Deciphering the Role and Signaling Pathways of PKCα in Luminal A Breast Cancer Cells.

Protein kinase C (PKC) comprises a family of highly related serine/threonine protein kinases involved in multiple signaling pathways, which control cell proliferation, survival, and differentiation. The role of PKCα in cancer has been studied for many years. However, it has been impossible to establish whether PKCα acts as an oncogene or a tumor suppressor. Here, we analyzed the importance of PKCα in cellular processes such as proliferation, migration, or apoptosis by inhibiting its gene expression in a luminal A breast cancer cell line (MCF-7). Differential expression analysis and phospho-kinase arrays of PKCα-KD vs. PKCα-WT MCF-7 cells identified an essential set of proteins and oncogenic kinases of the JAK/STAT and PI3K/AKT pathways that were down-regulated, whereas IGF1R, ERK1/2, and p53 were up-regulated. In addition, unexpected genes related to the interferon pathway appeared down-regulated, while PLC, ERBB4, or PDGFA displayed up-regulated. The integration of this information clearly showed us the usefulness of inhibiting a multifunctional kinase-like PKCα in the first step to control the tumor phenotype. Then allowing us to design a possible selection of specific inhibitors for the unexpected up-regulated pathways to further provide a second step of treatment to inhibit the proliferation and migration of MCF-7 cells. The results of this study suggest that PKCα plays an oncogenic role in this type of breast cancer model. In addition, it reveals the signaling mode of PKCα at both gene expression and kinase activation. In this way, a wide range of proteins can implement a new strategy to fine-tune the control of crucial functions in these cells and pave the way for designing targeted cancer therapies.

Identification of an interferon-stimulated gene, isg15, involved in host immune defense against viral infections in gilthead seabream (Sparus aurata L.).

Interferons (IFNs) play a key role in the innate immunity of vertebrates against viral infections by inducing hundreds of IFN-stimulated genes (ISGs), such as isg15. Isg15 is an ubiquitin-like protein, which can conjugate cellular and viral proteins in a process called ISGylation, although it can also act as a cytokine-like protein. Gilthead seabream (Sparus aurata L.) is an important asymptomatic carrier of viral haemorrhagic septicaemia virus (VHSV) and nodavirus, representing a threat to other co-cultivated susceptible species. In order to better understand virus-host interactions in this fish species, this study addresses the identification and molecular characterization of seabream isg15 (sb-isg15). In addition, the modulation of transcript levels of sb-isg15 was analysed in SAF-1 cells and seabream acidophilic granulocytes (AGs) stimulated in vitro with different pathogen-associated molecular patterns (PAMPs) or inoculated with VHSV and striped jack nervous necrosis virus (SJNNV). The full-length cDNA of sb-isg15 gene, encoding a predicted protein of 155 amino acids, was identified and seen to share the same characteristics as other fish and mammalian isg15 genes. Here we report the clear induction of sb-isg15 transcript levels in SAF-1 cells and AGs stimulated with toll-like receptor (TLR) ligands, such as polyinosinic:polycytidylic acid (poly I:C) or genomic DNA from Vibrio anguillarum (VaDNA), respectively. Furthermore, VHSV and SJNNV inoculation induced a significant degree of sb-isg15 transcription in SAF-1 cells and AGs. However, the relative levels of viral RNA transcription showed that SJNNV replication seems to be more efficient than VHSV in both in vitro systems. Interestingly, sb-isg15 transcript induction elicited by VaDNA was reduced in VHSV- and SJNNV-inoculated AGs, suggesting an interference prompted by the viruses against the type I IFN system. Taken together, these findings support the use of seabream AGs as a valuable experimental system to study virus-host interactions, in which sb-isg15 seems to play an important role.

Prostaglandin E2 promotes M2 polarization of macrophages via a cAMP/CREB signaling pathway and deactivates granulocytes in teleost fish.

The profile of prostaglandin (PG) production is determined by the differential expression of the enzymes involved in their production and degradation. Although the production of PGE2 by fish leukocytes has been relatively well studied in several fish species, knowledge of how its production is regulated, its biological activities and the signaling pathways activated by this PG is scant or even contradictory. In this work we show that in the teleost fish gilthead seabream (Sparus aurata L.) macrophages regulate PGE2 release mainly by inducing the expression of the genes encoding the enzymes responsible for its synthesis, while acidophilic granulocytes (AGs) not only induce these genes quickly after activation but also inhibit the expression of the genes encoding the enzymes responsible for PGE2 degradation at later time points. In addition, treatment of macrophages with PGE2 promoted their M2 polarization, which is characterized by high expression levels of interleukin-10, mannose-receptor c-type 1 and arginase 2 genes. In sharp contrast, PGE2 promoted the deactivation of AGs, since it decreased the production of reactive oxygen species and the expression of genes encoding pro-inflammatory cytokines. These differences are the result of the alternative signaling pathways used by PGE2 in macrophages and AGs, a cAMP/CREB signaling pathway operating in macrophages, but not in AGs, downstream of PGE2. Our data identify for the first time a role for professional phagocyte-derived-PGE2 in the resolution of inflammation in fish and highlight key differences in the PGE2 signaling pathway in macrophages and granulocytes.

Peroxisome proliferator-activated receptors alpha and beta mediate the anti-inflammatory effects of the cyclopentenone prostaglandin 15-deoxy-Δ12,14-PGJ2 in fish granulocytes.

Prostaglandins (PGs) are highly reactive small lipophilic molecules derived from polyunsaturated fatty acids of the cell membrane and play a key role in the resolution of inflammation processes. 15-deoxy-Δ12,14-PGJ2 (15dPGJ2) is a cyclopentenone PG (CyPG) of the J series with anti-inflammatory, anti-proliferative and pro-apoptotic effects. This CyPG can signal through: (i) the PGD2 receptor (DP2) and peroxisome proliferator-activated receptor γ (PPARγ) or (ii) by covalent binding to protein nucleophiles, such as, thiols groups of cysteine, lysine or histidine via a Michael addition reaction, modifying its structure and function. In this work we show that acidophilic granulocytes (AGs) of gilthead seabream (Sparus aurata L.), the functional equivalent to mammalian neutrophils, constitutively expressed ppara, pparb and pparg genes, the latter showing the highest expression and up-regulation when stimulated by bacterial DNA. In addition, we tested the ability of 15dPGJ2, and its biotinylated analog, as well as several PPARγ ligands, to modulate reactive oxygen species (ROS) and/or cytokines production during a Toll like receptor (TLR)-mediated granulocyte response. Thus, 15dPGJ2 was able to significantly decrease bacterial DNA-induced ROS production and transcript levels of pparg, interleukin-1ß (il1b) and prostaglandin-endoperoxide synthase 2 (ptgs2). In contrast, its biotinylated analog was less potent and a higher dose was required to elicit the same effects on ROS production and cytokine expression. In addition, different PPARγ agonists were able to mimic the effects of 15dPGJ2. Conversely, the PPARγ antagonist T007097 abolished the effect of 15dPGJ2 on DNA bacterial-induced ROS production. Surprisingly, transactivation assays revealed that both 15dPGJ2 and its biotinylated analog signaled via Pparα and Pparß, but not by Pparγ. These results were further confirmed by HPLC/MS analysis, where Pparß was identified as an interactor of biotin-15dPGJ2 in naïve and DNA-stimulated leukocytes. Taken together, our data show that 15dPGJ2 acts both through Ppar activation and covalent binding to proteins in fish granulocytes and identify for the first time in vertebrates a role for Pparα and Pparß in the resolution of inflammation mediated by 15dPGJ2.

The molecular, functional and phylogenetic characterization of PGE2 receptors reveals their different roles in the immune response of the teleost fish gilthead seabream (Sparus aurata L.).

Prostaglandin E2 (PGE2) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE2 signaling, as well as the pathways activated downstream, are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE2, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE2 receptors in the regulation of the immune responses.

The role of prostaglandins in the regulation of fish immunity.

Prostaglandins (PGs) play a key role in the development of the immune response. These molecules are produced by a variety of cells including leukocytes, macrophages being the most important source of PGs for the innate immune system. PGs can elicit a variety of effects depending on the inflammatory milieu but also on the presence of specific PG receptors in many cell types. The biosynthetic pathways of prostaglandins, their production by cells of the immune system and their functions as immune-modulators, have been poorly studied in fish. This review is based on the available published data and our own experience and highlights recent advances in our understanding of the role for PGs in the regulation of fish immunity. The evolutionary conservation of their functions in fish is proof of their importance as a regulator of the immune response.

Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins.

Inflammasomes are cytosolic molecular platforms that alert the immune system about the presence of infection. Here we report that zebrafish guanylate-binding protein 4 (Gbp4), an IFNγ-inducible GTPase protein harbouring a C-terminal CARD domain, is required for the inflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo. Despite the presence of the CARD domain, Gbp4 requires the universal inflammasome adaptor Asc for mediating its antibacterial function. In addition, the GTPase activity of Gbp4 is indispensable for inflammasome activation and ST clearance. Mechanistically, neutrophils are recruited to the infection site through the inflammasome-independent production of the chemokine (CXC motif) ligand 8 and leukotriene B4, and then mediate bacterial clearance through the Gbp4 inflammasome-dependent biosynthesis of prostaglandin D2. Our results point to GBPs as key inflammasome adaptors required for prostaglandin biosynthesis and bacterial clearance by neutrophils and suggest that transient activation of the inflammasome may be used to treat bacterial infections.

Professional phagocytic granulocyte-derived PGD2 regulates the resolution of inflammation in fish.

Prostaglandins (PGs) play a key role in the development on the immune response through the regulation of both pro- and anti-inflammatory processes. PGD(2) can be either pro- or anti-inflammatory depending on the inflammatory milieu. Prostaglandin D synthase (PGDS) is the enzyme responsible for the conversion of PGH(2) to PGD(2). In mammals, two types of PGDS synthase have been described, the hematopoietic (H-PGDS) and the lipocalin (L-PGDS). In the present study we describe the existence of two orthologs of the mammalian L-PGDS (PGDS1 and PGDS2) in the gilthead seabream and characterize their gene expression profiles and biological activity. The results showed a dramatic induction of the gene coding for PGDS1 in acidophilic granulocytes (AGs), which are functionally equivalent to mammalian neutrophils, after a prolonged in vitro activation with different pathogen associated molecular patterns (PAMPs). In contrast PGDS2 was not expressed in these cells. The functional relevance of the induction of PGDS1 in AGs was confirmed by the ability of these cells to release PGD(2) upon PAMP stimulation. To gain further insight into the role of PGD(2) in the resolution of inflammation in fish, we examined the ability of PGD(2) or its cyclopentenone derivates (cyPGs) to modulate the main functional activities of AGs. It was found that both PGD(2) and cyPGs inhibited the production of reactive oxygen species and downregulated the transcript levels of the gene encoding interleukin-1ß. Taken together, these results demonstrate that the use of PGD(2) and its metabolites in the resolution of inflammation was established before the divergence of fish from tetrapods more than 450 million years ago and support a critical role for granulocytes in the resolution of inflammation in vertebrates.