Biomedical subjects

Stefania Catalano

Publications and source records attributed to Stefania Catalano.

13 recordsLinked to original sources

PubMed2007

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-beta 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 (Delta4) enhanced FasL mRNA, protein content and promoter activity in TM4 cells. The treatment with N(6),2'-O-dibutyryladenosine-3'-5'-cyclic monophosphate [(Bu)(2)cAMP] (simulating FSH action), that is well known to stimulate aromatase activity in Sertoli cells, amplified Delta4 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 ERalpha 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.

Androstenedione↗

PubMed2006

Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells.

The aim of the present study was to provide new mechanistic insight into the growth arrest and apoptosis elicited by peroxisome proliferator-activated receptor (PPAR)gamma in breast cancer cells. We ascertained that PPARgamma mediates the inhibition of cycle progression in MCF7 cells exerted by the specific PPARgamma agonist rosiglitazone [BRL4653 (BRL)], because this response was no longer notable in the presence of the receptor antagonist GW9662. We also provided evidence that BRL is able to up-regulate mRNA and protein levels of the tumor suppressor gene p53 and its effector p21(WAF1/Cip1) in a time- and dose-dependent manner. Moreover, in transfection experiments with deletion mutants of the p53 gene promoter, we documented that the nuclear factor-kappaB sequence is required for the transcriptional response to BRL. Interestingly, EMSA showed that PPARgamma binds directly to the nuclear factor-kappaB site located in the promoter region of p53, and chromatin immunoprecipitation experiments demonstrated that BRL increases the recruitment of PPARgamma on the p53 promoter sequence. Next, both PPARgamma and p53 were involved in the cleavage of caspases-9 and DNA fragmentation induced by BRL, given that GW9662 and an expression vector for p53 antisense blunted these effects. Our findings provide evidence that the PPARgamma agonist BRL promotes the growth arrest and apoptosis in MCF7 cells, at least in part, through a cross talk between p53 and PPARgamma, which may be considered an additional target for novel therapeutic interventions in breast cancer patients.

Base Pairing↗

PubMed2006

Peroxisome proliferator-activated receptor (PPAR)gamma is expressed by human spermatozoa: its potential role on the sperm physiology.

The peroxisome proliferation-activated receptor gamma (PPARgamma) 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 PPARgamma. The functionality of the receptor was evidenced by 15-deoxy-12,14-prostaglandin J(2) (PGJ2) and rosiglitazone (BRL) PPARgamma-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 PPARgamma on human sperm was confirmed by an irreversible PPARgamma antagonist, GW9662. Our findings provide evidence that human sperm express a functional PPARgamma whose activation influences sperm physiology. In conclusion, the presence of PPARgamma 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.

Acrosome Reaction↗

PubMed2005

Estrogen receptor alpha binds to peroxisome proliferator-activated receptor response element and negatively interferes with peroxisome proliferator-activated receptor gamma 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) gamma-mediated transcriptional activity remain to be elucidated. The aim of the present study was to provide new insight into the crosstalk between ERalpha and PPARgamma 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 ERalpha to influence PPAR response element-mediated transcription. A possible direct interaction between ERalpha and PPARgamma was ascertained by co-immunoprecipitation 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 ERalpha binds to PPAR response element and represses its transactivation. Moreover, we have documented the physical and functional interactions of ERalpha and PPARgamma, which also involve the p85 regulatory subunit of PI3K. Interestingly, ERalpha and PPARgamma 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 17beta-estradiol and BRL49653 on MCF7 cell proliferation. CONCLUSION: ERalpha physically associates with PPARgamma and functionally interferes with PPARgamma signaling. This crosstalk could be taken into account in setting new pharmacologic strategies for breast cancer disease.

Breast Neoplasms↗

PubMed2005

Leptin secretion by human ejaculated spermatozoa.

INTRODUCTION: Leptin action is a dynamic area of investigation that continues to broaden beyond the basic lipostatic model originally envisaged. Here, we show that leptin is expressed in and secreted from human ejaculated spermatozoa. METHODS: By RT-PCR, Western blot, and immunofluorescence techniques, we have demonstrated that human sperm express leptin. RIA method evidenced leptin secretion. Phosphatidyl-inositol-kinase-3 (PI3K)/Akt pathway was examined by PI3K activity assay and Western blot. Leptin and insulin regulation of glycogen synthesis was evaluated by glycogen synthase activity (GSA). RESULTS: The large differences of leptin secretion between uncapacitated and capacitated sperm suggest a functional role for leptin in capacitation. Indeed, in uncapacitated sperm, leptin enhances both cholesterol efflux and protein tyrosine phosphorylation. In uncapacitated sperm, both insulin and leptin increased PI3K activity, Akt S473, and glycogen synthase kinase-3 S9 phosphorylation. Interestingly, during capacitation, concomitantly to the massive release of both hormones, we observed a strong reduction in the phosphorylation of glycogen synthase kinase-3 S9, kinase downstream of Akt that regulates the glycogen synthase. Our results from GSA showed that the enzymatic activity was significantly higher in uncapacitated than in capacitated sperm. Particularly, in uncapacitated sperm, GSA appeared to depend on the hormones concentration, because the enzymatic activity was stimulated at low doses, whereas it was inhibited at high doses. Moreover, both leptin and insulin regulate in autocrine fashion sperm glycogen synthesis. CONCLUSION: Leptin secretion by sperm suggests that the male gamete may be able to modulate its metabolism independently by systemic leptin. These data open new considerations about leptin significance in male fertility.

Blotting, Southern↗

PubMed2004

Autocrine regulation of insulin secretion in human ejaculated spermatozoa.

A striking feature of insulin expression is its almost complete restriction to beta-cells of the pancreatic islet in normal mammals. Here we show that insulin is expressed in and secreted from human ejaculated spermatozoa. Both insulin transcript and protein were detected. In addition, the large differences in insulin secretion, assessed by RIA, between noncapacitated and capacitated sperm suggest a role for insulin in capacitation. Insulin had an oscillatory secretory pattern involving glucose dose-dependent increases and significant decreases during the blockage of an insulin autocrine effect. It appears that the effect of glucose on the fertilizing ability of sperm is mediated by glucose metabolism through the pentose phosphate pathway. Then we evaluated the autocrine effect of sperm insulin on glucose metabolism by studying the activity of glucose-6-phosphate dehydrogenase, the key rate-limiting enzyme in the pentose phosphate pathway. The simultaneous decrease in both insulin release and glucose-6-phosphate dehydrogenase activity induced by blocking the autocrine insulin effect with three different procedures (blockage of insulin release by nifedipine, immune neutralization of the released insulin by antiinsulin serum, and blockage of an insulin intracellular effector such as phosphotidylinositol 3-kinase by wortmannin) strongly suggests a physiological role of sperm insulin on these two events. Insulin secretion by spermatozoa may provide an autocrine regulation of glucose metabolism based on their energetic needs independent of systemic insulin. In conclusion, these data open a new area of study in male reproduction.

Autocrine Communication↗

PubMed2004

Leptin induces, via ERK1/ERK2 signal, functional activation of estrogen receptor alpha in MCF-7 cells.

Leptin is a hormone with multiple biological actions, produced predominantly by adipose tissue. In humans, plasma levels correlate with total body fat, and high concentrations occur in obese women. Among its functions, leptin is able to stimulate normal and tumor cell growth. We demonstrated that leptin induces aromatase activity in MCF-7 cells evidencing its important role in enhancing in situ estradiol production and promoting estrogen-dependent breast cancer progression. Estrogen receptor alpha (ERalpha), which plays an essential role in breast cancer development, can be transcriptionally activated in a ligand-independent manner. Taking into account that unliganded ERalpha is an effector of mitogen-activated protein kinase (MAPK) signal and that leptin is able, via Janus kinase, to activate the Ras-dependent MAPK pathway, in the present study we investigate the ability of leptin to transactivate ERalpha. We provided evidence that leptin is able to reproduce the classic features of ERalpha transactivation in a breast cancer cell line: nuclear localization, down-regulation of its mRNA and protein levels, and up-regulation of a classic estrogen-dependent gene such as pS2. Transactivation experiments with a transfected reporter gene for nuclear ER showed an activation of ERalpha either in MCF-7 or in HeLa cells. Using a dominant negative ERK2 or the MAPK inhibitor PD 98059, we showed that leptin activates the ERalpha through the MAPK pathway. The N-terminal transcriptional activation function 1 appears essential for the leptin response. Finally, it is worth noting that leptin exposure potentates also the estradiol-induced activation of ERalpha. Thus, we are able to demonstrate that the amplification of estrogen signal induced by leptin occurs through an enhancing in situ E(2) production as well as a direct functional activation of ERalpha.

Adipose Tissue↗

PubMed2004

Low calcium intake is associated with decreased adrenal androgens and reduced bone age in premenarcheal girls in the last pubertal stages.

In 50 premenarcheal girls selected from the lowest and highest end of the calcium-intake distribution of a large population sample, we evaluated bone mineral density (BMD), together with the following hormonal-metabolic parameters: androstenedione (ASD), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), testosterone (T), estradiol (E2), the apparent free fractions of T (AFTC) and E2 (AFEC), osteocalcin (OC), parathyroid hormone (PTH), and 25-hydroxyvitamin D. Dietary calcium was assessed by 3-day food records, and BMD was measured at ultradistal (ud) and proximal (pr) radial sites, using dual-energy X-ray absorptiometry. Calcium intake, which was below the recommended levels set for the Italian population and below the recommended daily allowance (RDA) in both subgroups of girls, did not show any apparent relationship with ud- and pr- BMD. However, despite the similar chronological age of the two premenarcheal groups, in the low-calcium consumers, we found lower bone age, delayed pubertal development, and lower circulating adrenal androgens. Of interest, in girls who had a low calcium intake, PTH levels were significantly higher. In all premenarcheals, we observed that DHEA, T, and AFTC were positively correlated with bone age and with bone density at both radial sites. Even though bone density at the two radial sites did not show any apparent relationship to calcium consumption, the increased mean PTH in the girls with low calcium intake seems to underscore the hormonal attempt in maintaining calcium homeostasis. In conclusion, low calcium intake and reduced levels of adrenal androgens, leading to decreased bone age and delayed pubertal development, indicate a link between calcium intake, the hormonal milieu, and skeletal maturation.

Absorptiometry, Photon↗

PubMed2004

Estrogen receptor (ER)alpha and ER beta are both expressed in human ejaculated spermatozoa: evidence of their direct interaction with phosphatidylinositol-3-OH kinase/Akt pathway.

Human and animal models have evidenced how estrogen insufficiency is associated with abnormal spermatogenesis and male infertility. We previously demonstrated that estradiol is able to influence both capacitation and acrosome reaction in human ejaculated spermatozoa. It remains to be elucidated whether the biochemical changes induced by estradiol, in a rapid nongenomic way, are mediated by a single estrogen receptor (ER) or by the two ER subtypes, ER alpha and ER beta. In the present study, we have first demonstrated the concomitant expression of ER beta and ER alpha in human ejaculated spermatozoa. By RT-PCR and Southern blot, transcripts of both ERs were detected. Western blot analysis showed ER alpha and ER beta proteins at the same size as the "classical" ERs. The localization of ER alpha and ER beta with the immunocytochemistry shows a differential distribution of the two ER subtypes, the former being prevalently located in the midpiece, but the latter being in the tail. Estradiol has been associated with sperm longevity; however, the mechanism through which estradiol acts in sperm survival was never investigated. Upon estradiol exposure, we observed an enhanced phosphorylation of the proteins involved in the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway like PDK1, Akt, GSK-3, Bcl-2, together with ERK1/2, which was also involved in cell survival signals. Moreover, such phosphorylations were reduced in the presence of ICI 182, 780, addressing the role of estradiol and ERs in sperm survival. For instance we have provided, for the first time, a different interaction of the two ERs with the PI3K/Akt pathway, because ER alpha interacts with the p55 regulatory subunit of PI3K, whereas ER beta interacts with Akt1. However, it still remains to be elucidated whether the functional role of each of the ER subtypes in sperm survival signaling is redundant or distinct.

Blotting, Southern↗

PubMed2003

Oxidative stress in diabetes-induced endothelial dysfunction involvement of nitric oxide and protein kinase C.

Reactive oxygen species (ROS) formation plays a major role in diabetes-induced endothelial dysfunction, though the molecular mechanism(s) involved and the contribution of nitric oxide (NO) are still unclear. This study using bovine retinal endothelial cells was aimed at assessing (i) the role of oxygen-dependent vs. NO-dependent oxidative stress in the endothelial cell permeability alterations induced by the diabetic milieu and (ii) whether protein kinase C (PKC) activation ultimately mediates these changes. Superoxide, lipid peroxide, and PKC activity were higher under high glucose (HG) vs. normal glucose throughout the 30 d period. Nitrite/nitrate and endothelial NO synthase levels increased at 1 d and decreased thereafter. Changes in monolayer permeability to 125I-BSA induced by 1 or 30 d incubation in HG or exposure to advanced glycosylation endproduct were reduced by treatment with antioxidants or PKC inhibitors, whereas NO blockade prevented only the effect of 1 d HG. HG-induced changes were mimicked by a PKC activator, a superoxide generating system, an NO and superoxide donor, or peroxynitrite (attenuated by PKC inhibition), but not a NO donor. The short-term effect of HG depends on a combined oxidative and nitrosative stress with peroxynitrite formation, whereas the long-term effect is related to ROS generation; in both cases, PKC ultimately mediates permeability changes.

Animals↗

PubMed2003

Leptin enhances, via AP-1, expression of aromatase in the MCF-7 cell line.

Leptin, a product of adipocytes, is involved in the regulation of body weight and results strongly correlated to body fat content. An excess of fat mass represents a breast cancer risk factor particularly in postmenopausal women, where estrogen production by adipose tissue through its own aromatase activity stimulates tumor progression. Leptin stimulates estrogen production through the increase of aromatase expression and activity in human luteinized granulosa cells and adipose stromal cells. In the present study, we have examined the possible link that exists between leptin and breast cancer, focusing our attention on the direct effect of leptin on aromatase activity, which may enhance estrogen production and induce tumor cell growth stimulation. We have shown that leptin enhances aromatase mRNA expression, aromatase content, and its enzymatic activity in MCF-7. Aromatase expression appears to be regulated by tissue-specific promoter. It has been demonstrated that promoters II and 1.3 are the major promoters that drive aromatase expression in MCF-7. Transient transfection experiments using vector containing human aromatase promoters II and 1.3 sequence fused with luciferase reporter gene demonstrated that leptin is able to activate this promoter. In the presence of either mitogen-activated protein kinase inhibitor PD 98059 or ERK2 dominant negative as well as in the presence of STAT3 dominant negative, the stimulatory effects of leptin on aromatase promoter, enzymatic activity, and aromatase protein content were inhibited. Functional studies of mutagenesis and electrophoretic mobility shift assay revealed that the AP-1 motif is important in determining the up-regulatory effects induced by leptin on aromatase expression in MCF-7.

Androstenedione↗

PubMed2003

Triiodothyronine decreases the activity of the proximal promoter (PII) of the aromatase gene in the mouse Sertoli cell line, TM4.

Estrogens and thyroid hormones play a significant role in regulating functions and development of the testis. The synthesis of estrogens from androgens is catalyzed by the enzyme complex termed aromatase, which in the testis displays an age-related cellular compartmentalization, primarily in Sertoli cells in immature animals, whereas in adults it is expressed in Leydig and germ cells. T3 induces a precocious terminal differentiation of prepubertal Sertoli cells together with a dramatic decrease of their aromatase activity. In the present work, we have examined the mechanism by which T3 exerts this inhibitory action on aromatase expression. As an experimental model, we used the mouse Sertoli cell line TM4, which conserves a large spectrum of functional features present in immature Sertoli cells. For instance, after revealing the presence of aromatase by immunocytochemistry and measuring its enzymatic activity, we confirmed in this cell line the functional events previously characterized in primary cultures of immature rat Sertoli cells: 1) a strong stimulation of aromatase activity by dibutyryl-cAMP [(Bu)2cAMP] (simulating FSH action); and 2) the inhibition of aromatase activity by incubation with T3 under basal condition and after (Bu)2cAMP stimulation. After identifying promoter II as the regulatory region located immediately upstream of the transcriptional initiation site in the TM4 cell line by rapid amplification of cDNA ends analysis, we conducted experiments to examine the molecular mechanism by which thyroid hormones modulate aromatase gene expression in this cell line. TM4 cells were transfected with plasmids containing different segments of the rat promoter II sequence ligated to a luciferase reporter gene. Analysis of the activities of these promoter fusions demonstrated that T3 inhibits basal and (Bu)2cAMP-stimulated activity of the aromatase promoter. This effect was not revealed in T3-treated cells transfected with construct in which the steroidogenic factor-1 (SF-1) response element was mutated. These results indicate that the inhibitory effect of T3 requires the integrity of the SF-1 response element and are further supported in the EMSA. The EMSA experiments demonstrated that thyroid hormone/thyroid receptor alpha1 complex (TH/TRalpha1) is able to compete with SF-1 in binding to oligonucleotides containing an SF-1 motif, an element essential for the activity of the PII aromatase promoter. The findings suggest that the binding of the thyroid hormone/thyroid receptor alpha1 complex to the SF-1 motif is the molecular mechanism by which T3 exerts an inhibitory effect on aromatase gene expression in the TM4 cell line.

Animals↗

PubMed2003

Towards a physiological role for cytochrome P450 aromatase in ejaculated human sperm.

BACKGROUND: Advances in the definition of the function and the mechanism of estrogen action in different tissues have come from human and animal models of estrogen insufficiency. Recently we have demonstrated that aromatase is present and biologically active in human ejaculated sperm, suggesting that autonomous estradiol sperm production may influence sperm functions. In the present study we investigate a possible physiological role for enzymatically active P450 aromatase in human ejaculated sperm. METHODS AND RESULTS: To confirm the presence of mRNA coding for P450 aromatase, total RNA isolated from human sperm underwent RT-PCR and then Southern blot analysis. In non-capacitating medium, we observed that only estradiol and aromatizable steroids were able to increase sperm motility/migration; concomitantly they enhanced protein tyrosine phosphorylation and increased p-44/42 extracellular signal-regulated kinase activity. When we tested acrosin activity, it emerged that estradiol and aromatizable androgens were also able to induce the acrosome reaction evaluated by two different cytological staining techniques (triple-stain and fluorescein isothiocyanate-Pisum sativum agglutinin). All these events were enhanced by the 2'-O-dibutyryladenosine-3',5'-cyclic monophosphate and inhibited in the presence of the specific aromatase inhibitor, letrozole. CONCLUSIONS: From this study, it appears that a link exists between the locally produced estradiol (from ejaculated sperm), sperm capacitation and the acrosome reaction. The induction of both events by aromatizable androgens in the absence of exogenous mediators suggests that estrogen biosynthesis in ejaculated sperm is a process that may influence the intrinsic sperm fertilizing capability.

Acrosin↗