Emilia Middea

Estrogen Receptor α Binds to Peroxisome Proliferator–Activated Receptor Response Element and Negatively Interferes with Peroxisome Proliferator–Activated Receptor γ Signaling in Breast Cancer Cells

Purpose: The molecular mechanisms involved in the repressive effects exerted by estrogen receptors (ER) on peroxisome proliferator–activated receptor (PPAR) γ–mediated transcriptional activity remain to be elucidated. The aim of the present study was to provide new insight into the crosstalk between ERα and PPARγ pathways in breast cancer cells. Experimental Design: Using MCF7 and HeLa cells as model systems, we did transient transfections and electrophoretic mobility shift assay and chromatin immunoprecipitation studies to evaluate the ability of ERα to influence PPAR response element–mediated transcription. A possible direct interaction between ERα and PPARγ was ascertained by coimmunoprecipitation assay, whereas their modulatory role in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway was evaluated by determining PI3K activity and AKT phosphorylation. As a biological counterpart, we investigated the growth response to the cognate ligands of both receptors in hormone-dependent MCF7 breast cancer cells. Results: Our data show for the first time that ERα binds to PPAR response element and represses its transactivation. Moreover, we have documented the physical and functional interactions of ERα and PPARγ, which also involve the p85 regulatory subunit of PI3K. Interestingly, ERα and PPARγ pathways have an opposite effect on the regulation of the PI3K/AKT transduction cascade, explaining, at least in part, the divergent response exerted by the cognate ligands 17β-estradiol and BRL49653 on MCF7 cell proliferation. Conclusion: ERα physically associates with PPARγ and functionally interferes with PPARγ signaling. This crosstalk could be taken into account in setting new pharmacologic strategies for breast cancer disease.

Human male gamete endocrinology: 1alpha, 25-dihydroxyvitamin D3 (1,25(OH)2D3) regulates different aspects of human sperm biology and metabolism

BackgroundA wider biological role of 1alpha,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, in tissues not primarily related to mineral metabolism was suggested. Recently, we evidenced the ultrastructural localization the 1,25(OH)2D3 receptor in the human sperm. However, the 1,25(OH)2D3 action in human male reproduction has not yet been clarified.Methods and ResultsBy RT-PCR, Western blot and Immunofluorescence techniques, we demonstrated that human sperm expresses the 1,25(OH)2D3 receptor (VDR). Besides, 25(OH)D3-1 alpha-hydroxylase, evidenced by Western blot analysis, indicated that in sperm 1,25(OH)2D3 is locally produced, highlighting the potential for autocrine-paracrine responses. 1,25(OH)2D3 through VDR, increased intracellular Ca2+ levels, motility and acrosin activity revealing an unexpected significance of this hormone in the acquisition of fertilizing ability. In sperm, 1,25(OH)2D3 through VDR, reduces triglycerides content concomitantly to the increase of lipase activity. Rapid responses stimulated by 1,25(OH)2D3 have been observed on Akt, MAPK and GSK3 implying that this secosteroid is involved in different sperm signalling pathways.ConclusionOur data extended the role of 1,25(OH)2D3 beyond its conventional physiological actions, paving the way for novel therapeutic opportunities in the treatment of the male reproduction disorders.

Peroxisome proliferator‐activated receptor (PPAR)γ is expressed by human spermatozoa: Its potential role on the sperm physiology

The peroxisome proliferation‐activated receptor gamma (PPARγ) is mainly expressed in the adipose tissue and integrates the control of energy, lipid, and glucose homeostasis. The present study, by means of RT‐PCR, Western blot, and immunofluorescence techniques, demonstrates that human sperm express the PPARγ. The functionality of the receptor was evidenced by 15‐deoxy‐12,14‐prostaglandin J2 (PGJ2) and rosiglitazone (BRL) PPARγ‐agonists that were tested on capacitation, acrosome reaction, and motility. Both treatments also increase AKT phosphorylations and influence glucose and lipid metabolism in sperm. The specificity of PGJ2 and BRL effects through PPARγ on human sperm was confirmed by an irreversible PPARγ antagonist, GW9662. Our findings provide evidence that human sperm express a functional PPARγ whose activation influences sperm physiology. In conclusion, the presence of PPARγ in male gamete broadens the field of action of this nuclear receptor, bringing us to look towards sperm as an endocrine mobile unit independent of the systemic regulation. J. Cell. Physiol. 209: 977–986, 2006.

Expression of nuclear insulin receptor substrate 1 in breast cancer

Background: Insulin receptor substrate 1 (IRS-1), a cytoplasmic protein transmitting signals from the insulin and insulin-like growth factor 1 receptors, has been implicated in breast cancer. Previously, it was reported that IRS-1 can be translocated to the nucleus and modulate oestrogen receptor α (ERα) activity in vitro. However, the expression of nuclear IRS-1 in breast cancer biopsy specimens has never been examined. Aims: To assess whether nuclear IRS-1 is present in breast cancer and non-cancer mammary epithelium, and whether it correlates with other markers, especially ERα. Parallel studies were carried out for the expression of cytoplasmatic IRS-1. Methods: IRS-1 and ERα expression was assessed by immunohistochemical analysis. Data were evaluated using Pearson’s correlation, linear regression and receiver operating characteristic analysis. Results: Median nuclear IRS-1 expression was found to be low in normal mammary epithelial cells (1.6%) and high in benign tumours (20.5%), ductal grade 2 carcinoma (11.0%) and lobular carcinoma (∼30%). Median ERα expression in normal epithelium, benign tumours, ductal cancer grade 2 and 3, and lobular cancer grade 2 and 3 were 10.5, 20.5, 65.0, 0.0, 80 and 15%, respectively. Nuclear IRS-1 and ERα positively correlated in ductal cancer (p<0.001) and benign tumours (p<0.01), but were not associated in lobular cancer and normal mammary epithelium. In ductal carcinoma, both nuclear IRS-1 and ERα negatively correlated with tumour grade, size, mitotic index and lymph node involvement. Cytoplasmic IRS-1 was expressed in all specimens and positively correlated with ERα in ductal cancer. Conclusions: A positive association between nuclear IRS-1 and ERα is a characteristic for ductal breast cancer and marks a more differentiated, non-metastatic phenotype.

Fibronectin and type IV collagen activate ERalpha AF-1 by c-Src pathway: effect on breast cancer cell motility

The expression of estrogen receptor alpha (ERα) is generally associated with a less invasive and aggressive phenotype in breast carcinoma. In an attempt to understand the role of ERα in regulating breast cancer cells invasiveness, we have demonstrated that cell adhesion on fibronectin (Fn) and type IV Collagen (Col) induces ERα-mediated transcription and reduces cell migration in MCF-7 and in MDA-MB-231 cell lines expressing ERα. Analysis of deleted mutants of ERα indicates that the transcriptional activation function (AF)-1 is required for ERα-mediated transcription as well as for the inhibition of cell migration induced by cell adhesion on extracellular matrix (ECM) proteins. In addition, the nuclear localization signal region and some serine residues in the AF-1 of the ERα are both required for the regulation of cell invasiveness as we have observed in HeLa cells. It is worth noting that c-Src activation is coincident with adhesion of cells to ECM proteins and that the inhibition of c-Src activity by PP2 or the expression of a dominant-negative c-Src abolishes ERα-mediated transcription and partially reverts the inhibition of cell invasiveness in ERα-positive cancer cells. These findings address the integrated role of ECM proteins and ERα in influencing breast cancer cell motility through a mechanism that involves c-Src and seems not to be related to a specific cell type.

Human sperm physiology: Estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ) influence sperm metabolism and may be involved in the pathophysiology of varicocele-associated male infertility†

The mechanisms by which varicocele affects fertility remain undetermined. Estrogens play a key role in the human male reproduction and human sperm expresses the estrogen receptors (ERs) and aromatase. In this study, by Western blotting we evidenced the ERs content concomitantly in healthy sperm and in oligoastenoteratozoospermic (OAT) samples without and with varicocele. In varicocele a strong reduction of the ERβ was observed, while the ERα was almost absent. Besides, transmission electron microscopy (TEM) confirmed the reduction of ERs expression in “varicocele” sperm, indicating that varicocele has a detrimental effect on sperm structure at molecular level. To further define the estrogen significance in male gamete and the pathophysiology of varicocele we investigated both the expression of ERα and ERβ in normal and pathologic sperm samples as well as we evaluated estradiol (E2) action on lipid and glucose sperm metabolism. Responses to E2 treatments on cholesterol efflux, protein tyrosine phosphorylations, motility, and acrosin activity in varicocele sperm were reduced or absent. The evaluation of the triglycerides content, lipase and acyl‐CoA dehydrogenase activities, suggest that E2 exerts a lipolytic effect on human sperm metabolism. Concerning glucose metabolism, it appears that E2 induces G6PDH activity concomitantly to the insulin secretion. In “varicocele” sperm, the E2 did not induce energy expenditure. OAT sperm had E2‐responsiveness but in a lesser extent with respect healthy sperm. This study discovered a novel role for E2/ERs in human sperm physiology, since they modulate sperm metabolism and new detrimental effects related to the pathophysiology of the varicocele condition. J. Cell. Physiol. 226: 3403–3412, 2011.

17β-Estradiol enhances α5 integrin subunit gene expression through ERα–Sp1 interaction and reduces cell motility and invasion of ERα-positive breast cancer cells

In breast tumors the expression of estrogen receptor alpha (ERα) is known to be associated with a more favorable prognosis. ERα expression has been reported to reduce the metastatic potential of breast cancer cells. Recently, we have observed that extracellular matrix proteins activate ERα and that both liganded and unliganded receptor modulate cell invasiveness acting at nuclear level. To explain the mechanisms by which ERα regulates cell adhesion, we have evaluated the expression of α5β1 integrin, prevalently expressed in stationary cells, in response to 17β-estradiol (E2). Here we show that E2/ERα increases the expression of integrin α5β1 through Sp1-mediated binding to a GC-rich region located upstream of an ERE half-site in the 5′ flanking region of the α5 gene forming a ternary ERα–Sp1-DNA complex. Estrogen responsiveness of the α5 gene promoter, as observed in HeLa cells, underlies a general mechanism of regulation which is not strictly linked to the cell type. Our data reveal novel insight into the molecular mechanisms sustaining the reduced invasiveness of ERα expressing cells demonstrating that α5β1 integrin expression is related to the maintenance of the stationary status of the cells, counteracting E2/ERα capability to enhance breast cancer cell migration and invasion.

Fas ligand expression in TM4 sertoli cells is enhanced by estradiol “in situ” production†

The testis is an immunologically privileged site of the body where Sertoli cells work on to favor local immune tolerance by testicular autoantigens segregation and immunosuppressive factors secretion. Fas/Fas Ligand (FasL) system, expressed prevalently in Sertoli cells, has been considered to be one of the central mechanisms in testis immunological homeostasis. In different cell lines it has been reported that the proapoptotic protein FasL is regulated by 17‐β estradiol (E2). Thus, using as experimental model mouse Sertoli cells TM4, which conserve a large spectrum of functional features present in native Sertoli cells, like aromatase activity, we investigated if estradiol “in situ” production may influence FasL expression. Our results demonstrate that an aromatizable androgen like androst‐4‐ene‐3,17‐dione (Δ4) enhanced FasL mRNA, protein content and promoter activity in TM4 cells. The treatment with N6,2′‐O‐dibutyryladenosine‐3′‐5′‐cyclic monophosphate [(Bu)2cAMP] (simulating FSH action), that is well known to stimulate aromatase activity in Sertoli cells, amplified Δ4 induced FasL expression. Functional studies of mutagenesis, electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays revealed that the Sp‐1 motif on FasL promoter was required for E2 enhanced FasL expression in TM4 cells. These data let us to recruit FasL among those genes whose expression is up‐regulated by E2 through a direct interaction of ERα with Sp‐1 protein. Finally, evidence that an aromatizable androgen is able to increase FasL expression suggests that E2 production by aromatase activity may contribute to maintain the immunoprivilege status of Sertoli cells. J. Cell. Physiol. 211: 448–456, 2007.