Floxed Il1rl2 Locus with mCherry Reporter Element Reveals Distinct Expression Patterns of the IL-36 Receptor in Barrier Tissues.

IL-36 cytokines are emerging as beneficial in immunity against pathogens and cancers but can also be detrimental when dysregulated in autoimmune and autoinflammatory conditions. Interest in targeting IL-36 activity for therapeutic purposes is rapidly growing, yet many unknowns about the functions of these cytokines remain. Thus, the availability of robust research tools is essential for both fundamental basic science and pre-clinical studies to fully access outcomes of any manipulation of the system. For this purpose, a floxed Il1rl2, the gene encoding the IL-36 receptor, mouse strain was developed to facilitate the generation of conditional knockout mice. The targeted locus was engineered to contain an inverted mCherry reporter sequence that upon Cre-mediated recombination will be flipped and expressed under the control of the endogenous Il1rl2 promoter. This feature can be used to confirm knockout in individual cells but also as a reporter to determine which cells express the IL-36 receptor IL-1RL2. The locus was confirmed to function as intended and further used to demonstrate the expression of IL-1RL2 in barrier tissues. Il1rl2 expression was detected in leukocytes in all barrier tissues. Interestingly, strong expression was observed in epithelial cells at locations in direct contact with the environment such as the skin, oral mucosa, the esophagus, and the upper airways, but almost absent from epithelial cells at more inward facing sites, including lung alveoli, the small intestine, and the colon. These findings suggest specialized functions of IL-1RL2 in outward facing epithelial tissues and cells. The generated mouse model should prove valuable in defining such functions and may also facilitate basic and translational research.

Inhibiting endothelial cell Mst1 attenuates acute lung injury in mice.

Background: Lung endothelium plays a pivotal role in the orchestration of inflammatory and injury responses to acute pulmonary insults. Mammalian sterile 20-like kinase 1 (Mst1), a mammalian homolog of Hippo, is a serine/threonine kinase that is ubiquitously expressed in many tissues and has been shown to play an important role in the regulation of apoptosis, inflammation, stress responses, and organ growth. While Mst1 exhibits high expression in the lung, its involvement in the endothelial response to pulmonary insults remains largely unexplored. Methods: Mst1 activity was assessed in lung endothelium by western blot. Mst1 endothelial specific knockout mice and a pharmacological inhibitor were employed to assess the effects of Mst1 on homeostatic and lipopolysaccharide (LPS)-induced endothelial responses. Readouts for these studies included various assays, including NF-κB activation and levels of various inflammatory cytokines and adhesion molecules. The role of Mst1 in lung injury was evaluated in a LPS-induced murine model of acute lung injury (ALI). Results: Mst1 phosphorylation was significantly increased in lung endothelial cells after exposure to tumor necrosis factor (TNF)-alpha (TNF-α) and mouse lung tissues after LPS exposure. Overexpression of full length Mst1 or its kinase domain promoted nuclear factor kappaB (NF-κB) activation through promoting JNK and p38 activation, whereas dominant negative forms of Mst1 (DN-Mst1) attenuated endothelial responses to TNF-α and interleukin-1ß. Consistent with this, targeted deletion of Mst1 in lung endothelium reduced lung injury to LPS in mice. Similarly, wild-type mice were protected from LPS-induced lung injury following treatment with a pharmacological inhibitor of Mst1/2. Conclusions: Our findings identified Mst1 kinase as a key regulator in the control of lung EC activation and suggest that therapeutic strategies aimed at inhibiting Mst1 activation might be effective in the prevention and treatment of lung injury to inflammatory insults.

Epigenetic regulation of vascular smooth muscle cell phenotypic switch and neointimal formation by PRMT5.

AIMS: Phenotypic transition of vascular smooth muscle cells (VSMCs) from a contractile to a synthetic state is involved in the development of cardiovascular diseases, including atherosclerosis, hypertension, and post-angioplasty restenosis. Arginine methylation catalyzed by protein arginine methyltransferases (PRMTs) has been implicated in multiple cellular processes, however, its role in VSMC biology remains undetermined. The objective of this study was to determine the role of PRMTs in VSMC phenotypic switch and vascular remodelling after injury. METHODS AND RESULTS: Our results show that PRMT5 is the most abundantly expressed PRMT in human aortic SMCs, and its expression is up-regulated in platelet-derived growth factor (PDGF)-stimulated VSMCs, human atherosclerotic lesions, and rat carotid arteries after injury, as determined by western blot and immunohistochemical staining. PRMT5 overexpression inhibits the expression of SMC marker genes and promotes VSMC proliferation and migration, while silencing PRMT5 exerts the opposite effects. Mechanistically, we found that PRMT5 overexpression led to histone di-methylation of H3R8 and H4R3, which in turn attenuates acetylation of H3K9 and H4, thus limiting recruitment of the SRF/myocardin complexes to the CArG boxes of SMC marker genes. Furthermore, both SMC-specific deletion of PRMT5 in mice and local delivery of lentivirus expressing shPRMT5 to rat carotid arteries significantly attenuated neointimal formation after injury. Likewise, pharmacological inhibition of PRMT5 by EPZ015666 markedly inhibited carotid artery ligation-induced neointimal formation in mice. CONCLUSIONS: Our results identify PRMT5 as a novel regulator in VSMC phenotypic switch and suggest that inhibition of PRMT5 may represent an effective therapeutic strategy for proliferative vascular diseases.

Protein disulfide isomerase A1 regulates breast cancer cell immunorecognition in a manner dependent on redox state.

Oxidoreductase protein disulphide isomerases (PDI) are involved in the regulation of a variety of biological processes including the modulation of endoplasmic reticulum (ER) stress, unfolded protein response (UPR), ER­mitochondria communication and the balance between pro­survival and pro­death pathways. In the current study the role of the PDIA1 family member in breast carcinogenesis was investigated by measuring ROS generation, mitochondrial membrane disruption, ATP production and HLA­G protein levels on the surface of the cellular membrane in the presence or absence of PDIA1. The results showed that this enzyme exerted pro­apoptotic effects in estrogen receptor (ERα)­positive breast cancer MCF­7 and pro­survival in triple negative breast cancer (TNBC) MDA­MB­231 cells. ATP generation was upregulated in PDIA1­silenced MCF­7 cells and downregulated in PDIA1­silenced MDA­MB­231 cells in a manner dependent on the cellular redox status. Furthermore, MCF­7 and MDA­MB­231 cells in the presence of PDIA1 expressed higher surface levels of the non­classical human leukocyte antigen (HLA­G) under oxidative stress conditions. Evaluation of the METABRIC datasets showed that low PDIA1 and high HLA­G mRNA expression levels correlated with longer survival in both ERα­positive and ERα­negative stage 2 breast cancer patients. In addition, analysis of the PDIA1 vs. the HLA­G mRNA ratio in the subgroup of the living stage 2 breast cancer patients exhibiting low PDIA1 and high HLA­G mRNA levels revealed that the longer the survival time of the ratio was high PDIA1 and low HLA­G mRNA and occurred predominantly in ERα­positive breast cancer patients whereas in the same subgroup of the ERα­negative breast cancer mainly this ratio was low PDIA1 and high HLA­G mRNA. Taken together these results provide evidence supporting the view that PDIA1 is linked to several hallmarks of breast cancer pathways including the process of antigen processing and presentation and tumor immunorecognition.

Suppression of µ1 subunit of the adaptor protein complex 2 reduces dengue virus release.

Dengue virus (DENV) requires clathrin-mediated endocytosis for its entry into the cells where the adaptor protein complex (AP) is vital for the clathrin-coated vesicle formation. The role of AP-2 was previously examined in the early stages of DENV infection; however, the role of AP-2 in the late stage of DENV infection was not determined. The µ1 subunit of AP-2 (AP2M1) is one of the most important cytoplasmic carrier domains in clathrin-mediated endocytosis and the phosphorylation of this subunit by the kinase enzyme, AP-2 associated protein kinase 1 (AAK1), stimulates clathrin and supports the cell surface receptor incorporation. In the present study, we primarily aimed to investigate the role of AP2M1 by gene silencing approach as well as using naked DENV RNA transfection into AP2M1 knockdown cells. Secondarily, an inhibitor of AAK1, sunitinib was used to investigate whether AAK1 could influence the virus production in DENV-infected Huh7 cells. The knockdown of AP2M1 in the DENV-infected Huh7 cells displayed a reduction in the viral titer at 24 h post-infection. Furthermore, experiments were conducted to bypass the DENV internalization using a naked DENV RNA transfection into the AP2M1 knockdown cells. Higher intracellular DENV RNA, DENV E protein, and intracellular virion were observed, whereas the extracellular virion production was comparably less than that of control. Treatment with sunitinib in DENV-infected Huh7 cells was able to reduce extracellular virion production and was consistent with all four serotypes of DENV. Therefore, our findings demonstrate the role of AP2M1 in the exocytosis step of DENV replication leading to infectious DENV production and the efficacy of sunitinib in suppressing virus production during the infection with different serotypes of DENV.

Sirtuin Family Members Selectively Regulate Autophagy in Osteosarcoma and Mesothelioma Cells in Response to Cellular Stress.

The class III NAD+ dependent deacetylases-sirtuins (SIRTs) link transcriptional regulation to DNA damage response and reactive oxygen species generation thereby modulating a wide range of cellular signaling pathways. Here, the contribution of SIRT1, SIRT3, and SIRT5 in the regulation of cellular fate through autophagy was investigated under diverse types of stress. The effects of sirtuins' silencing on cell survival and autophagy was followed in human osteosarcoma and mesothelioma cells exposed to DNA damage and oxidative stress. Our results suggest that the mitochondrial sirtuins SIRT3 and 5 are pro-proliferative under certain cellular stress conditions and this effect correlates with their role as positive regulators of autophagy. SIRT1 has more complex role which is cell type specific and can affect autophagy in both positive and negative ways. The mitochondrial sirtuins (SIRT3 and SIRT5) affect both early and late stages of autophagy, whereas SIRT1 acts mostly at later stages of the autophagic process. Investigation of potential crosstalk between SIRT1, SIRT3, and SIRT5 revealed several feedback loops and a significant role of SIRT5 in regulating SIRT3 and SIRT1. Results presented here support the notion that sirtuin family members play important as well as differential roles in the regulation of autophagy in osteosarcoma vs. mesothelioma cells exposed to DNA damage and oxidative stress, and this can be exploited in increasing the response of cancer cells to chemotherapy.

Coat protein complex I facilitates dengue virus production.

Dengue hemorrhagic fever is a life-threatening disease caused by the dengue virus (DENV). After DENV enters into host cells, it replicates to generate viral particles to infect other cells. DENV exploits components of the cellular trafficking pathway to achieve effective virion production. Understanding of the proteins required for this trafficking process is essential for revealing the pathogenesis of DENV infection. Coat protein complex and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), two host protein families in the cellular trafficking pathway, were investigated to elucidate their respective roles during DENV infection. Coat proteins (COPI and COPII) and SNAREs (STX 5 and NSF) were knocked down in a DENV-infected Huh7 cells by RNA interference. Depletion of COPI and COPII, but not of STX5 and NSF, decreased DENV production in DENV-infected Huh7 cells. DENV proteins, including DENV C, prM, E, and NS1, were significantly reduced in COPI-silenced DENV-infected Huh7 cells, when compared to those of control cells. COPI also facilitated DENV production in an endothelial cell line and in all DENV serotypes, indicating the importance of coat protein complex in facilitating DENV infection.

Adaptor Protein 1A Facilitates Dengue Virus Replication.

Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication.