Conventional progesterone receptors (PR) B and PRA are expressed by human chondrocytes and can be involved in the pathogenesis of osteoarthritis.

To evaluate the expression, location and role of progesterone receptors (PRs) A and B in human chondrocytic cell lines, Western blotting, real time PCR analyses, transmission electron microscopy and immunogold assays were performed. By transfection and co-transfection assays, the influence of progesterone (OHPg) on estrogen receptor alpha (ERα) promoter activity was investigated. MTT and pAKT documented OHPg effects on chondrocytes survival. The PR-B and PR-A were both observed in human chondrocytes. The PR-B was evidenced both in the nucleus and in the cytosol of the cells. OHPg, through PR-B, induced ERα expression by acting at the ER promoter level affecting chondrocytes survival. We reported for the first time the expression of PRs in human chondrocytes. Interestingly, we described a novel mechanism via progesterone induction of ERα, which may explain, at least in part, the dramatic rise in OA prevalence among postmenopausal women.

Effects of Resveratrol on p66Shc phosphorylation in cultured prostate cells.

There is increasing evidence that diet plays a crucial role in age-related diseases and cancer. Oxidative stress is a conceivable link between diet and diseases, thus food antioxidants, counteracting the damage caused by oxidation, are potential tools for fight age-related diseases and cancer. Resveratrol (RSV), a polyphenolic antioxidant from grapes, has gained enormous attention particularly because of its ability to induce growth arrest and apoptosis in cancer cells, and it has been proposed as both chemopreventive and therapeutic agent for cancer and other diseases. Even though the effects of RSV have been studied in prostate cancer cells and animal models, little is known about its effects on normal cells and tissues. To address this issue, we have investigated the effects of RSV on EPN cells, a human non-transformed prostate cell line, focusing on the relationship between RSV and p66Shc, a redox enzyme whose activities strikingly intersect those of RSV. p66Shc activity is regulated by phosphorylation of serine 36 (Ser36) and has been related to mitochondrial oxidative stress, apoptosis induction, regulation of cell proliferation and migration. Here we show that RSV inhibits adhesion, proliferation and migration of EPN cells, and that these effects are associated to induction of dose- and time-dependent p66Shc-Ser36 phosphorylation and ERK1/2 de-phosphorylation. Moreover, we found that RSV is able to activate also p52Shc, another member of the Shc protein family. These data show that RSV affects non-transformed prostate epithelial cells and suggest that Shc proteins may be key contributors of RSV effects on prostate cells.

Sperm metabolism in pigs: a role for peroxisome proliferator-activated receptor gamma (PPARγ).

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor expressed predominantly in adipose tissue, also implicated in energy homeostasis. In this study, we used western blotting and immunofluorescence techniques to demonstrate for the first time that pig spermatozoa express PPARγ. To investigate the functional role of PPARγ in pig sperm, we evaluated its action on different events that characterize the biology of sperm cells, i.e. motility, capacitation, viability and acrosome reaction, using the PPARγ-agonist 15-deoxy-12,14-prostaglandin J2 (PGJ2). In responses to PGJ2 treatment, motility, cholesterol efflux and tyrosine phosphorylation were increased, which broadens the role of PPARγ from that previously described in the literature, as it also acts to improve sperm functionality. To further our understanding of the significance of PPARγ in pig sperm, we focused its effects on lipid and glucose metabolism. Evaluation of triglyceride content and lipase, acyl-CoA dehydrogenase and G6PDH activities suggests that PPARγ induces energy expenditure in pig spermatozoa. These data represent a meaningful advance in the field of sperm energy metabolism. Taken together, our results demonstrate for the first time that PPARγ is expressed by pig sperm, thus improving its functionalities in terms of motility, capacitation, acrosome reaction, survival and metabolism.

Progesterone through progesterone receptors affects survival and metabolism of pig sperm.

Progesterone receptors (PR) through interaction with the specific ligand progesterone (PRG), play a central coordinate role in different reproductive events. In this study conventional PR were identified in boar spermatozoa by Western blotting. Immunofluorescence techniques indicate that PR are located at sperm acrosomal region, suggesting a possible role in the oocyte fertilization events. Treatment with 17-hydroxyprogesterone (17-OHP) enhanced viability and induced cholesterol efflux, serine and tyrosine phosphorylation, p-Bcl2, p-Akt that are known hallmarks of capacitation in sperm. The analysis of the triglyceride contents, lipase and acyl-CoA dehydrogenase activities, as well as the G6PDH activity, was conducted so as to address whether there was an increase in energy expenditure via 17-OHP through the PR. Taken together these results, particularly the 17-OHP action on boar sperm lipid and glucose metabolism, give emphasis to the role of PR in sperm physiology within the oviductal environment. Moreover the present study highlights, the effect of PRG via PR on boar sperm survival, renewing the role of this hormone in male reproduction as candidate for boar fertility. Thus the present research contributes to further elucidating the role of progesterone on the physiological regulation of the most relevant spermatozoa functions for a successful fertilization.

Bergapten induces ER depletion in breast cancer cells through SMAD4-mediated ubiquitination.

ERα function is crucial for the development of normal mammary gland as well as in the process of progression of breast cancer cells. Signals that target receptor levels contribute to regulate estrogens effects in the cells. An intricate cross-regulation has been documented between ERα and TGF-ß down-stream molecules: SMAD2, SMAD3, and SMAD4, that can bind ERα and regulate their signaling. Thus, identification of natural anticancer drugs able to influence the latter molecule might provide alternative choices for breast cancer treatment. Taking into account our previous published data we wanted to study the effect of 5-Methoxypsoralen (bergapten) on ERα and on TGF-ß pathway. We reported that bergapten, a coumarin containing compound, effectively depletes ERα in MCF-7 breast cancer sensitive cells and in tamoxifen-resistant clone. The decrease of ERα protein after bergapten treatment results from the ubiquitine-proteasome pathway as demonstrated by the use of MG-132. IP experiments with ER antibody, demonstrated that the protein has physical interaction with SMAD4 and poly-ubiquitine and the amount of ubiquitinated receptor, linked to SMAD4, is greater under bergapten. The crucial role played by SMAD4, in this process, emerges from the observation that in breast cancer cells, silencing of SMAD4, resulted in increased expression of endogenous ERα in both control and bergapten-treated cells, compared to wild- type cells. The same results were confirmed in siRNA TGF-ß RII cells. The results suggest a novel negative regulation of ERα by TGF-ß/SMAD4 in breast cancer cells and indicate that the SMAD4 protein is involved in the degradation of ERα induced by bergapten. We propose that bergapten may efficiently act as a natural antitumoral agent, able to deplete ERα from breast cancer tamoxifen-sensitive and resistant cells, thereby retraining the effect of membrane signals targeting ERα and in such way its mitogenic potentiality.

Expression of cyclooxygenase-1 (COX-1) and COX-2 in human male gametes from normal patients, and those with varicocele and diabetes: a potential molecular marker for diagnosing male infertility disorders.

Rising rates of varicocele and diabetes mellitus (DM) pose a significant problem to human fertility. Recent studies have pointed out the impact of cyclooxygenase (COX) in the regulation of testicular function and male fertility. Prominent COX-2 expression has been described recently in the testes of infertile patients, but little is known about the role and identity of COX isoforms in human sperm under certain disease states such as varicocele and DM. We therefore examined the expression profile and ultrastructural localization of COX-1 and COX-2 concomitantly in semen samples from healthy donors, and patients with varicocele and DM. Using Western blotting assay, 'varicocele' and 'diabetic' sperm showed enhanced COX isoforms expression with respect to the 'healthy' sperm. Immunogold labeling revealed human sperm anatomical regions containing COX-1 and COX-2, confirming their increased expression in pathological samples. Our data demonstrate that both COX isoforms are upregulated in the spermatozoa of varicocele and diabetic patients, suggesting the harmful effect of the diseases also at the sperm molecular level, going beyond the abnormal morphology described to date. In conclusion, COX enzymes may possess a biological relevance in the pathogenesis and/or maintenance of male factor infertility associated with varicocele and DM, and may be considered additional molecular markers for the diagnosis of male infertility disorders.

Conventional progesterone receptors (PR) B and PRA are expressed in human spermatozoa and may be involved in the pathophysiology of varicocoele: a role for progesterone in metabolism.

The physiological roles of intracellular progesterone (PRG) receptors (PRs) have been studied intensively in female mammals, while their functions in male are scarce. Conventional PRs were evidenced in our study by Western blotting, concomitantly in healthy spermatozoa and in oligoasthenoteratozoospermic samples without and with varicocoele. Transmission electron microscopy revealed the presence of the PRs on the membrane as well as in the nucleus, mitochondria and flagellum. A reduced expression of the PRs was observed only in varicocoele spermatozoa. Responses to PRG treatment on cholesterol efflux, tyrosine phosphorylation, src and Akt activities, acrosin activity and acrosome reaction in varicocoele spermatozoa were reduced or absent. To further investigate PRG significance in human male gamete, we focused its action on lipid and glucose metabolism. The evaluation of the triglycerides content, lipase and acyl-CoA dehydrogenase activities suggests that PRG through the PRs exerts a lipolytic effect on human spermatozoa. An increase in glucose-6-phosphate dehydrogenase activity was also obtained, evidencing a role for PRG on glucose metabolism. In 'varicocoele' spermatozoa, the PRG did not induce energy consumption. The action of PRs on sperm metabolism is a novel finding that renews the importance of PRG in male fertility. Our results showed that varicocoele may lead to male factor infertility by a mechanism involving a decreased PR expression in human spermatozoa that evidences a detrimental effect on spermatozoa at the molecular level, going beyond the abnormal sperm morphology described to date.

Evidence that bergapten, independently of its photoactivation, enhances p53 gene expression and induces apoptosis in human breast cancer cells.

Psoralens (5-MOP and 8-MOP), a class of naturally occurring compounds, in combination with ultraviolect light are potent modulators of epidermal cell growth and differentiation. For a long time, photo-chemotherapy has been used in the treatment of psoriasis where it can reduce the number of cycling keratinocytes and decrease the IGF-1 receptors. However, the molecular mechanism of PUVA therapy remains unclear. In this study, we have evaluated, for the first time, in MCF-7 and SKBR-3 breast cancer cells the effects of 5-MOP (Bergapten), independently of its photoactivation, on the signalling pathways involved in cell cycle arrest and in apoptosis. Drug treatment induced a block in the G0/G1 phase and increased mRNA and protein levels of p53 and p21waf. These data correlate with a functional activation of caspase 8/caspase 9 together with DAPI staining and DNA ladder. Bergapten can transactivate p53 gene promoter in these cells and site-direct mutagenesis studies showed that the binding sequence of the nuclear factor NF-Y on p53 promoter is required for 5-MOP responsiveness. Besides, Bergapten increases NF-Y nuclear translocation through p38 MAPK activation. The same treatment impairs the PI3Kinase/AKT survival signal, in hormone-dependent MCF-7 cells even in the presence of IGF-I/E2 mitogenic factors. Here, we demonstrated that Bergapten, independently on the exposure to UV, generates membrane signalling pathways able to address apoptotic responses in breast cancer cells and to counteract the stimulatory effect of IGF-I/E2 on estrogen-receptor positive MCF-7 cell growth and progression.

Progesterone receptor B recruits a repressor complex to a half-PRE site of the estrogen receptor alpha gene promoter.

In the present study, we demonstrate that elevated levels of the progesterone receptor (PR)-B isoform in breast cancer cells induces down-regulation of estrogen receptor (ER) alpha mRNA and protein content, causing concomitant repression of the estrogen-regulated genes insulin receptor substrate 1, cyclin D1, and pS2, addressing a specific effect of PR/PR-B on ERalpha gene transcription. ERalpha gene promoter activity was drastically inhibited by PR-B overexpression. Promoter analysis revealed a transcriptionally responsive region containing a half-progesterone response element (PRE) site located at -1757 bp to -1752 bp. Mutation of the half-PRE down-regulated the effect induced by PR/PR-B overexpression. Moreover chromatin immunoprecipitation analyses revealed an increase of PR bound to the ERalpha-regulatory region encompassing the half-PRE site, and the recruitment of a corepressor complex containing nuclear receptor corepressor (NCoR) but not silencing mediator of retinoid and thyroid hormone receptor and DAX1, concomitantly with hypoacetylation of histone H4 and displacement of RNA polymerase II. Furthermore, NCoR ablation studies demonstrated the crucial involvement of NCoR in the down-regulatory effects due to PR-B overexpression on ERalpha protein and mRNA. We also demonstrated that the ERalpha regulation observed in MCF-7 cells depended on PR-B expression because PR-B knockdown partially abrogates the feedback inhibition of ERalpha levels after estrogenic stimulus. Our study provides evidence for a mechanism by which overexpressed PR-B is able to actively repress ERalpha gene expression.

Breast cancer: from estrogen to androgen receptor.

To investigate the link existing between androgens and human breast cancer, the hormonal milieu present in pre- and post-menopausal women has been translated in an in vitro model utilizing a hormone dependent breast cancer cell line MCF-7 exposed to DHEA, DHEAS, androstenediol, T, DHT with or w/o E(2). DHEAS and androstenediol stimulate the growth of MCF-7 cell line but reduce cell proliferation induced by E(2) (1 nM). T and DHT (1-100 nM) instead inhibit MCF-7 cell proliferation independently on E(2) presence. When we focused our study on the most powerful androgen, DHT alone (100 nM) consistently inhibits MCF-7 cell proliferation by 50% of the basal growth rate and counteracts E(2) proliferative action by 68%. These data correlate well with cell cycle analysis showing an enhanced number of cells in G(0)/G(1) phase after 6 days of DHT treatment. Upon prolonged DHT exposure, Western blotting analysis shows a markedly increased AR content, while immunohistochemistry indicates that it was mostly translocated into the nucleus. So we assumed that the enhanced activation of the AR might inhibit MCF-7 cells proliferation. This assumption is corroborated by the fact that the inhibitory effects induced by DHT on MCF-7 cell proliferation are abrogated in the presence of hydroxyflutamide. Therefore to better investigate the role of AR in inhibiting E(2) action at genomic level, MCF-7 cells were transiently cotransfected with the reporter plasmid XETL carrying firefly luciferase sequence under the control of an estrogen responsive element and the full length AR or with an AR carrying a mutation (Cis 574-->Arg 574) which abolishes its binding to DNA. The over-expression of the AR markedly decreases E(2) signalling which furthermore appears inhibited by simultaneous exposure to DHT but reversed by addition of hydroxyflutamide. The inhibitory effect was no longer noticeable when MCF-7 cells were cotransfected with XETL and the mutant AR. Taken together these data demonstrate that gonadal androgens antagonize MCF-7 proliferation induced by E(2). This seems to be related to the inhibitory effects of the over-expressed AR on E(2) genomic action.

Intercodon dinucleotides affect codon choice in plant genes.

In this work, 710 CDSs corresponding to over 290 000 codons equally distributed between Brassica napus, Arabidopsis thaliana, Lycopersicon esculentum, Nicotiana tabacum, Pisum sativum, Glycine max, Oryza sativa, Triticum aestivum, Hordeum vulgare and Zea mays were considered. For each amino acid, synonymous codon choice was determined in the presence of A, G, C or T as the initial nucleotide of the subsequent triplet; data were statistically analysed under the hypothesis of an independent assortment of codons. In 33.4% of cases, a frequency significantly (P: = 0.01) different from that expected was recorded. This was mainly due to a pervasive intercodon TpA and CpG deficiency. As a general rule, intercodon TpAs and CpGs were preferably replaced by CpAs and TpGs, respectively. In several instances, codon frequencies were also modified to avoid homotetramer and homotrimer formation, to reduce intercodon ApCs downstream (1,2) GG or AG dinucleotides, as well as to increase GpA or ApG intercodons under certain contexts. Since TpA, CpG and homotetra(tri)mer deficiency directly or indirectly accounted for 77% of significant variation in the codon frequency, it can be concluded that codon usage mirrors precise needs at the DNA structure level. Plant species exhibited a phylogenetically-related adaptation to structural constraints. Codon usage flexibility was reflected in strikingly different arrays of optimum codons for probe design.

Sustained versus transient cyclic AMP intracellular levels: effect on thyrotropin-dependent growth of thyroid cells.

Cyclic AMP (cAMP) is the second messenger that stimulates growth and differentiation of thyroid cells, which are dependent upon thyrotropin for the initiation of the cell cycle. Treatment of thyroid cells with phosphodiesterase inhibitors, such as 1-methyl-3-isobutylxanthine (IBMX), RO-20-1724, or aminophylline, induces persistent levels of cAMP and blocks cell proliferation. IBMX-treated cells are arrested at the G1-S border, but removal of the drug allows cell growth to resume. The inhibiting effect of IBMX is dose dependent, and the phase of the cell cycle is irrelevant. These data indicate that prolonged and steady accumulation of cAMP blocks the cell cycle in thyroid cells.

In the thyroid cells proliferation, differentiated and metabolic functions are under the control of different steps of the cyclic AMP cascade.

In the course of studies to elucidate the complex network of interactions controlling FRTL5 cell proliferation, thyroid stimulating hormone (TSH)-independent mutants (M cells), have been obtained from FRTL5 cells by chemical mutagenesis. In the present studies, the role of TSH on the proliferation and on differentiated and metabolic functions in these mutant cells have been investigated and compared to their response to insulin-like growth factor I (IGF-I). The addition of IGF-I to M cells leads to normal stimulation of DNA synthesis. However, inspite of the fact that mutant cells display normal TSH receptors, TSH is unable to stimulate the proliferation of the M cells. Nevertheless, TSH is able to increase intracellular levels of cAMP leading to regulation of TSH function in the M cells. On the other hand, TSH does not influence iodide transport and actin filaments depolimerization in these cells. However, aminoacid transport, stimulated in wild-type FRTL5 cells by both TSH and IGFs, is under the control of IGFs but not of TSH in the mutant cells. Neither TSH or IGF-I modified the expression of c-fos proto-oncogene in the M cells, probably because of high constitutive expression. These data suggest that a crucial signalling step(s) required for TSH induced mitogenesis is impaired in the M cells, and that this signalling step is not required for IGF-I induced mitogenesis.