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Toll-like receptors ( TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer ( PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 ( IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa. [ABSTRACT FROM AUTHOR]

Toll-Like receptors ( TLRs) are a family of evolutionary conserved transmembrane proteins that recognize highly conserved molecules in pathogens. TLR-expressing cells represent the first line of defence sensing pathogen invasion, triggering innate immune responses and subsequently priming antigen-specific adaptive immunity. In vitro and in vivo studies on experimental cancer models have shown both anti- and pro-tumoural activity of different TLRs in prostate cancer, indicating these receptors as potential targets for cancer therapy. In this review, we highlight the intriguing duplicity of TLR stimulation by pathogens: their protective role in cases of acute infections, and conversely their negative role in favouring hyperplasia and/or cancer onset, in cases of chronic infections. This review focuses on the role of TLRs in the pathophysiology of prostate infection and cancer by exploring the biological bases of the strict relation between TLRs and prostate cancer. In particular, we highlight the debated question of how reliable mutations or deregulated expression of TLRs are as novel diagnostic or prognostic tools for prostate cancer. So far, the anticancer activity of numerous TLR ligands has been evaluated in clinical trials only in organs other than the prostate. Here we review recent clinical trials based on the most promising TLR agonists in oncology, envisaging a potential application also in prostate cancer therapy. [ABSTRACT FROM AUTHOR]

Upon stimulation by histamine, human vascular endothelial cells (EC) shed a soluble form of tumour necrosis factor receptor 1 (sTNFR1) that binds up free TNF, dampening the inflammatory response. Shedding occurs through proteolytic cleavage of plasma membrane-expressed TNFR1 catalysed by TNF-α converting enzyme (TACE). Surface expressed TNFR1 on EC is largely sequestered into specific plasma membrane microdomains, the lipid rafts/caveolae. The purpose of this study was to determine the role of these domains in TACE-mediated TNFR1 shedding in response to histamine. Human umbilical vein endothelial cells derived EA.hy926 cells respond to histamine via H1 receptors to shed TNFR1. Both depletion of cholesterol by methyl-β-cyclodextrin and small interfering RNA knockdown of the scaffolding protein caveolin-1 (cav-1), treatments that disrupt caveolae, reduce histamine-induced shedding of membrane-bound TNFR1. Moreover, immunoblotting of discontinuous sucrose gradient fractions show that TACE, such as TNFR1, is present within low-density membrane fractions, concentrated within caveolae, in unstimulated EA.hy926 endothelial cells and co-immunoprecipitates with cav-1. Silencing of cav-1 reduces the levels of both TACE and TNFR1 protein and displaces TACE, from low-density membrane fractions where TNFR1 remains. In summary, we show that endothelial lipid rafts/caveolae co-localize TACE to surface expressed TNFR1, promoting efficient shedding of sTNFR1 in response to histamine. [ABSTRACT FROM AUTHOR]

Although numerous markers have been helpful in isolating and enriching spermatogonial stem cells (SSCs), such as Thy-1 and GFRα-1, no specific marker for this cell type has been identified so far. A 400-bp regulatory region of the stimulated by retinoic acid gene 8 ( Stra8) promoter was reported to direct gene expression into SSCs and we have recently generated a new transgenic mouse model harboring the enhanced green fluorescent protein (EGFP) downstream of this Stra8 promoter. In this study, a detailed analysis of the EGFP expression pattern in the testis was carried out, showing that the transgene was expressed in meiotic and postmeiotic germ cells and not in undifferentiated germ cells. These findings were supported by confocal microscopy and flow cytometric analyses, and do not agree with the previous report concerning the 400-bp Stra8 promoter activity. Microsc. Res. Tech. 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The highly polarized structure and function of mammalian spermatozoa dictate that these cells compartmentalize specific metabolic and signaling pathways to regions where they are needed. Fas was initially identified as membrane receptor for pro-apoptotic signals, has been recently recognized as a molecule with pleiotropic functions. In this article, we provide evidence of a peculiar Fas localization: it is closely associated to the perinucleus, mainly at the level of the inner acrosomal membrane, as well as in the inner compartment of mitochondria. Immunoelectron microscopy and Western blot analysis indicated that intracellular Fas was associated with mitochondria in mouse epididymal spermatozoa. Accordingly, also in human ejaculated sperm, immunofluorescence analysis showed Fas localized in the middle piece of sperm flagellum where mitochondria are grouped. The potential functional implications of these findings are discussed. Microsc. Res. Tech., 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

We have investigated the role of NAADP-mediated Ca2+ mobilization in endothelin (ET) signaling via endothelin receptor subtype A (ETA) and endothelin receptor subtype B (ETB) in rat peritubular smooth muscle cells. Microinjection and extracellular application of NAADP were both able to elicit Ca2+ release which was blocked by inhibitory concentrations of NAADP, by impairing Ca2+ uptake in acidic stores with bafilomycin, and by thapsigargin. Ca2+ release in response to selective ETB stimulation was abolished by inhibition of NAADP signaling through the same strategies, while these treatments only partially impaired ETA-dependent Ca2+ signaling, showing that transduction of the ETB signal is dependent on NAADP. In addition, we show that lipid rafts/caveolae contain ETA, ETB, and NAADP/cADPR generating enzyme CD38 and that stimulation of ETB receptors results in increased CD38 activity; interestingly, ETB- (but not ETA-) mediated Ca2+ responses were antagonized by disruption of lipid rafts/caveolae with methyl-β-cyclodextrin. These data demonstrate a primary role of NAADP in ETB-mediated Ca2+ signaling and strongly suggest a novel role of lipid rafts/caveolae in triggering ET-induced NAADP signaling. J. Cell. Physiol. 216: 396–404, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

When chronically stimulated with agonists of contraction, smooth muscle cells (SMCs) undergo cell hypertrophy, a process defined as increase in size and potentiation of the contractile phenotype in the absence of proliferation. Hypertrophic response has long been associated to a number of pathologies of the cardiovascular and respiratory systems. We have investigated the phenotypic and functional response of SMCs to long-term treatment with endothelin. Our model was primary cultures of peritubular smooth muscle cells (PSMC) a testicular cell type target of locally produced endothelin and characterized by an unusual phenotypic stability when cultured in simple medium in complete absence of serum. We report the following responses of PSMC to 4-day exposure to ET-1: (i) increased protein synthesis without induction of cell proliferation; (ii) increase in cell size (evaluated by means of flow cytometry) and increased expression of SM-α-actin, desmin, caldesmon and calponin, markers of the contractile phenotype. In experiments of selective stimulation of either ETA or ETB receptor subtypes, both proved to be involved in inducing the observed hypertrophic responses. The hypertrophic cells exhibit the ultrastructural features of differentiated SMCs and are capable of calcium mediated contractile response when acutely stimulated with ET-1 specifically through ETA and/or ETB receptors, as evaluated by calcium imaging and scanning electron microscopy. These observations demonstrate that engagement of ET receptors is capable of inducing potentiation of the contractile phenotype and functional hypertrophy of PSMC. J. Cell. Physiol. 212: 264–273, 2007. © 2007 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis represents a fundamental process during fetal/post-natal testis development. Therefore pro- and anti-apoptotic proteins are essential to regulate testis physiology. c-FlipL is a known inhibitor of caspase 8/10 activity; in this study its perinatal expression in mouse male germ cells was investigated. In testis sections and seminiferous tubule whole mount c-FlipL was found to be expressed in undifferentiated spermatogonia and to co-localize with germ stem cells markers. In vivo investigations in the vitamin-A deficient mouse, lacking differentiated germ cells, confirmed c-FlipL expression in undifferentiated spermatogonia. Further analyses showed Fas expression but no significant caspase 8/10 activity when c-FlipL was highly expressed. Altogether these data suggest that c-Flip may control the survival rate of undifferentiated spermatogonia. [Copyright &y& Elsevier]

Peritubular smooth muscle cells (PSMC) from rat testis in primary serum-free cultures unexpectedly undergo contraction and subsequent cell hypertrophy in response to the growth factor PDGF-BB, remaining stationary. The present study investigates the transduction pathways involved in the observed paradoxical upregulation of the differentiated phenotype and induction of hypertrophy in PSMC. PI3K, ERK, JNK, and p38 kinases, known to mediate PDGF-BB signaling in the canonic dedifferentiative and proliferative response of smooth muscle cells (SMC) were rapidly activated by PDGF-BB but only p38 remained activated after 2-day stimulation. Immunofluorescence and immunoblotting experiments showed that in 4-day treatment: (i) continuous inhibition of PI3K, of ERK, of JNK, failed to inhibit either cell enlargement and formation of prominent α-SM actin containing stress fibers or the typical increase in α-SM actin; (ii) when stimulated in the presence of the p38 inhibitor SB203580 both responses were significantly inhibited and cytofluorimetric analysis of cell size showed a remarkable reduction of the hypertrophic response. PDGF-BB was also found to activate the small GTPase RhoA and inhibition of Rho-dependent kinase ROCK by Y27632 counteracted the effects of PDGF-BB similarly to SB203580. Both the transcription factor ATF2 and the nucleosomal kinase MSK1, downstream targets of p38, were activated by PDGF-BB, but p38 inhibitor SB203580 inhibited only the phosphorylation of MSK1 which appeared unaffected by ROCK inhibitor Y27632. In concluding, p38 and the Rho-ROCK system were found to play prominent, probably independent roles in the upregulation of PSMC differentiated phenotype and induction of hypertrophy by PDGF-BB. J. Cell. Physiol. 207: 123–131, 2006. © 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The article discusses c-Flip expression and function in fetal mouse genocytes. c-Flip is expressed in fetal male mouse gonads and its expression is developmentally regulated. Fas-dependent apoptosis in fetal testes is dependent on c-Flip levels. Initiator caspase-10 is expressed in fetal male mouse gonad and its activity is developmentally regulated.

Investigates the signal transduction mechanisms involved in Fas induction. Pretreatment of Sertoli cells with specific inhibitors of ERKs and P38 at all concentrations tested; Regulation of Fas expression to avoid indiscriminate cell death; Significance of understanding the molecular mechanisms activated by cytokines.