Neuronal p58IPK Protects Retinal Ganglion Cells Independently of Macrophage/Microglia Activation in Ocular Hypertension.

p58IPK is a multifaceted endoplasmic reticulum (ER) chaperone and a regulator of eIF2α kinases involved in a wide range of cellular processes including protein synthesis, ER stress response, and macrophage-mediated inflammation. Systemic deletion of p58IPK leads to age-related loss of retinal ganglion cells (RGC) and exacerbates RGC damage induced by ischemia/reperfusion and increased intraocular pressure (IOP), suggesting a protective role of p58IPK in the retina. However, the mechanisms remain elusive. Herein, we investigated the cellular mechanisms underlying the neuroprotection action of p58IPK using conditional knockout (cKO) mouse lines where p58IPK is deleted in retinal neurons (Chx10-p58IPK cKO) or in myeloid cells (Lyz2-p58IPK cKO). In addition, we overexpressed p58IPK by adeno-associated virus (AAV) in the retina to examine the effect of p58IPK on RGC survival after ocular hypertension (OHT) in wild type (WT) mice. Our results show that overexpression of p58IPK by AAV significantly improved RGC survival after OHT in WT mice, suggesting a protective effect of p58IPK on reducing RGC injury. Conditional knockout of p58IPK in retinal neurons or in myeloid cells did not alter retinal structure or cellular composition. However, a significant reduction in the b wave of light-adapted electroretinogram (ERG) was observed in Chx10-p58IPK cKO mice. Deletion of p58IPK in retinal neurons exacerbates RGC loss at 14 days after OHT. In contrast, deficiency of p58IPK in myeloid cells increased the microglia/macrophage activation but had no effect on RGC loss. We conclude that deletion of p58IPK in macrophages increases their activation, but does not influence RGC survival. These results suggest that the neuroprotective action of p58IPK is mediated by its expression in retinal neurons, but not in macrophages. Therefore, targeting p58IPK specifically in retinal neurons is a promising approach for the treatment of neurodegenerative retinal diseases including glaucoma.

Porcine circovirus type 2 exploits cap to inhibit PKR activation through interaction with Hsp40.

Porcine circovirus type 2 is the main pathogen of porcine circovirus disease, which has caused enormous economic losses to the pig industry worldwide. The PKR signaling pathway is important for the cellular antiviral response, but its role in the process of PCV2 infection is unknown. In this study, we first found that dsRNA was produced and that PKR was activated in PCV2 infection. However, interestingly, the activation of PKR was inhibited when the Cap protein was exogenously expressed in PAMs, and this inhibition was reversed by the expression of DNAJC7. The interaction between Cap and DNAJC7 was confirmed by laser confocal microscopy, coimmunoprecipitation and GST pull-down, and it was found that PCV2 infection or the expression of Cap protein could induce DNAJC7 to migrate to the nucleus and release P58IPK, an inhibitor of PKR activation. Downregulating the expression of DNAJC7 by a specific inhibitor or recombinant lentivirus-mediated shRNA, inhibited the replication of the PCV2 genome and the production of virions, which was consistent with the increase of DNAJC7 expression in multiple tissues of weaned piglets infected with PCV2. These data indicate that although PKR was activated by PCV2 infection, the activation was inhibited by Cap through an interaction with DNAJC7. These results help to understand the molecular mechanism of immune escape after PCV2 infection.

p58IPK Is an Endogenous Neuroprotectant for Retinal Ganglion Cells.

p58IPK is an endoplasmic reticulum (ER)-resident chaperone playing a critical role in facilitating protein folding and protein homeostasis. Previously, we have demonstrated that p58IPK is expressed broadly in retinal neurons including retinal ganglion cells (RGCs) and loss of p58IPK results in age-related RGC degeneration. In the present study, we investigate the role of p58IPK in neuroprotection by in vitro and in vivo studies using primary RGC culture and two well-established disease-relevant RGC injury models: retinal ischemia/reperfusion (I/R) and microbead-induced ocular hypertension. Our results demonstrate that in both in vivo models, p58IPK -/- mice exhibit significantly increased RGC loss compared to wild type (WT) mice. In vitro, p58IPK-deficient RGCs show reduced viability and are more susceptible to cell death induced by the ER stress inducer tunicamycin (TM). Overexpression of p58IPK by adeno-associated virus (AAV) significantly diminishes TM-induced cell death in both WT and p58IPK -/- RGCs. Interestingly, we find that loss of p58IPK leads to reduced mRNA expression, but not the protein level, of mesencephalic astrocyte-derived neurotrophic factor (MANF), a neurotrophic factor that resides in the ER. Treatment with recombinant MANF protein protects R28 retinal neural cells and mouse retinal explants from TM-induced cell death. Taken together, our study suggests that p58IPK functions as an endogenous neuroprotectant for RGCs. The mechanisms underlying p58IPK's neuroprotective action and the potential interactions between p58IPK and MANF warrant future investigation.

Experimental Nonalcoholic Steatohepatitis and Liver Fibrosis Are Ameliorated by Pharmacologic Activation of Nrf2 (NF-E2 p45-Related Factor 2).

BACKGROUND & AIMS: Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis. METHODS: Nrf2+/+ and Nrf2-/- C57BL/6 mice were fed a high-fat plus fructose (HFFr) or regular chow diet for 16 weeks or 30 weeks, and then treated for the final 6 weeks, while still being fed the same HFFr or regular chow diets, with either TBE-31 or dimethyl sulfoxide vehicle control. Measures of whole-body glucose homeostasis, histologic assessment of liver, and biochemical and molecular measurements of steatosis, endoplasmic reticulum (ER) stress, inflammation, apoptosis, fibrosis, and oxidative stress were performed in livers from these animals. RESULTS: TBE-31 treatment reversed insulin resistance in HFFr-fed wild-type mice, but not in HFFr-fed Nrf2-null mice. TBE-31 treatment of HFFr-fed wild-type mice substantially decreased liver steatosis and expression of lipid synthesis genes, while increasing hepatic expression of fatty acid oxidation and lipoprotein assembly genes. Also, TBE-31 treatment decreased ER stress, expression of inflammation genes, and markers of apoptosis, fibrosis, and oxidative stress in the livers of HFFr-fed wild-type mice. By comparison, TBE-31 did not decrease steatosis, ER stress, lipogenesis, inflammation, fibrosis, or oxidative stress in livers of HFFr-fed Nrf2-null mice. CONCLUSIONS: Pharmacologic activation of Nrf2 in mice that had already been rendered obese and insulin resistant reversed insulin resistance, suppressed hepatic steatosis, and mitigated against NASH and liver fibrosis, effects that we principally attribute to inhibition of ER, inflammatory, and oxidative stress.

Overexpression of p58ipk protects neuroblastoma against paraquat-induced toxicity.

BACKGROUND: Paraquat (PQ) is a powerful pathologic pesticide that contribute to the neurotoxicity, however, the pathogenic mechanism between them was unclear. The aims of this study were to explore the underlying mechanism of PQ-induced toxicity and then make potential contribute to such neuronal diseases therapy. METHODS: Human cell line SH-SY5Y was pretreated with a set concentrations of PQ to detect the cell apoptosis and the expression of related genes and proteins. Next, pcDNA 3.1-p58ipk or si-p58ipk was transfected the PQ-induced cells to detect the cytotoxicity. RESULTS: PQ significantly increased the cell apoptosis as well as the expression of p58ipk and CHOP, but decreased the expression of pAKT. p58ipk suppression resulted in an increase of cell apoptosis and CHOP expression, but the expression of pAKT was significantly decreased in PQ-induced SH-SY5Y cells. However, overexpressed p58ipk led to an opposite result. CONCLUSION: The results indicated that the expression of p58ipk was related to the toxicity level of PQ-induced cells and the mechanism between them was that p58ipk regulated the toxicity might through regulating the endoplasmic reticulum stress (ER-stress) and then regulating cell apoptosis. Further studies take emphasize on the effect of ER-stress on neuron system and explore ER-stress-related therapy are important on the treatment of neurodegenerative disease.

Identification of p58IPK as a novel neuroprotective factor for retinal neurons.

PURPOSE: Endoplasmic reticulum (ER)-resident chaperone protein p58(IPK) plays a vital role in regulation of protein folding and biosynthesis. The goal of this study was to examine the role of p58(IPK) in retinal neuronal cells under normal and stressed conditions. METHODS: Retinal expression of p58(IPK), retinal morphology, apoptosis, ER stress, and apoptotic gene expression were examined in p58(IPK) knockout (KO) and/or wild-type (WT) mice with or without intravitreal injection of N-methyl-D-aspartic acid (NMDA). In in vitro experiments, differentiated R28 retinal neuronal cells transduced with adenovirus encoding p58(IPK) (Ad-p58(IPK)) or control virus (Ad-LacZ) were exposed to tunicamycin (TM) or hydrogen peroxide (H2O2). Levels of ER stress, apoptosis, and cell survival were evaluated. RESULTS: Chaperone protein p58(IPK) is expressed predominantly in retinal ganglion cells (RGC), inner retinal neurons, and the photoreceptor inner segments. Mice lacking p58(IPK) exhibited increased CHOP expression and loss of RGCs with aging (8-10 months). Intravitreal injection of NMDA induced retinal ER stress and increased p58(IPK) expression in WT mice; this resulted in greater ER stress and enhanced RGC apoptosis in p58(IPK) KO mice. In cultured R28 cells, overexpression of p58(IPK) significantly reduced eIF2α phosphorylation, decreased CHOP expression, and alleviated the activation of caspase-3 and PARP. Overexpression of p58(IPK) also protected against oxidative and ER stress-induced cell apoptosis. Furthermore, p58(IPK) downregulated the proapoptotic gene Bax and upregulated the antiapoptotic gene Bcl-2 expression in stressed R28 cells. CONCLUSIONS: Our study has demonstrated a protective role of p58(IPK) in retinal neurons, which may act in part through a mechanism involving modulation of ER homeostasis and apoptosis, particularly under conditions of cellular stresses.

Deletion of P58(IPK), the Cellular Inhibitor of the Protein Kinases PKR and PERK, Causes Bone Changes and Joint Degeneration in Mice.

OBJECTIVE: Protein kinase-like endoplasmic reticulum kinase (PERK) and protein kinase R (PKR) are implicated in endoplasmic reticulum stress-induced arthritis and pro-inflammatory cytokine-mediated cartilage degradation in vitro, respectively. We determined whether knockout of the cellular inhibitor of PERK and PKR, P58(IPK) causes joint degeneration in vivo and whether these molecules are activated in human osteoarthritis (OA). MATERIALS AND METHODS: Sections of knee joints from P58(IPK)-null and wild-type mice aged 12-13 and 23-25 months were stained with toluidine blue and scored for degeneration using the osteoarthritis research society international (OARSI) system. Bone changes were assessed by radiology and high-resolution micro-computed tomography of hind limbs. Sections from the medial tibial plateaus of two human knees, removed in total knee replacement surgery for OA, were immunolabelled for phosphorylated PERK and PKR and P58(IPK). RESULTS: Knockout mice exhibited narrower tibiae (p = 0.0031) and smaller epiphyses in tibiae (p = 0.0004) and femora (p = 0.0214). Older knockout mice had reduced total volume inside the femoral periosteal envelope (p = 0.023), reduced tibial (p = 0.03), and femoral (p = 0.0012) bone volumes (BV) and reduced femoral BV fraction (p = 0.025). Compared with wild-types, younger P58(IPK)-null mice had increased OARSI scores in medial femoral condyles (p = 0.035). Thirty four percent of null mice displayed severe joint degeneration with complete articular cartilage loss from the medial compartment and heterotopic chondro-osseous tissue in the medial joint capsule. Phosphorylated PERK and PKR were localized throughout human osteoarthritic tibial plateaus but, in particular, in areas exhibiting the most degeneration. There was limited expression of P58(IPK). CONCLUSION: This study is the first to reveal a critical role for P58(IPK) in maintaining joint integrity in vivo, implicating the PKR and PERK stress signaling pathways in bony changes underlying the pathogenesis of joint degeneration.

Characterization of porcine P58IPK gene and its up-regulation after H1N1 or H3N2 influenza virus infection.

BACKGROUND: The 58-kDa inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (P58IPK) is a cellular protein that is activated during influenza virus infection. Although the function of human P58IPK has been studied for a long time, porcine P58IPK (pP58IPK) has little been studied except for its cloning. OBJECTIVE: In this study, we aimed to investigate the characteristics of the pP58IPK gene, determine its subcellular localization, and find its expression change during H1N1 or H3N2 infection. STUDY DESIGN: First, the sequence and structure of pP58IPK were analyzed. Second, pP58IPK gene was cloned into pEGFP-N1 and pEGFP-C1 vectors, respectively, which were transfected into cells to determine its subcellular localization. Third, Lung tissues of piglets from H1N1 infected, H3N2 infected and control groups were analyzed using histopathology, real-time PCR, and immunohistochemistry. RESULTS: The sequence and structure of pP58IPK was highly similar to the counterpart of human. pP58IPK protein distributed only in the cytoplasm. Lung tissues of piglets infected by H1N1 or H3N2 appeared obvious pathological changes, and the expression of pP58IPK in both mRNA and protein level was up-regulated by approximate 1.5-fold in piglets infected by H1N1 or H3N2 comparing with control piglets. CONCLUSIONS: We analyzed the characteristics of the pP58IPK gene, constructed a phylogenetic tree, determined its subcellular localization, and investigated its expression changes during H1N1 or H3N2 infection. The fundamental data accumulated in this study provides a potential medical model for investigating the function of P58IPK during influenza A viruses infection.