cells, were expressed throughout the early postpartum period, whereas only the short PDE4D isoforms (PDE4D1 and PDE4D2) were transcriptionally regulated by FSH. Unexpectedly, the immunoblot data also revealed that the soluble PDE4 activities were mainly related to the long PDE4D isoforms and that short PDE4D1 was predominantly particulate. The subcellular distribution and expression of PDE4D proteins were unaffected by the developmental status of the Sertoli cells. Only the expression of short PDE4D1 appeared to be upregulated by FSH and only in 20-day-old Sertoli cells, which suggests phenotype-dependent differential regulation of Pde4d1 mRNA translation. Resensitization of the cAMP response to FSH in 20-day-old Sertoli cells was also associated with the highest FSH-induced transient increase in both soluble and particulate PDE4 activities, which suggests developmental changes in the PKA-mediated upregulation of the catalytic activities of long PDE4D. Such alterations may be involved in the phenotype-dependent alterations in FSH receptor coupling with its associated G proteins in rat Sertoli cells.

Alterations in proteoglycan synthesis selectively impair FSH-induced particulate cAMP-phosphodiesterase 4 (PDE4) activation in immature rat Sertoli cells.

FSH-induced upregulation of cAMP-PDE4 activities was decreased in cultured Sertoli cells when alteration of cell proteoglycans (PGs) metabolism was simultaneously induced either by para-nitrophenyl beta-d-xyloside (PNPX) or by sodium chlorate. This effect was restricted to the particulate PDE4 activities and its timing was consistent with the half-life of Sertoli cell PGs. It did not result from alterations in Pde4d variants expression, the major FSH-regulated PDE4 in Sertoli cells. Moreover, lack of changes in the particulate levels of major immunoreactive 75 kDa and 90 kDa PDE4D proteins, corresponding likely to short PDE4D1 and long PDE4D3/D8/D9 isoforms respectively, suggested that the decrease in FSH-stimulated of PDE4 activities in chlorate- and PNPX-treated cells at the end of the 24-h incubation period resulted from the increased reversal of the activated particulate PDE4(D) activities back to unstimulated levels. By controlling FSH-stimulated particulate PDE4 inactivation through a still unknown mechanism (sustained activation of PKA or reduction of phosphoprotein phosphatase activities), cell PGs could be involved in the alteration of cAMP response to FSH accompanying the transition of Sertoli cells from proliferative to non-proliferative differentiated state.