Oncostatin M and STAT3 Signaling Pathways Support Human Trophoblast Differentiation by Inhibiting Inflammatory Stress in Response to IFNγ and GM-CSF.

Interleukin-6 (IL-6) superfamily cytokines play critical roles during human pregnancy by promoting trophoblast differentiation, invasion, and endocrine function, and maintaining embryo immunotolerance and protection. In contrast, the unbalanced activity of pro-inflammatory factors such as interferon gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) at the maternal-fetal interface have detrimental effects on trophoblast function and differentiation. This study demonstrates how the IL-6 cytokine family member oncostatin M (OSM) and STAT3 activation regulate trophoblast fusion and endocrine function in response to pro-inflammatory stress induced by IFNγ and GM-CSF. Using human cytotrophoblast-like BeWo (CT/BW) cells, differentiated in villous syncytiotrophoblast (VST/BW) cells, we show that beta-human chorionic gonadotrophin (ßhCG) production and cell fusion process are affected in response to IFNγ or GM-CSF. However, those effects are abrogated with OSM by modulating the activation of IFNγ-STAT1 and GM-CSF-STAT5 signaling pathways. OSM stimulation enhances the expression of STAT3, the phosphorylation of STAT3 and SMAD2, and the induction of negative regulators of inflammation (e.g., IL-10 and TGFß1) and cytokine signaling (e.g., SOCS1 and SOCS3). Using STAT3-deficient VST/BW cells, we show that STAT3 expression is required for OSM to regulate the effects of IFNγ in ßhCG and E-cadherin expression. In contrast, OSM retains its modulatory effect on GM-CSF-STAT5 pathway activation even in STAT3-deficient VST/BW cells, suggesting that OSM uses STAT3-dependent and -independent mechanisms to modulate the activation of pro-inflammatory pathways IFNγ-STAT1 and GM-CSF-STAT5. Moreover, STAT3 deficiency in VST/BW cells leads to the production of both a large amount of ßhCG and an enhanced expression of activated STAT5 induced by GM-CSF, independently of OSM, suggesting a key role for STAT3 in ßhCG production and trophoblast differentiation through STAT5 modulation. In conclusion, our study describes for the first time the critical role played by OSM and STAT3 signaling pathways to preserve and regulate trophoblast biological functions during inflammatory stress.

Investigating a new C2-symmetric testosterone dimer and its dihydrotestosterone analog: Synthesis, antiproliferative activity on prostate cancer cell lines and interaction with CYP3A4.

The synthesis of a 17α-linked C2-symmetric testosterone dimer and its dihydrotestosterone analog is reported. The dimers were synthesized using a short five-step reaction sequence with 28% and 38% overall yield for the testosterone and dihydrotestosterone dimer, respectively. The dimerization reaction was achieved by an olefin metathesis reaction with 2nd generation Hoveyda-Grubbs catalyst. The dimers and their corresponding 17α-allyl precursors were tested for the antiproliferative activity on androgen-dependent (LNCaP) and androgen-independent (PC3) prostate cancer cell lines. The effects on cells were compared with that of the antiandrogen cyproterone acetate (CPA). The results showed that the dimers were active on both cell lines, with an increased activity towards androgen-dependent LNCaP cells. However, the testosterone dimer (11) was fivefold more active than the dihydrotestosterone dimer (15), with an IC50 of 11.7 µM vs. 60.9 µM against LNCaP cells, respectively, and more than threefold more active than the reference drug CPA (IC50 of 40.7 µM). Likewise, studies on the interaction of new compounds with drug-metabolizing cytochrome P450 3A4 (CYP3A4) showed that 11 was a fourfold stronger inhibitor than 15 (IC50 of 3 µM and 12 µM, respectively). This suggests that changes in the chemical structure of sterol moieties and the manner of their linkage could largely affect both the antiproliferative activity of androgen dimers and their crossreactivity with CYP3A4.

Relevance of iNOS expression in tumor growth and maintenance of cancer stem cells in a bladder cancer model.

The expression of inducible nitric oxide (NO) synthase (iNOS) in human bladder cancer (BC) is a poor prognostic factor associated with invasion and tumor recurrence. Here, we evaluated the relevance of iNOS expression in BC progression and in cancer stem cell (CSC) maintenance in a murine BC model. Also, iNOS expression and CSC markers were analyzed in human BC samples. iNOS inhibitors (L-NAME or 1400W) or shRNA were used on murine BC model with different iNOS expressions and invasiveness grades: MB49 (iNOS+, non-muscle invasive (NMI)) and MB49-I (iNOS++, muscle invasive (MI)), in order to analyzed cell proliferation, tumor growth, angiogenesis, number of CSC, and pluripotential marker expression. iNOS, SOX2, Oct4, and Nanog expressions were also analyzed in human BC samples by qPCR and immunohistochemistry. iNOS inhibtion reduced parameters associated with tumor progression and reduced the number of CSC, wich resulted higher in MB49-I than in MB49, in concordance with the higher expression of SOX2, Oct4, and Nanog. The expression of SOX2 was notoriously diminished, when iNOS was inhibited only in the MI cell line. Similar results were observed in human samples, where MI tumors expressed higher levels of iNOS and pluripotential genes, in comparison to NMI tumors with a positive correlation between those and iNOS, suggesting that iNOS expression is associated with CSC. iNOS plays an important role in BC progression and CSC maintenance. Its inhibition could be a potential therapeutic target to eradicate CSC, responsible for tumor recurrences. KEY MESSAGES: • iNOS expression is involved in bladder tumor development, growth, and angiogenesis. • iNOS expression is involved in bladder cancer stem cell generation and maintenance, playing an important role regulating their self-renewal capacity, especially in muscle invasive murine bladder cancer cells. • iNOS expression is higher in human muscle invasive tumors, in association with a high expression of pluripotential genes, especially of SOX2.

Leukemia inhibitory factor regulates the activation of inflammatory signals in macrophages and trophoblast cells.

The pleiotropic cytokine leukemia inhibitory factor (LIF) is a key gestational factor known to establish dynamic cellular and molecular cross talk at the feto-maternal interface. Previously, we described the regulatory role of the LIF-trophoblast-IL10 axis in the process of macrophage deactivation in response to pro-inflammatory cytokines. However, the direct regulatory effects of LIF in macrophage and trophoblast cell function remains elusive. In this study, we aimed to examine whether and how LIF regulates the behavior of macrophages and trophoblast cells in response to pro-inflammatory stress factors. We found that LIF modulated the activating effects of interferon-gamma (IFNγ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in macrophages and trophoblast cells by reducing the phosphorylation levels of signal transducer and activator of transcription-1 (Stat1) and -5 (Stat5). Cell activation with IFNγ inhibited cell invasion and migration but this immobilizing effect was abrogated when macrophages and trophoblast cells were deactivated with LIF; macrophage cell motility restitution could in part be explained by the positive effects of LIF in Stat3 activation and matrix metalloproteinase 9 (MMP-9) expression. Pharmacological inhibition of Stat1 and Stat3 indicated that IFNγ-induced Stat1 activation mediated macrophage motility inhibition, and that cell motility in IFNγ-activated macrophages is restored via LIF-induced Stat3 activation and Stat1 inhibition. Moreover, IFNγ-induced TNFα gene expression was also abrogated by LIF through Stat1 inhibition and Stat3 activation. Finally, we have found that cell invasion of trophoblast cells is inhibited when they were cocultured with GM-CSF-differentiated, IFNγ-stimulated macrophages. This effect, however, was inhibited when macrophages were exposed to LIF. Overall, this in vitro study reveals for the first time the anti-inflammatory and pro-gestational activities of LIF by acting directly on macrophages and trophoblast cells.

Molecular therapy with derivatives of amino benzoic acid inhibits tumor growth and metastasis in murine models of bladder cancer through inhibition of TNFα/NFΚB and iNOS/NO pathways.

Muscle-invasive bladder cancer (MIBC) is an aggressive form of urothelial bladder carcinoma (UBC) with poorer outcomes compared to the non-muscle invasive form (NMIBC). Higher recurrent rates and rapid progression after relapse in UBC is known to be linked with chronic inflammation. Here, the preclinical murine models of NMIBC (MB49) and MIBC (MB49-I) were used to assess the antitumor effects of DAB-1, an anti-inflammatory aminobenzoic acid derivative we have developed in order to target cancer-related inflammation. A subchronic toxicity study on cancer-free mice shown that DAB-1 treatment did not affect normal mouse development or normal function of vital organs. In mice bearing MB49-I tumors, whole body accumulation of the radioconjugate [131I]DAB-1 was higher than in control mice, the main sites of [131I]DAB-1 accumulation being the liver (34%), the intestines (21%), and the tumors (18%). In vivo molecular therapy of ectopic and orthotopic tumors indicated that treatment with DAB-1 efficiently inhibited tumor growth, metastasis formation, and mortality rate. The antitumor efficacy of DAB-1 was associated with strong decreased tumor cell proliferation and iNOS expression in tumor tissues and deactivation of macrophages from tumor-bearing mice. Mechanistic investigations revealed that DAB-1 efficiently inhibited i) TNFα/NFΚB and IL6/STAT3 signaling pathways activation; ii) TNFα-induced NO production by decreasing NFΚB transcriptional activation and functional iNOS expression; and iii) cellular proliferation with minimal or no effects on cell mortality or apoptosis. In conclusion, this study provides preclinical and biological/mechanistic data highlighting the potential of DAB-1 as a safe and efficient therapeutic agent for the treatment of patients with NMIBC and MIBC.

Identification of an anti-inflammatory derivative with anti-cancer potential: The impact of each of its structural components on inflammatory responses in macrophages and bladder cancer cells.

Inflammation plays a crucial role in many types of cancer and is known to be involved in their initiation and promotion. As such, it is presently recognized as an important risk factor for several types of cancers such as bladder, prostate and breast cancers. The discovery of novel anti-inflammatory compounds can have a huge implication not only for the treatment of cancer but also as preventive and protective treatment modalities. We have recently identified a new compound (1) that presents interesting anti-inflammatory activity. In order to better understand its biological action, we have divided the molecule in its basic components and verified their respective contribution towards the anti-inflammatory response of the whole molecule. We have discovered that only the combination of the maleimide function together with the tert-butyloxycarbonylhydrazinamide function lead to important anti-inflammatory properties. The main derivative 1 can decrease the activating effects of INFγ or IL6 on human (hMϕs) macrophages by 38% or by 64% at a concentration of 10 µM as indicated by a decrease of STAT1 or STAT3 activation. The expression of pro-inflammatory markers CD40 and MHCII in INFγ stimulated hMϕs were reduced by 87% and 49%, respectively with a 3 h pretreatment of 1 at 10 µM. The cell motility assay revealed that 1 at 10 µM can reduce relative cell motility induced by IL6 by 92% in comparison with the untreated control hMϕ monolayers. Compound 1 reduced by 91% the inflammatory response induced by the cytokines (INFγ + TNFα) in the macrophage-like J774A.1 cells at a concentration of 25 µM, as measured by the detection of NO production with the Griess reagent. Furthermore, upon removal of the tert-butyloxycarbonyl protective group the unprotected derivative as a hydrochloride salt (1A) retains interesting anti-inflammatory activity and was found to be less toxic than the parent compound (1).

The activating effect of IFN-γ on monocytes/macrophages is regulated by the LIF-trophoblast-IL-10 axis via Stat1 inhibition and Stat3 activation.

Interferon gamma (IFN-γ) and leukemia inhibitory factor (LIF) are key gestational factors that may differentially affect leukocyte function during gestation. Because IFN-γ induces a pro-inflammatory phenotype in macrophages and because trophoblast cells are principal targets of LIF in the placenta, we investigated whether and how soluble factors from trophoblast cells regulate the effects of IFN-γ on macrophage activation. IFN-γ reduces macrophage motility, but enhances Stat1 activation, pro-inflammatory gene expression and cytotoxic functions. Soluble factors from villous cytotrophoblasts (vCT+LIF cells) and BeWo cells (BW/ST+LIF cells) that were differentiated in the presence of LIF inhibit macrophage Stat1 activation but inversely sustain Stat3 activation in response to IFN-γ. vCT+LIF cells produce soluble factors that induce Stat3 activation; this effect is partially abrogated in the presence of neutralizing anti-interleukin 10 (IL-10) antibodies. Moreover, soluble factors from BW/ST+LIF cells reduce cell proliferation but enhance the migratory responses of monocytes. In addition, these factors reverse the inhibitory effect of IFN-γ on monocyte/macrophage motility. BW/ST+LIF cells also generate IFN-γ-activated macrophages with enhanced IL-10 expression, but reduced tumor-necrosis factor alpha (TNF-α), CD14 and CD40 expression as well as impaired cytotoxic function. Additional assays performed in the presence of neutralizing anti-IL-10 antibodies and exogenous IL-10 demonstrate that reduced macrophage cytotoxicity and proliferation, but increased cell motility result from the ability of trophoblast IL-10 to sustain Stat3 activation and suppress IFN-γ-induced Stat1 activation. These in vitro studies are the first to describe the regulatory role of the LIF-trophoblast-IL-10 axis in the process of macrophage activation in response to pro-inflammatory cytokines.

Involvement of Akt isoforms in chemoresistance of endometrial carcinoma cells.

OBJECTIVE: In tumors, upstream regulation of Akt is affected by oncogenic events which lead to its constitutive activation and promote cell survival. Since studies have demonstrated that the three Akt isoforms exhibit different physiological functions, Akt isoforms may contribute differently in chemoresistance. The objective of the study was to determine the role of each Akt isoforms in chemoresistance. METHODS: We stably transfected the chemoresistant KLE endometrial carcinoma cells with specific shRNAs for Akt1, Akt2 or Akt3. Alternatively, we stably transfected the chemosensitive Hec-1-A endometrial carcinoma cells, in which no Akt activity is detected, with constitutively active Akt expression vectors for each isoform. RESULTS: We demonstrated that Akt1 and Akt2 downregulation by RNAi highly sensitizes KLE cells to cisplatin by inducing the activation of pro-apoptotic factors such as the cleavage of caspases-3, -6, -9 and PARP; downregulation of all Akt isoforms leads to increased sensitivity to doxorubicin while only Akt1-2 downregulation increases taxol sensitivity. Proliferation of Akt1, and mostly Akt2 deficient cells was affected by cisplatin treatment. Constitutive Akt1 or Akt2 expression led to an increased resistance to apoptosis. Akt isoforms have been shown to influence migration in other cancer cells. We showed that Akt2 blocks cell motility, while Akt1-3 had less effect on our endometrial cancer cell models. CONCLUSION: Our findings highlight the contribution of Akt1 and Akt2 in the molecular mechanisms that govern chemoresistance of endometrial carcinomas. Furthermore, Akt isoform-specific transfectants will provide a strong model to determine the involvement of each Akt isoform in tumor progression and metastasis.

CD40 pathway activation reveals dual function for macrophages in human endometrial cancer cell survival and invasion.

Reproductive malignancies are a major cause of cancer death in women worldwide. CD40 is a TNF receptor family member, which upon activation may mediate tumor regression. However, despite the great potential of CD40 agonists, their use as a therapeutic option for reproductive cancers has never been investigated. Because CD40 ligation is a potent pathway of macrophage activation, an in vitro model of pro-inflammatory type-1 (Mϕ-1) and anti-inflammatory type-2 (Mϕ-2) macrophages was developed to determine whether and how macrophage CD40 pathway activation might influence endometrial tumor cell behavior. Analysis of tumor growth kinetic in the endometrial cancer xenograft model indicates that, when injected once into the growing tumors, CD40-activated Mϕ-1 greatly reduced, while CD40-activated Mϕ-2 increased tumor size when compared to control isotype-activated Mϕ-1 and Mϕ-2, respectively. In vitro assays indicated that CD40-activated Mϕ-2 increased cell viability but failed to promote cell invasion. CD40-activated Mϕ-1, in contrast, decreased cell survival but greatly increased cell invasion in tumor cells less susceptible to cell death by apoptosis; they also induced the expression of some pro-inflammatory genes, such as IL-6, LIF, and TNF-α, known to be involved in tumor promotion and metastasis. The presence of IFN-γ is minimally required for CD40-activated Mϕ-1 to promote tumor cell invasion, a process that is mediated in part through the activation of the PI3K/Akt2 signaling pathway in tumor cells. From these results, we speculate that some functions of CD40 in tumor-associated Mϕs might limit the therapeutic development of CD40 agonists in endometrial cancer malignancies.

Cisplatin increases B-cell-lymphoma-2 expression via activation of protein kinase C and Akt2 in endometrial cancer cells.

Human carcinomas often show resistance to cisplatin and Bcl-2 is associated with resistance to cisplatin. However, Bcl-2 regulation on cisplatin treatment in human cancers is unknown. Here, we show a novel mechanism by which cisplatin treatment promotes resistance by increasing the expression of Bcl-2 mRNA. Bcl-2 mRNA and protein expression was increased in cisplatin-resistant endometrial cancer cell lines (KLE and HEC-1-A), but not in cisplatin-sensitive cell line (Ishikawa). Cisplatin-mediated increase in Bcl-2 expression was blocked by combination with either actinomycin D or cycloheximide. In addition, Bcl-2 inhibition by HA14-1 led to increased cisplatin-induced apoptosis in KLE and HEC-1-A, whereas Bcl-2 overexpression in Ishikawa led to decreased cisplatin-induced apoptosis. Inhibition of protein kinase C (PKC) activity prevented cisplatin-dependant increase in Bcl-2 mRNA, and induced apoptosis in KLE cells. Furthermore, PKC inhibition was associated with decreased Akt and NF-κB activity. Cells stably expressing shRNA for Akt isoforms revealed that Akt2 was involved in cisplatin-dependant increase in Bcl-2 and apoptosis. Overexpression of Akt2 in Akt2-deficient cells led to increased Bcl-2 expression on cisplatin treatment. Our data suggest a novel regulation pathway of Bcl-2 by cisplatin, via the activation of PKC and Akt2, which has a profound impact on resistance to cisplatin-induced apoptosis in endometrial cancer cells.

Characterization of two distinct populations of epididymosomes collected in the intraluminal compartment of the bovine cauda epididymis.

During their transit along the epididymis, mammalian spermatozoa acquire new proteins that are necessary for their acquisition of forward motility and fertility. By using the bovine model, we previously showed that small membranous vesicles named epididymosomes are secreted in the epididymal intraluminal compartment. Epididymosomes from caput and cauda are different, and interact sequentially with the transiting spermatozoa. In fact, selected proteins of epididymosomes are transferred to different subcompartments of the maturing spermatozoa. In this study, we investigate the possibility that different subpopulations of epididymosomes are present in the caudal portion of the epididymis. Through the use of discontinuous sucrose gradient ultracentrifugation, we isolated two distinct populations that differ in their protein and lipid compositions. Although they have similar diameters, the ultrastructural appearance of these two populations was very different. The low-density (Ld) vesicles are enriched in cholesterol, sphingomyelin, and ganglioside M1, suggesting the existence of detergent-resistant membrane domains or rafts. The high-density (Hd) vesicles show a high protein concentration, including ACTB and VAMP8. When each subpopulation of biotinylated cauda epididymosomes was coincubated with caput spermatozoa, a subset of biotinylated proteins was transferred to the sperm; the Ld and Hd vesicles transferring the same pattern of proteins. In vitro competition assays of protein transferred from Ld or Hd epididymosomes to sperm confirm the similarity in the selected transferred proteins. Electrospray tandem mass spectrometry (ES-MS/MS) analysis of proteins associated with the two populations of vesicles confirm the epididymal origin of some of them, the possible involvement of others in transmembrane signaling systems, and the identification of proteins for which functions in sperm physiology remain to be determined. Mass spectrometry analysis also revealed that ELSPBP1 and GBB2 were transferred from epididymosomes to spermatozoa. Results are discussed with regard to the functions of these two cauda epididymosome populations in sperm physiology.

Compartmentalization of proteins in epididymosomes coordinates the association of epididymal proteins with the different functional structures of bovine spermatozoa.

Epididymosomes are small membranous vesicles secreted by epithelial cells within the luminal compartment of the epididymis. In bovine, many proteins are associated with epididymosomes, and some of them, such as the glycosylphosphatidylinositol (GPI)-anchored protein P25b, macrophage migration inhibitory factor (MIF), and aldose reductase (AKR1B1), are transferred to spermatozoa during the epididymal maturation process. P25b is associated with detergent-resistant membrane (DRM) domains of epididymal spermatozoa, whereas MIF and AKR1B1 are cytosolic proteins associated with detergent-soluble fractions. In this study, we tested the hypothesis that DRM domains are also present in the epididymosomes and that P25b DRM-associated proteins in these vesicles are transferred to the DRMs of spermatozoa. The presence of DRMs in epididymosomes was confirmed by their insolubility in cold Triton X-100 and their low buoyant density in sucrose gradient. Furthermore, DRMs isolated from epididymosomes are characterized by the exclusive presence of ganglioside GM1 and by high levels of cholesterol and sphingomyelin. Biochemical analysis indicated that P25b is linked to DRM in epididymosomes, whereas MIF and AKR1B1 are completely excluded from these membrane domains. Proteolytic treatment of epididymosomes and immunoblotting studies showed that P25b is affected by trypsin or pronase proteolysis. In contrast, MIF and AKR1B1 are not degraded by proteases, suggesting that they are localized within epididymosomes. Interaction studies between epididymosomes and epididymal spermatozoa demonstrated that P25b is transferred from the DRM of epididymosomes to the DRM of the caput epididymal spermatozoa as a GPI-anchored protein. Together, these data suggest that specific localization and compartmentalization of proteins in the epididymosomes coordinate the association of epididymal proteins with the different functional structures of spermatozoa.

Seminal plasma proteins regulate the association of lipids and proteins within detergent-resistant membrane domains of bovine spermatozoa.

Maturing spermatozoa acquire full fertilization competence by undergoing major changes in membrane fluidity and protein composition and localization. In epididymal spermatozoa, several proteins are associated with cholesterol- and sphingolipid-enriched detergent-resistant membrane (DRM) domains. These proteins dissociate from DRM in capacitated sperm cells, suggesting that DRM may play a role in the redistribution of integral and peripheral proteins in response to cholesterol removal. Since seminal plasma regulates sperm cell membrane fluidity, we hypothesized that seminal plasma factors could be involved in DRM disruption and redistribution of DRM-associated proteins. Our results indicate that: 1) the sperm-associated proteins, P25b and adenylate kinase 1, are linked to DRM of epididymal spermatozoa, but were exclusively associated with detergent-soluble material in ejaculated spermatozoa; 2) seminal plasma treatment of cauda epididymal spermatozoa significantly lowered the content of cholesterol and the ganglioside, GM1, in DRM; and 3), seminal plasma dissociates P25b from DRM in epididymal spermatozoa. We found that the seminal plasma protein, Niemann-Pick C2 protein, is involved in cholesterol and GM1 depletion within DRM, then leading to membrane redistribution of P25b that occurs in a very rapid and capacitation-independent manner. Together, these data suggest that DRM of ejaculated spermatozoa are reorganized by specific seminal plasma proteins, which induce lipid efflux as well as dissociation of DRM-anchored proteins. This process could be physiologically relevant in vivo to allow sperm survival and attachment within the female reproductive tract and to potentiate recognition, binding, and penetration of the oocyte.

Epididymosomes are involved in the acquisition of new sperm proteins during epididymal transit.

During epididymal transit, spermatozoa acquire new proteins. Some of these newly acquired proteins behave as integral membrane proteins, including glycosylphosphatidylinositol (GPI)-anchored proteins. This suggests that the secreted epididymal proteins are transferred to spermatozoa by an unusual mechanism. Within the epididymal lumen, spermatozoa interact with small membranous vesicles named epididymosomes. Many proteins are associated with epididymosomes and the protein composition of these vesicles varies along the excurrent duct and differs from soluble intraluminal proteins. Some epididymosome-associated proteins have been identified and their functions in sperm maturation hypothesized. These include P25b, a zona pellucida binding protein, macrophage migration inhibitory factor, enzymes of the polyol pathway, HE5/CD52, type 5 glutathione peroxidase, and SPAM1 or PH-20. The electrophoretic patterns of proteins associated to epididymosomes are complex and some of these proteins are transferred to defined surface domains of epididymal spermatozoa. Epididymosomes collected from different epididymal segments interact differently with spermatozoa. This protein transfer from epididymosomes to spermatozoa is time-dependent, temperature-dependent and pH-dependent, and is more efficient in the presence of zinc. Some proteins are segregated to lipid raft domains of epididymosomes and are selectively transferred to raft domains of the sperm plasma membrane. Some evidence is presented showing that epididymosomes are secreted in an apocrine manner by the epididymal epithelial cells. In conclusion, epididymosomes are small membranous vesicles secreted in an apocrine manner in the intraluminal compartment of the epididymis and play a major role in the acquisition of new proteins by the maturing spermatozoa.

Requirement of oxidation-dependent CD40 homodimers for CD154/CD40 bidirectional signaling.

It is well established that the CD154/CD40 interaction is required for T cell-dependent B cell differentiation and maturation. However, the early molecular and structural mechanisms that orchestrate CD154 and CD40 signaling at the T cell/APC contact site are not well understood. We demonstrated that CD40 engagement induces the formation of disulfide-linked (dl) CD40 homodimers that predominantly associate with detergent-resistant membrane microdomains. Mutagenesis and biochemical analyses revealed that (a) the integrity of the detergent-resistant membranes is necessary for dl-CD40 homodimer formation, (b) the cytoplasmic Cys(238) of CD40 is the target for the de novo disulfide oxidation induced by receptor oligomerization, and (c) dl-CD40 homodimer formation is required for CD40-induced interleukin-8 secretion. Stimulation of CD154-positive T cells with staphylococcal enterotoxin E superantigen that mimics nominal antigen in initiating cognate T cell/APC interaction revealed that dl-CD40 homodimer formation is required for interleukin-2 production by T cells. These findings indicate that dl-CD40 homodimer formation has a physiological role in regulating bidirectional signaling.

Comparison between epididymosomes collected in the intraluminal compartment of the bovine caput and cauda epididymidis.

During their transit along the epididymidis, mammalian spermatozoa acquire new proteins involved in the acquisition of male gamete fertilizing ability. We previously described membranous vesicles called epididymosomes, which are secreted in an apocrine manner by the epididymal epithelium. Some selected proteins associated with epididymosomes are transferred to spermatozoa during epididymal transit. The present study compared epididymosomes collected from caput epididymal fluid with vesicles from the cauda epididymidis in the bull. Two-dimensional gel electrophoresis revealed major differences in protein composition of epididymosomes isolated from the caput and cauda epididymidis. LC-QToF analysis of major protein spots as well as Western blot analysis confirmed the differences in proteins associated with these two populations of epididymosomes. Biotinylated proteins associated with caput and cauda epididymosomes also revealed differences. When incubated with caput epididymal spermatozoa, epididymosomes prepared from these two segments transferred different protein patterns. By contrast, cauda epididymosomes transferred the same pattern of proteins to spermatozoa from the caput and cauda epididymidis. Transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa decreased in a dose-dependent manner when biotinylated epididymosomes were diluted with unbiotinylated vesicles. Caput epididymosomes added in excess were unable to inhibit transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa. Following transfer of biotinylated proteins from cauda epididymosomes to caput spermatozoa, addition of unbiotinylated cauda epididymosomes was unable to displace already transferred biotinylated proteins. These results established that epididymosomes from caput and cauda epididymidis have different protein composition and interact differently with maturing spermatozoa.

Role of exosomes in sperm maturation during the transit along the male reproductive tract.

Even tough differentiated spermatozoa are unable of transcriptional or translational activity; the sperm surface undergoes major modifications in macromolecules composition during the transit along the male reproductive tract. This is the result of sequential, well orchestrated interactions between the male reproductive tract secretions and the transiting male gamete. This is particularly true when spermatozoa transit along the epididymis. The epididymis is a long convoluted tubules in which the spermatozoa leaving the testis have to transit. The unraveled epididymal tubule can be as long as 80 m in stallion, and the transit time of spermatozoa is of 3-12 days depending on the species. The epididymis is usually divided in three segments: the caput (proximal part), the corpus, and cauda. While the cauda epididymides acts as a sperm reservoir, the caput and corpus are responsible for sperm maturation. This means that, under androgen control, the epididymal epithelium secretes proteins that will interact sequentially with sperm surface. Some of the sperm proteins acquired during maturation along the excurrent duct behave as integral membrane proteins. In fact, some epididymal originating proteins are glycosylphosphatidylinositol (GPI)-anchored to the sperm plasma membrane. Our laboratory has shown that some of these proteins are secreted in an apocrine manner by the epididymal epithelium and are associated to exosomes, called epididymosomes. Epididymosomes are rich in sphingomyelin and are characterized by a high cholesterol/phospholipids ratio. Many proteins are associated to epididymosomes, some of which are selectively transferred to spermatozoa during the epididymal transit. We have identified some of these exosomes associated proteins transferred to the maturing spermatozoa. These include two enzymes involved in the polyol pathway: an aldose reductase and a sorbitol dehydrogenase. A cytokine named MIF (macrophage migration inhibitory factor) is another protein associated to exosomes who is transferred to spermatozoa during the epididymal transit. We hypothesized that both the polyol pathway and MIF secreted in an apocrine fashion by the epididymal epithelium modulate sperm motility during the transit along the male reproductive tract. Finally, P25b, belonging to a family of sperm surface proteins (P26h/P34H) necessary for the binding to the surface of the egg, is also acquired through the interaction between epididymosomes and the male gamete. In vitro studies have defined the conditions of protein transfer when epididymal spermatozoa are co-incubated with epididymosomes. The transfer of selected proteins to specific membrane domains of spermatozoa is saturable, temperature and pH-dependent, being optimal at pH 6.5. The presence of zinc in the incubation medium, but not of calcium neither magnesium, significantly increases the efficiency of protein transfer. These results show that exosomes play a role in sperm epididymal maturation which is an essential event to produce male gametes with optimal fertilizing ability.

Requirement of the extracellular cysteine at position six for CD40/CD40 dimer formation and CD40-induced IL-8 expression.

We recently showed that oligomerization of CD40 molecules on cell surface leads to disulfide-linked CD40/CD40 dimer formation, an event that is necessary for CD40-induced B7-2 expression in human B cells. Here, we demonstrate that CD40/CD40 dimers formation also occurs in different cell types such as T24 bladder cancer cells and CD40-transfected HEK 293 cells. Disulfide bonds mediate the formation of CD40/CD40 homodimers in CD40-activated cells. To determine the potential residue(s) involved in disulfide bonds formation and subsequent CD40-induced IL-8 expression, we generated a CD40 mutant in which the extracellular cysteine 6 was replaced by a glutamine (CD40-C6Q). CD40-induced IL-8 mRNA expression and protein synthesis were studied in stably transfected HEK 293 cells that were sorted out along with similar levels of expression of wild type (CD40-WT) and CD40-C6Q molecules. In contrast to cells expressing CD40-WT protein, disulfide-linked CD40/CD40 dimer formation was completely abolished in HEK 293 cells expressing CD40-C6Q proteins. Abolishment of disulfide-linked CD40/CD40 dimers in these transfected cells was sufficient to inhibit CD40-induced mRNA expression and secretion of IL-8. This study identifies the extracellular cysteine 6 of CD40 molecules as a potential molecular target to disrupt the expression of CD40-induced pro-inflammatory cytokines by epithelial cells.

CD40/CD40 homodimers are required for CD40-induced phosphatidylinositol 3-kinase-dependent expression of B7.2 by human B lymphocytes.

Preformed CD40/CD40 homodimers were initially observed on human Burkitt lymphoma cell lines, normal B cells, and transitional bladder carcinoma cell lines. However, the nature and the biological relevance of these homodimers have not yet been investigated. In the present study, we demonstrated that Epstein-Barr virus-transformed B cells and CD40-transfected HEK 293 cells constitutively expressed disulfide-linked CD40/CD40 homodimers at low levels. Oligomerization of CD40 leads to a rapid and significant increase in the disulfide-linked CD40/CD40 homodimer formation, a response that could be prevented using a thiol-alkylating agent. Formation of CD40/CD40 homodimers was found to be absolutely required for CD40-mediated activation of phosphatidylinositol 3-kinase, which, in turn regulated B7.2 expression. In contrast, CD40 monomers provided the minimal signal emerging from CD40, activating p38 MAP kinase and inducing homotypic B cell adhesion. CD40/CD40 homodimer formation was totally independent of TRAF1/2/3/5 associations with the threonine at position 254 in the cytoplasmic tail of the CD40 molecules. This finding may be vital to better understanding the molecular mechanisms that govern cell signaling triggered by CD40/CD154 interactions.

Lipid raft-dependent and -independent signaling through HLA-DR molecules.

Lipid rafts are plasma membrane microdomains that are highly enriched in signaling molecules and that act as signal transduction platforms for many immune receptors. The involvement of these microdomains in HLA-DR-induced signaling is less well defined. We examined the constitutive presence of HLA-DR molecules in lipid rafts, their possible recruitment into these microdomains, and the role of these microdomains in HLA-DR-induced responses. We detected significant amounts of HLA-DR molecules in the lipid rafts of EBV(+) and EBV(-) B cell lines, monocytic cell lines, transfected HeLa cells, tonsillar B cells, and human monocytes. Localization of HLA-DR in these microdomains was unaffected by the deletion of the cytoplasmic domain of both the alpha and beta chains. Ligation of HLA-DR with a bivalent, but not a monovalent, ligand resulted in rapid tyrosine phosphorylation of many substrates, especially Lyn, and activation of ERK1/2 MAP kinase. However, the treatment failed to induce further recruitment of HLA-DR molecules into lipid rafts. The HLA-DR-induced signaling events were accompanied by the induction of cell-cell adhesion that could be inhibited by PTK and Lyn but not ERK1/2 inhibitors. Disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) resulted in the loss of membrane raft association with HLA-DR molecules, inhibition of HLA-DR-mediated protein tyrosine phosphorylation and cell-cell adhesion. MbetaCD did not affect the activation of ERK1/2, which was absent from lipid rafts. These results indicate that although all the HLA-DR-induced events studied are dependent on HLA-DR dimerization, some require the presence of HLA-DR molecules in lipid rafts, whereas others do not.