Oral and anal microbiome from HIV-exposed individuals: role of host-associated factors in taxa composition and metabolic pathways.

Evidence indicates that the microbiome plays a significant role in HIV immunopathogenesis and associated complications. This study aimed to characterize the oral and anal microbiome of Men who have Sex with Men (MSM) and Transgender Women (TGW), with and without HIV. One hundred and thirty oral and anal DNA-derived samples were obtained from 78 participants and subjected to shotgun metagenomics sequencing for further microbiome analysis. Significant differences in the microbiome composition were found among subjects associated with HIV infection, gender, sex behavior, CD4+ T-cell counts, antiretroviral therapy (ART), and the presence of HPV-associated precancerous anal lesions. Results confirm the occurrence of oncogenic viromes in this high HIV-risk population. The oral microbiome in HIV-associated cases exhibited an enrichment of bacteria associated with periodontal disease pathogenesis. Conversely, anal bacteria showed a significant decrease in HIV-infected subjects (Coprococcus comes, Finegoldia magna, Blautia obeum, Catenibacterium mitsuokai). TGW showed enrichment in species related to sexual transmission, which concurs that most recruited TGW are or have been sex workers. Prevotella bivia and Fusobacterium gonidiaformans were positively associated with anal precancerous lesions among HIV-infected subjects. The enrichment of Holdemanella biformis and C. comes was associated with detectable viral load and ART-untreated patients. Metabolic pathways were distinctly affected by predominant factors linked to sexual behavior or HIV pathogenesis. Gene family analysis identified bacterial gene signatures as potential prognostic and predictive biomarkers for HIV/AIDS-associated malignancies. Conclusions: Identified microbial features at accessible sites are potential biomarkers for predicting precancerous anal lesions and therapeutic targets for HIV immunopathogenesis.

Tristetraprolin promotes survival of mammary progenitor cells by restraining TNFα levels.

Tristetraprolin (TTP) is an RNA binding protein that destabilizes mRNAs of factors involved in proliferation, invasiveness, and inflammation. Disruption of the gene that codes for TTP (Zfp36) led to severe arthritis, autoimmunity, cachexia and dermatitis in mice. It has been shown that these phenotypes were mostly due to excessive TNFα levels in the affected tissues. We have previously reported that TTP expression is required for lactation maintenance. Our results indicated that conditional MG TTP-KO female mice displayed early involution due to the untimely induction of pro-inflammatory pathways led mostly by TNFα overexpression. Here we show that reducing TTP levels not only affects the fully differentiated mammary gland, but also harms morphogenesis of this tissue by impairing the progenitor cell population. We found that Zfp36 expression is linked to mammary stemness in human and mice. In addition, diminishing TTP expression and activity induced apoptosis of stem-like mouse mammary cells, reduced its ability to form mammospheres in culture and to develop into complete glands when implanted into cleared mammary fat pads in vivo. Our results show that survival of the stem-like cells is compromised by increased levels of inflammatory cytokines and stimulation of signaling cascades involving NFκB, STAT3 and MAPK-p38 activation. Moreover, TNFα overexpression and the consequent p38 phosphorylation would be the leading cause of progenitor cell death upon TTP expression restriction. Taken together, our results reveal the relevance of TTP for the maintenance of the mammary progenitor cell compartment by maintaining local TNFα levels at bay.

A major role for Nrf2 transcription factors in cell transformation by KSHV encoded oncogenes.

Kaposi's sarcoma (KS) is the most common tumor in AIDS patients. The highly vascularized patient's skin lesions are composed of cells derived from the endothelial tissue transformed by the KSHV virus. Heme oxygenase-1 (HO-1) is an enzyme upregulated by the Kaposi´s sarcoma-associated herpesvirus (KSHV) and highly expressed in human Kaposi Sarcoma (KS) lesions. The oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR) is expressed by the viral genome in infected cells. It is involved in KS development, HO-1 expression, and vascular endothelial growth factor (VEGF) expression. vGPCR induces HO-1 expression and HO-1 dependent transformation through the Ga13 subunit of heterotrimeric G proteins and the small GTPase RhoA. We have found several lines of evidence supporting a role for Nrf2 transcription factors and family members in the vGPCR-Ga13-RhoA signaling pathway that converges on the HO-1 gene promoter. Our current information assigns a major role to ERK1/2MAPK pathways as intermediates in signaling from vGPCR to Nrf2, influencing Nrf2 translocation to the cell nucleus, Nrf2 transactivation activity, and consequently HO-1 expression. Experiments in nude mice show that the tumorigenic effect of vGPCR is dependent on Nrf2. In the context of a complete KSHV genome, we show that the lack of vGPCR increased cytoplasmic localization of Nrf2 correlated with a downregulation of HO-1 expression. Moreover, we also found an increase in phospho-Nrf2 nuclear localization in mouse KS-like KSHV (positive) tumors compared to KSHV (negative) mouse KS-like tumors. Our data highlights the fundamental role of Nrf2 linking vGPCR signaling to the HO-1 promoter, acting upon not only HO-1 gene expression regulation but also in the tumorigenesis induced by vGPCR. Overall, these data pinpoint this transcription factor or its associated proteins as putative pharmacological or therapeutic targets in KS.

A Non-Coding RNA Network Involved in KSHV Tumorigenesis.

Regulatory pathways involving non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and long non-coding RNAs (lncRNA), have gained great relevance due to their role in the control of gene expression modulation. Using RNA sequencing of KSHV Bac36 transfected mouse endothelial cells (mECK36) and tumors, we have analyzed the host and viral transcriptome to uncover the role lncRNA-miRNA-mRNA driven networks in KSHV tumorigenesis. The integration of the differentially expressed ncRNAs, with an exhaustive computational analysis of their experimentally supported targets, led us to dissect complex networks integrated by the cancer-related lncRNAs Malat1, Neat1, H19, Meg3, and their associated miRNA-target pairs. These networks would modulate pathways related to KSHV pathogenesis, such as viral carcinogenesis, p53 signaling, RNA surveillance, and cell cycle control. Finally, the ncRNA-mRNA analysis allowed us to develop signatures that can be used to an appropriate identification of druggable gene or networks defining relevant AIDS-KS therapeutic targets.

Autophagy in Viral Development and Progression of Cancer.

Autophagy is a complex degradative process by which eukaryotic cells capture cytoplasmic components for subsequent degradation through lysosomal hydrolases. Although this catabolic process can be triggered by a great variety of stimuli, action in cells varies according to cellular context. Autophagy has been previously linked to disease development modulation, including cancer. Autophagy helps suppress cancer cell advancement in tumor transformation early stages, while promoting proliferation and metastasis in advanced settings. Oncoviruses are a particular type of virus that directly contribute to cell transformation and tumor development. Extensive molecular studies have revealed complex ways in which autophagy can suppress or improve oncovirus fitness while still regulating viral replication and determining host cell fate. This review includes recent advances in autophagic cellular function and emphasizes its antagonistic role in cancer cells.

KSHV G-protein coupled receptor vGPCR oncogenic signaling upregulation of Cyclooxygenase-2 expression mediates angiogenesis and tumorigenesis in Kaposi's sarcoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) vGPCR is a constitutively active G protein-coupled receptor that subverts proliferative and inflammatory signaling pathways to induce cell transformation in Kaposi's sarcoma. Cyclooxygenase-2 (COX-2) is an inflammatory mediator that plays a key regulatory role in the activation of tumor angiogenesis. Using two different transformed mouse models and tumorigenic full KSHV genome-bearing cells, including KSHV-Bac16 based mutant system with a vGPCR deletion, we demostrate that vGPCR upregulates COX-2 expression and activity, signaling through selective MAPK cascades. We show that vGPCR expression triggers signaling pathways that upregulate COX-2 levels due to a dual effect upon both its gene promoter region and, in mature mRNA, the 3'UTR region that control mRNA stability. Both events are mediated by signaling through ERK1/2 MAPK pathway. Inhibition of COX-2 in vGPCR-transformed cells impairs vGPCR-driven angiogenesis and treatment with the COX-2-selective inhibitory drug Celecoxib produces a significant decrease in tumor growth, pointing to COX-2 activity as critical for vGPCR oncogenicity in vivo and indicating that COX-2-mediated angiogenesis could play a role in KS tumorigenesis. These results, along with the overexpression of COX-2 in KS lesions, define COX-2 as a potential target for the prevention and treatment of KSHV-oncogenesis.

Regulation of the Expression of Heme Oxygenase-1: Signal Transduction, Gene Promoter Activation, and Beyond.

Significance: Heme oxygenase-1 (HO-1) is a ubiquitous 32-kDa protein expressed in many tissues and highly inducible. They catalyze the degradation of the heme group and the release of free iron, carbon monoxide, and biliverdin; the latter converted to bilirubin by biliverdin reductase. Its role in the regulation of cellular homeostasis is widely documented. Studying regulation of HO-1 expression is important not only to understand the life of healthy cells but also the unbalances in cell metabolism that lead to disease. Recent Advances: The regulation of its enzymatic activity depends heavily upon changes in expression studied mainly at the transcriptional level. Current knowledge regarding HO-1 gene expression focuses primarily on transcription factors such as Nrf2 (nuclear factor erythroid 2-related factor 2), AP-1 (activator protein-1), and hypoxia-inducible factor, which collect signal transduction pathway information at the HO-1 gene promoter. Understanding of gene expression regulation is not limited to transcription factor activity but also involves an extended range of post- or cotranscriptional regulated events. Critical Issues: In addition to the regulation of gene promoter activity, alternative splicing, alternative polyadenylation, and regulation of messenger RNA stability play critical roles in changes in HO-1 gene expression levels, involving specific factors, proteins, and microRNAs. All potential targets for diagnosis or treatment of diseases are related to HO-1 dysregulation. Future Directions: Unbalances in the tightly regulated gene expression mechanisms lead to cell transformation and cancer development. Knowledge of these events and signal transduction cascades triggered by oncogenes in which HO-1 plays a critical role is of upmost importance for research in this field.

R-spondin3 Is Associated with Basal-Progenitor Behavior in Normal and Tumor Mammary Cells.

R-spondin3 (RSPO3) is a member of a family of secreted proteins that enhance Wnt signaling pathways in diverse processes, including cancer. However, the role of RSPO3 in mammary gland and breast cancer development remains unclear. In this study, we show that RSPO3 is expressed in the basal stem cell-enriched compartment of normal mouse mammary glands but is absent from committed mature luminal cells in which exogenous RSPO3 impairs lactogenic differentiation. RSPO3 knockdown in basal-like mouse mammary tumor cells reduced canonical Wnt signaling, epithelial-to-mesenchymal transition-like features, migration capacity, and tumor formation in vivo Conversely, RSPO3 overexpression, which was associated with some LGR and RUNX factors, highly correlated with the basal-like subtype among patients with breast cancer. Thus, we identified RSPO3 as a novel key modulator of breast cancer development and a potential target for treatment of basal-like breast cancers.Significance: These findings identify RSPO3 as a potential therapetuic target in basal-like breast cancers.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/16/4497/F1.large.jpg Cancer Res; 78(16); 4497-511. ©2018 AACR.

Role of 3'-5'-cyclic adenosine monophosphate on the epidermal growth factor dependent survival in mammary epithelial cells.

Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions.

An interplay between the p38 MAPK pathway and AUBPs regulates c-fos mRNA stability during mitogenic stimulation.

Mitogen-activated protein kinase (MAPK) pathways constitute key regulatory elements linking extracellular stimuli to nuclear gene expression. Immediate-early responsive genes (IEGs) of the activator protein 1 (AP-1) family, such as fos, achieve peak expression levels shortly after cells are stimulated with growth factors and sharply decrease thereafter. Several AU-rich binding proteins (AUBPs), including HuR (Hu-antigen R, Elav-like protein 1, ELAVL1) and KSRP (far upstream element-binding protein 2, KHSRP) bind to a fos AU-rich element (ARE) present in the 3'-UTR (untranslated region) of fos mRNA regulating its stability by a still poorly defined mechanism. We show in the present study that, whereas HuR binds and stabilizes transcribed reporter mRNAs bearing the fos 3'-UTR, KSRP counteracts this effect. Furthermore, we found that fos mRNA stability and HuR phosphorylation status are dependent on the activity of p38 MAPK in both epithelial cells and fibroblasts upon proliferative stimulation. Analysing PPI (protein-protein interaction) networks, we performed a thorough query of interacting proteins for p38 MAPKs, HuR and other AUBPs upon growth factor stimulation. This revealed novel HuR interactors including inhibitors of protein phosphatase 2 (PP2A) activity. Over-expression of two of these interactors, pp32 and APRIL (acidic leucine-rich nuclear phosphoprotein 32 family member B, ANP32B) and pharmacological inhibition of PP2A stabilized a fos reporter mRNA. Our results indicate that p38 MAPK regulates fos mRNA decay by affecting the state of phosphorylation of HuR while controlling yet to be fully elucidated PP regulatory networks.

The use of alternative polyadenylation sites renders integrin ß1 (Itgb1) mRNA isoforms with differential stability during mammary gland development.

Integrins are heterodimeric cell-surface adhesion receptors that play a critical role in tissue development. Characterization of the full-length mRNA encoding the ß1 subunit (Itgb1) revealed an alternative functional cleavage and polyadenylation site that yields a new Itgb1 mRNA isoform 578 bp shorter than that previously reported. Using a variety of experimental and bioinformatic approaches, we found that the two Itgb1 isoforms are expressed at different levels in a variety of mouse tissues, including the mammary gland, where they are differentially regulated at successive developmental stages. The longer mRNA species is prevelant during lactation, whereas the shorter is induced after weaning. In 3D cultures, where expression of integrin ß1 protein is required for normal formation of acini, experimental blockade of the longer isoform induced enhanced expression of the shorter species which allowed normal morphological mammary differentiation. The short isoform lacks AU-rich motifs and miRNA target sequences that are potentially implicated in the regulation of mRNA stability and translation efficiency. We further determined that the AU-binding protein HuR appears to selectively stabilize the longer isoform in the mammary gland. In summary, the results of the present study identify a new regulatory instance involved in the fine-tuning of Itgb1 expression during mammary gland development and function.

Expression of heme oxygenase-1 in non-small cell lung cancer (NSCLC) and its correlation with clinical data.

While changes in heme oxygenase (HO-1) in lung cancer have already been reported, conflicting results were obtained for enzyme expression in human lung cancer specimens. Therefore, the aim of this work was to study HO-1 expression in a large collection of human lung cancer samples. For this purpose, we analyzed the expression of HO-1 in an organized tissue microarray (TMA) and investigated its correlation with clinicopathological data. Ninety-six percent of tumor samples were positive for HO-1, and the expression of HO-1 was significantly higher in cancerous than in non-cancerous tissues. Importantly, HO-1 expression correlated with advanced stages and lymph node involvement. Additionally, quantitative RT-PCR in 18 pairs of human lung carcinomas and their adjacent non-malignant tissues was performed. Our results demonstrate that HO-1 protein is upregulated in epithelial malignant cells in NSCLC and its expression is associated with higher stages of the disease. Additionally, different subcellular localization is observed between tumor and adjacent non-malignant tissues.

Tumor necrosis factor alpha induces LIF expression through ERK1/2 activation in mammary epithelial cells.

It has been reported that expression of tumor necrosis factor superfamily members occur at the onset of the mammary gland post-lactational involution. One of these proteins, tumor necrosis factor alpha (TNFalpha), is a major mediator of inflammation that is able to induce expression of several cytokines. Leukemia inhibitory factor (LIF) is an inflammatory cytokine that is induced and plays a fundamental role during post-lactational involution of the mammary gland. Therefore, our goal was to determine whether TNFalpha activity in the mammary epithelium might include regulation of LIF expression. This biological role would increase the significance of TNFalpha expression at the end of lactation. Our results show that TNFalpha was able to induce LIF transcription through ERK1/2 activation in a non-tumorigenic mouse mammary epithelial cell line, SCp2. We found that activation of TNFalpha receptor-2 (TNFR2) was specifically involved in triggering this signaling pathway. In addition, our data suggest the participation of AP-1 transcription factor family members in this pathway. We determined that TNFalpha treatment induced c-fos transcription, and blocking AP-1 activity resulted in a significant inhibition of TNFalpha-induced LIF expression. Finally, we found that TNFalpha was also able to trigger LIF expression and ERK1/2 activation in the mouse mammary gland in vivo. Therefore, our data suggest that TNFalpha may contribute to mammary gland involution by, among other activities, eliciting LIF expression through ERK1/2 and AP1 activation.

Bcl-XL mediates epidermal growth factor dependent cell survival in HC11 mammary epithelial cells.

Apoptosis is the predominant process controlling cell deletion during post-lactational mammary gland remodeling. The members of the Bcl-2 protein family, whose expression levels are under the control of lactogenic hormones, internally control this mechanism. Epidermal growth factor (EGF) belongs to a family of proteins that act as survival factors for mammary epithelial cells upon binding to specific membrane tyrosine kinase receptors. Expression of EGF peaks during lactation and dramatically decreases in the involuting mammary gland. Though it was suggested that the protective effect of EGF is mediated through the phosphatidylinositol-3-kinase (PI3K) or MEK/ERK kinases activities, little is known about the downstream mechanisms involved on the anti-apoptotic effect of EGF on mammary epithelial cells; particularly the identity of target genes controlling apoptosis. Here, we focused on the effect of EGF on the survival of mammary epithelial cells. We particularly aimed at the characterization of the signaling pathways that were triggered by this growth factor, impinge upon expression of Bcl-2 family members and therefore have an impact on the regulation of cell survival. We demonstrate that EGF provokes the induction of the anti-apoptotic isoform Bcl-XL and the phosphorylation and down-regulation of the pro-apoptotic protein Bad. The activation of JNK and PI3K/AKT signaling pathways promotes the induction of Bcl-XL while AKT activation also leads to Bad phosphorylation and down-regulation. This protective effect of EGF correlates mainly with the up-regulation of Bcl-XL than with the down-regulation of Bad. In fact, HC11 cells unable to express bcl-X, die even in the presence of EGF. In this context, Bcl-XL emerges as a key anti-apoptotic molecule critical for mediating EGF cell survival.

Programmed cell death and differential JNK, p38 and ERK response in a prenatal acute hypoxic hypoxia model.

We previously found that prenatal hypoxia induces a significant increase in the levels of active Caspase 3 at 60 min post-hypoxia (p-h) and in the number of TUNEL-positive pyknotic cells, which peaks at 6h p-h. The aim of this work was to study alterations in MAPKs pathways and the effect of specific inhibitors of the JNK (SP600125) and p38 (SB203580) pathways following acute hypoxia in chick optic lobe at embryonic day (ED) 12. To this end, JNK, p38 and ERK1-2 protein kinase expression levels were determined by Western blot in both their active and inactive forms, evaluated at successive p-h times. At 10 and 30 min p-h the P-JNK/JNK ratio was 1.912+/-0.341 and 1.920+/-0.304, respectively. Concomitantly, at 0 min p-h the P-p38/p38 ratio was 1.657+/-0.203. Lastly, the P-ERK/ERK ratio proving non-significant throughout. When inhibitors for JNK and p38 were used, we observed a decrease in the values of active Caspase 3 at 60 min p-h, which correlated with the control values in the parameters of TUNEL-positive cells at 6h p-h. Analysis for P-ATF-2 demonstrated an increase in hypoxic embryos compared to control ones which was reverted in a dose-dependent manner with the use of both inhibitors. All these results indicate that at ED 12, acute hypoxia might be differentially activating JNK and p38 pathways, without affecting the ERK pathway, which in turn would be activating Caspase 3, thus leading to cell death by apoptosis. Furthermore, JNK and p38 activation precede in time the programmed cell death induced by hypoxia.

Down-modulation of erbB2 activity is necessary but not enough in the differentiation of 3T3-L1 preadipocytes.

The high incidence of obesity-related pathologies, led to the study of the mechanisms involved in preadipose cell proliferation and differentiation. Here, we demonstrate that modulation of erbB2, plays a fundamental role during proliferation and adipogenic induction of preadipocytes. Using 3T3-L1 cells as model, we demonstrate that EGF (10 nM, 5 min) in addition to stimulate receptor tyrosine phosphorylation of both erbB2 and EGFR, is able to induce the heterodimer erbB2-EGFR. We treated proliferating 3T3-L1 cells with two inhibitors, AG 825 (IC(50) 0.35 microM, 54 times more selective for erbB2 than for EGFR, IC(50) 19 microM), and AG 879 (IC(50) of 1 microM for erbB2 versus 500 microM for EGFR). We found that both inhibited the proliferation on a dose-dependent basis, reaching a 30% maximal inhibition at 100 microM (P < 0.001) for AG825, and a 20% maximal inhibition at 10 microM (P < 0.001) for AG 879. These results involve erbB2 in 3T3-L1 proliferation. When studying the differentiation process, we found that the action of MIX-Dexa immediately activates MEK, JNK and p38 kinases. We observed that PD98059 and SP600125 (MEK-ERK and JNK inhibitors, respectively) added 1 h prior to the MIX-Dexa induction produced a decrease in erbB2 expression after 6 h, which is even greater than the one produced by the inducers, MIX-Dexa. This work supports erbB2 as a key factor in 3T3-L1 adipogenesis, acting mostly and not only during the proliferative phase but also during the differentiation through modulation of both its expression and activity.

The Galpha12/13 family of heterotrimeric G proteins and the small GTPase RhoA link the Kaposi sarcoma-associated herpes virus G protein-coupled receptor to heme oxygenase-1 expression and tumorigenesis.

Heme oxygenase-1 (HO-1), an inducible enzyme that metabolizes the heme group, is highly expressed in human Kaposi sarcoma lesions. Its expression is up-regulated by the G protein-coupled receptor from the Kaposi sarcoma-associated herpes virus (vGPCR). Although recent evidence shows that HO-1 contributes to vGPCR-induced tumorigenesis and vascular endothelial growth factor (VEGF) expression, the molecular steps that link vGPCR to HO-1 remain unknown. Here we show that vGPCR induces HO-1 expression and transformation through the Galpha(12/13) family of heterotrimeric G proteins and the small GTPase RhoA. Targeted small hairpin RNA knockdown expression of Galpha(12), Galpha(13), or RhoA and inhibition of RhoA activity impair vGPCR-induced transformation and ho-1 promoter activity. Knockdown expression of RhoA also reduces vGPCR-induced VEFG-A secretion and blocks tumor growth in a murine allograft tumor model. NIH-3T3 cells expressing constitutively activated Galpha(13) or RhoA implanted in nude mice develop tumors displaying spindle-shaped cells that express HO-1 and VEGF-A, similarly to vGPCR-derived tumors. RhoAQL-induced tumor growth is reduced 80% by small hairpin RNA-mediated knockdown expression of HO-1 in the implanted cells. Likewise, inhibition of HO-1 activity by chronic administration of the HO-1 inhibitor tin protoporphyrin IX to mice reduces RhoAQL-induced tumor growth by 70%. Our study shows that vGPCR induces HO-1 expression through the Galpha(12/13)/RhoA axes and shows for the first time a potential role for HO-1 as a therapeutic target in tumors where RhoA has oncogenic activity.

Neutrophil signaling pathways activated by bacterial DNA stimulation.

We have previously shown that bacterial DNA activates human neutrophils in a CpG-independent manner. In this study, we have characterized the signaling pathways involved in the activation mechanism. We found that p38 MAPK, ERK1/2, and JNK pathways, as well as the PI3K/Akt pathway, are activated by bacterial DNA. We also determined that bacterial DNA induces NF-kappaB and AP-1 activation. When analyzing the role of these pathways on neutrophil functions, we observed that up-regulation of CD11b triggered by bacterial DNA was decreased by pharmacological inhibitors of the p38 MAPK, ERK1/2, and JNK, whereas stimulation of IL-8 release was dependent on p38, ERK1/2, and NF-kappaB. Moreover, we found that IL-8 production was markedly enhanced by inhibition of JNK, suggesting that this pathway negatively modulates NF-kappaB-dependent transcription. We also observed that bacterial DNA stimulated IL-1R-associated kinase-1 kinase activity and its partial degradation. Finally, we determined that bacterial DNA stimulated CD11b up-regulation in TLR9(-/-) but not in MyD88(-/-) mouse neutrophils, supporting that bacterial DNA induces neutrophil activation through a TLR9-independent and MyD88-dependent pathway.

Different signaling pathways are involved in cardiomyocyte survival induced by a Trypanosoma cruzi glycoprotein.

We have recently reported that Trypanosoma cruzi infection protects cardiomyocytes against apoptosis induced by growth factor deprivation. Cruzipain, a major parasite antigen, reproduced this survival effect by a Bcl-2-dependent mechanism. In this study, we have investigated the molecular mechanisms of cruzipain-induced cardiomyocyte protection. Neonatal BALB/c mouse cardiac myocytes were cultured under minimum serum conditions in the presence of cruzipain or T. cruzi (Tulahuen strain). Some cultures were pretreated with the phosphatidylinositol 3-kinase (PI3K) inhibitor Ly294002 or specific inhibitors of the mitogen-activated protein kinase (MAPK) family members such as the mitogen-activated protein kinase kinase (MEK1) inhibitor PD098059, Jun N-terminal kinase (JNK) inhibitor SP600125, p38 MAPK inhibitor SB203580. Inhibition of PI3K and MEK1 but not JNK or p38 MAPK increased the apoptotic rate of cardiomyocytes treated with cruzipain. Phosphorylation of Akt, a major target of PI3K, and ERK1/2, MEK1-targets, was achieved at 15 min and 5 min, respectively. In parallel, these kinases were strongly phosphorylated by T. cruzi infection. In cultures treated with cruzipain, cleavage of caspase 3 was considerably diminished after serum starvation; Bcl-2 overexpression was inhibited by PD098059 but not by Ly294002, whereas Bad phosphorylation and Bcl-xL expression were increased and differentially modulated by both inhibitors. The results suggest that cruzipain exerts its anti-apoptotic property in cardiac myocytes at least by PI3K/Akt and MEK1/ERK1/2 signaling pathways. We further identified a differential modulation of Bcl-2 family members by these two signaling pathways.

Differential involvement of ERK1-2 and p38MAPK activation on Swiss 3T3 cell proliferation induced by prostaglandin F2alpha.

Prostaglandin F2alpha (PGF2alpha) induces cyclin D1 expression and DNA synthesis in Swiss 3T3 cells. In order to assess which signaling mechanisms are implicated in these processes, we have used both a pharmacological approach and interfering mutants. We demonstrate that PGF2alpha induces extracellular-signal-regulated kinase (ERK1-2) and p38MAPK activation, and inhibition of any of these signaling pathways completely blocks PGF2alpha-stimulated DNA synthesis. We also show that ERK1-2, but not p38MAPK activation is required to induce cyclin D1 expression, strongly suggesting that the concerted action of cyclin D1 gene expression and other events are required to induce complete phosphorylation of retinoblastoma protein and S-phase entry in response to PGF2alpha.