Induction of CIITA by IFN-γ in macrophages involves STAT1 activation byJAK and JNK

The induction of major histocompatibility complex (MHC) class II proteins by interferon gamma (IFN-γ) in macrophages play an important role during immune responses. Here we explore the signaling pathways involved in the induction by IFN-γ of the MHC II transactivator (CIIta) required for MHC II transcriptional activation. Cyclophilin A (CypA) is required for IFN-γ-dependent induction of MHC II in macrophages, but not when it is mediated by GM-CSF. The effect of CypA appears to be specific because it does not affect the expression of other molecules or genes triggered by IFN-γ, such as FcγR, NOS2, Lmp2, and Tap1. We found that CypA inhibition blocked the IFN-γ-induced expression of CIIta at the transcriptional level in two phases. In an early phase, during the first 2 h of IFN-γ treatment, STAT1 is phosphorylated at Tyrosine 701 and Serine 727, residues required for the induction of the transcription factor IRF1. In a later phase, STAT1 phosphorylation and JNK activation are required to trigger CIIta expression. CypA is needed for STAT1 phosphorylation in this last phase and to bind the CIIta promoter. Our findings demonstrate that STAT1 is required in a two-step induction of CIIta, once again highlighting the significance of cross talk between signaling pathways in macrophages

Differential voltage-dependent K+ channel responses during proliferation and activation in macrophages

Voltage-dependent K+ channels (VDPC) are expressed in most mammalian cells and involved in the proliferation and activation of lymphocytes. However, the role of VDPC in macrophage responses is not well established. This study was undertaken to characterize VDPC in macrophages and determine their physiological role during proliferation and activation. Macrophages proliferate until an endotoxic shock halts cell growth and they become activated. By inducing a schedule that is similar to the physiological pattern, we have identified the VDPC in non-transformed bone marrow-derived macrophages and studied their regulation. Patch clamp studies demonstrated that cells expressed outward delayed and inwardly rectifying K+ currents. Pharmacological data, mRNA, and protein analysis suggest that these currents were mainly mediated by Kv1.3 and Kir2.1 channels. Macrophage colony-stimulating factor-dependent proliferation induced both channels. Lipopolysaccharide (LPS)-induced activation differentially regulated VDPC expression. While Kv1.3 was further induced, Kir2.1 was down-regulated. TNF-alpha mimicked LPS effects, and studies with TNF-alpha receptor I/II double knockout mice demonstrated that LPS regulation mediates such expression by TNF-alpha-dependent and -independent mechanisms. This modulation was dependent on mRNA and protein synthesis. In addition, bone marrow-derived macrophages expressed Kv1.5 mRNA with no apparent regulation. VDPC activities seem to play a critical role during proliferation and activation because not only cell growth, but also inducible nitric-oxide synthase expression were inhibited by blocking their activities. Taken together, our results demonstrate that the differential regulation of VDPC is crucial in intracellular signals determining the specific macrophage response

Mesenchymal stromal cell engagement in cancer cell epithelial to mesenchymal transition

Due to coexistence of stromal and epithelial tumor cells, their dynamic interactions have been widely recognized as significant cellular components to the tumor tissue integrity. Initiation and outcome of epithelial to mesenchymal transition (EMT) in tumor cells are dependent on their interaction with adjacent or recruited mesenchymal stromal cells (MSCs). A plethora of mechanisms are involved in MSCs-controlled employment of the developmental processes of EMT that contribute to loss of epithelial cell phenotype and acquisition of stemness, invasiveness and chemoresistance of tumor cells. Interplay of MSCs with tumor cells, including interchange of soluble biomolecules, plasma membrane structures, cytoplasmic content, and organelles, is established through cell-cell contact and/or by means of paracrine signaling. The main focus of this review is to summarize knowledge about involvement of MSCs in cancer cell EMT. Understanding the underlying cellular and molecular mechanism involved in the interplay between MSCs and cancer EMT is essential for development of effective therapy approaches, which in combination with current treatments may improve the control of tumor progression. Developmental Dynamics 247:359-367, 2018

Transforming growth factor-, matrix metalloproteinases, and urokinase-type plasminogen activator interaction in the cancer epithelial to mesenchymal transition

Transforming growth factor- (TGF-) is a pleiotropic factor that acts as a tumor suppressor in the early stages, while it exerts tumor promoting activities in advanced stages of cancer development. One of the hallmarks of cancer progression is the capacity of cancer cells to migrate and invade surrounding tissues with subsequent metastasis to different organs. Matrix metalloproteinases (MMPs) together with urokinase-type plasminogen activator (uPA) and its receptor (uPAR), whose main original function described is the proteolytic degradation of the extracellular matrix, play key cellular roles in the enhancement of cell malignancy during cancer progression. TGF- tightly regulates the expression of several MMPs and uPA/uPAR in cancer cells, which in return can participate in TGF- activation, thus contributing to tumor malignancy. TGF- is one of the master factors in the induction of cancer-associated epithelial to mesenchymal transition (EMT), and recently both MMPs and uPA/uPAR have also been shown to be implicated in the cancer-associated EMT process. In this review, we analyze the main molecular mechanisms underlying MMPs and uPA/uPAR regulation by TGF-, as well as their mutual implication in the development of EMT in cancer cells. Developmental Dynamics 247:382-395, 2018

The epigenetic landscape of renal cancer

This is an accepted manuscript of an article published by Nature in Nature Reviews: Nephrology on 28/11/2016, available online: https://doi.org/10.1038/nrneph.2016.168 The accepted version of the publication may differ from the final published version.The majority of kidney cancers are associated with mutations in the von Hippel-Lindau gene and a small proportion are associated with infrequent mutations in other well characterized tumour-suppressor genes. In the past 15 years, efforts to uncover other key genes involved in renal cancer have identified many genes that are dysregulated or silenced via epigenetic mechanisms, mainly through methylation of promoter CpG islands or dysregulation of specific microRNAs. In addition, the advent of next-generation sequencing has led to the identification of several novel genes that are mutated in renal cancer, such as PBRM1, BAP1 and SETD2, which are all involved in histone modification and nucleosome and chromatin remodelling. In this Review, we discuss how altered DNA methylation, microRNA dysregulation and mutations in histone-modifying enzymes disrupt cellular pathways in renal cancers

Early steps regulationg proliferation and activation in macrophages

Macrophages are key regulators of immune system connecting innate and specific immune responses. Macrophages proliferate in presence of their growth factor, M-CSF. The addition of bacterial lipopolysacharide, LPS, induces macrophage activation and stops their proliferation engaging a pro-inflammatory response. The activation of ERK MAPK is required for both macrophage proliferation and activation. However, different time-course of ERK activation is displayed. Proliferation is a process dependent on early and short ERK activation, whereas LPS addition delays and elongates ERK activation inducing an inflammatory response. Proliferating or activating responses are balanced by the extent and duration of ERK phosphorylation that is regulated by mitogen kinase phosphatase MKP1 (DUSP1). MKP1 is induced by both M-CSF and LPS and its kinetics of induction is correlated with those of inactivation of MAPKs. The induction of MKP-1 by M-CSF or LPS is mediated by PKC-epsilon. Our studies in primary cultures of murine bone marrow derived macrophages, show that MKP-1 expression by both M-CSF and LPS is dependent on activation of Raf-1 kinase, and its interaction with PKC Õ. The time-course of activation of ERK is correlated with that of Raf-1 and MEK-1/2. The use of specific inhibitors and RNA of interference, has shown that ERK activation during proliferation is dependent on Raf-1 activation, whereas in response to inflamatory stimuli such as LPS an alternative pathway to Raf-1 to direct the activation of these kinases. Inhibition of Raf-1 activity causes a growth arrest. The cell cycle blockage at G1 phase correlated with increased expression of cyclin-dependent kinase (Cdks) inhibitors, p21Waf1 and p27Kip1. On the other hand, no effects were observed during macrophage activation as assessed by pro-inflammatory cytokine expression and induction of nitric oxide synthase following LPS stimulation. In addition, the transcriptional induction of MKP-1 phosphatase by both M-CSF and LPS is independent of ERK and p38 activation, but dependent on JNK activation as assessed using inhibitors. In consequence to inactivation of MKP-1, an elongation of other MAPKs activity, ERK and p38, is observed. Macrophages constitutively express JNK1 and JNK2 isoforms, while no JNK3 is detected. JNK1 is the main isoform involved in JNK activity. Using single knock-out mice for jnk1 and jnk2 genes, we have demonstrated that MKP-1 induction is mediated by JNK1 isoform. Moreover, JNK1 is also required for biosynthesis of proinflammatory cytokines (TNF-alpha, IL-1beta and IL-6) and for induction of nitric oxide synthase. This requirement is independent on JNK1 function as regulator of MKP-1 induction, as shown using knock-out mice for this phosphatase. These data indicate that Raf-1 is critical in ERK MAPK activation during macrophage proliferation whereas its absence does not compromise macrophage activation. Furthermore, Raf-1 is involved in the expression of MKP-1 phosphatase implicated in MAPK deactivation, through interaction with PKC-epsilon isoform. In addition, MKP-1 phosphatase expression is also dependent on JNK activity suggesting a selfregulation of MAPKs through induction of phosphatases. From different JNK isoforms, JNK1 is involved both in the expression of MKP-1 phosphatase and displays a direct role in the LPS-dependent macrophage activation

Early steps regulationg proliferation and activation in macrophages

Macrophages are key regulators of immune system connecting innate and specific immune responses. Macrophages proliferate in presence of their growth factor, M-CSF. The addition of bacterial lipopolysacharide, LPS, induces macrophage activation and stops their proliferation engaging a pro-inflammatory response. The activation of ERK MAPK is required for both macrophage proliferation and activation. However, different time-course of ERK activation is displayed. Proliferation is a process dependent on early and short ERK activation, whereas LPS addition delays and elongates ERK activation inducing an inflammatory response. Proliferating or activating responses are balanced by the extent and duration of ERK phosphorylation that is regulated by mitogen kinase phosphatase MKP1 (DUSP1). MKP1 is induced by both M-CSF and LPS and its kinetics of induction is correlated with those of inactivation of MAPKs. The induction of MKP-1 by M-CSF or LPS is mediated by PKC-epsilon.Our studies in primary cultures of murine bone marrow derived macrophages, show that MKP-1 expression by both M-CSF and LPS is dependent on activation of Raf-1 kinase, and its interaction with PKC Õ. The time-course of activation of ERK is correlated with that of Raf-1 and MEK-1/2. The use of specific inhibitors and RNA of interference, has shown that ERK activation during proliferation is dependent on Raf-1 activation, whereas in response to inflamatory stimuli such as LPS an alternative pathway to Raf-1 to direct the activation of these kinases. Inhibition of Raf-1 activity causes a growth arrest. The cell cycle blockage at G1 phase correlated with increased expression of cyclin-dependent kinase (Cdks) inhibitors, p21Waf1 and p27Kip1. On the other hand, no effects were observed during macrophage activation as assessed by pro-inflammatory cytokine expression and induction of nitric oxide synthase following LPS stimulation. In addition, the transcriptional induction of MKP-1 phosphatase by both M-CSF and LPS is independent of ERK and p38 activation, but dependent on JNK activation as assessed using inhibitors. In consequence to inactivation of MKP-1, an elongation of other MAPKs activity, ERK and p38, is observed. Macrophages constitutively express JNK1 and JNK2 isoforms, while no JNK3 is detected. JNK1 is the main isoform involved in JNK activity. Using single knock-out mice for jnk1 and jnk2 genes, we have demonstrated that MKP-1 induction is mediated by JNK1 isoform. Moreover, JNK1 is also required for biosynthesis of proinflammatory cytokines (TNF-alpha, IL-1beta and IL-6) and for induction of nitric oxide synthase. This requirement is independent on JNK1 function as regulator of MKP-1 induction, as shown using knock-out mice for this phosphatase. These data indicate that Raf-1 is critical in ERK MAPK activation during macrophage proliferation whereas its absence does not compromise macrophage activation. Furthermore, Raf-1 is involved in the expression of MKP-1 phosphatase implicated in MAPK deactivation, through interaction with PKC-epsilon isoform. In addition, MKP-1 phosphatase expression is also dependent on JNK activity suggesting a selfregulation of MAPKs through induction of phosphatases. From different JNK isoforms, JNK1 is involved both in the expression of MKP-1 phosphatase and displays a direct role in the LPS-dependent macrophage activation

Inorganic Nanoparticles and the Immune System: Detection, Selective Activation and Tolerance

The immune system is the responsible for body integrity and prevention of external invasion. On one side, nanoparticles are no triggers that the immune system is prepared to detect, on the other side it is known that foreign bodies, not only bacteria, viruses and parasites, but also inorganic matter, can cause various pathologies such as silicosis, asbestosis or inflammatory reactions. Therefore, nanoparticles entering the body, after interaction with proteins, will be either recognized as self-agents or detected by the immune system, encompassing immunostimulation or immunosuppression responses. The nature of these interactions seems to be dictated not specially by the composition of the material but by modifications of NP coating (composition, surface charge and structure). Herein, we explore the use of gold nanoparticles as substrates to carry multifunctional ligands to manipulate the immune system in a controlled manner, from undetection to immunostimulation. Murine bone marrow macrophages can be activated with artificial nanometric objects consisting of a gold nanoparticle functionalized with peptides. In the presence of some conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines were induced. The biochemical type of response depended on the type of conjugated peptide and was correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical NP conjugate design to either activate the immune system or hide from it, in order to reach their targets before being removed by phagocytes. Additionally, it opens up the possibility to modulate the immune response in order to suppress unwanted responses resulting from autoimmunity, or allergy or to stimulate protective responses against pathogens