Caspase-7 ablation modulates UPR,reprograms TRAF2-JNK apoptosis and protects T17M rhodopsin mice from severe retinal degeneration

The UPR is activated in the mouse retina expressing misfolded T17M rhodopsin (RHO) during autosomal dominant retinitis pigmentosa (ADRP) progression. Therefore, the goal of this study is to validate the UPR-induced caspase-7 as a new therapeutic target that modulates the UPR, reduces the level of apoptosis and protects the ADRP retina from retinal degeneration and light-induced damage. Mice were analyzed using ERG, SD-OCT and histology to determine the role of caspase-7 ablation. The results of these experiments demonstrate the significant preservation of photoreceptors and their function in T17M RHO CASP-7 retinas from P30 to P90 compared with control mice. These mice were also protected from the light-induced decline in the ERG responses and apoptosis. The RNA and protein analyses of T17M RHO+Csp7-siRNA, Tn+Csp7-siRNA 661W cells and T17M RHO CASP-7 retinas revealed that caspase-7 ablation reprograms the UPR and reduces JNK-induced apoptosis. This reduction is believed to occur through the downregulation of the mTOR and Hif1a proteins. In addition, decline in activated PARP1 was detected in T17M RHO CASP-7 retina. Altogether, our findings indicate that the targeting of caspase-7 in T17M RHO mice could be a feasible therapeutic strategy for advanced stages of ADRP.     

C/EBP homologous protein deficiency enhances hematopoietic stem cell function via reducing ATF3/ROS‐induced cell apoptosis

Hematopoietic stem cells (HSCs) reside in a quiescent niche to reserve their capacity of self‐renewal. Upon hematopoietic injuries, HSCs enter the cell cycle and encounter protein homeostasis problems caused by accumulation of misfolded proteins. However, the mechanism by which protein homeostasis influences HSC function and maintenance remains poorly understood. Here, we show that C/EBP homologous protein (CHOP), demonstrated previously to induces cell death upon unfolded protein response (UPR), plays an important role in HSCs regeneration. CHOP^−/− mice showed normal hematopoietic stem and progenitor cell frequencies in steady state. However, when treated with 5‐FU, CHOP deficiency resulted in higher survival rates, associated with an increased number of HSCs and reduced level of apoptosis. In serial competitive transplantation experiments, CHOP^−/− HSCs showed a dramatic enhancement of repopulation ability and a reduction of protein aggresomes. Mechanistically, CHOP deletion causes reduced ATF3 expression and further leads to decreased protein aggregation and ROS. In addition, CHOP^−/− HSCs exhibited an increased resistance to IR‐induced DNA damage and improved HSCs homeostasis and function in telomere dysfunctional (G3Terc ^−/−) mice. In summary, these findings disclose a new role of CHOP in the regulation of the HSCs function and homeostasis through reducing ATF3 and ROS signaling.     

A Highlights from MBoC Selection: Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop^−/− cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2α and promotes XIAP degradation through ATF4. Of interest, PERK''s down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.     

CHOP Mediates Endoplasmic Reticulum Stress-Induced Apoptosis in Gimap5-Deficient T Cells

Gimap5 (GTPase of the immunity-associated protein 5) has been linked to the regulation of T cell survival, and polymorphisms in the human GIMAP5 gene associate with autoimmune disorders. The BioBreeding diabetes-prone (BBDP) rat has a mutation in the Gimap5 gene that leads to spontaneous apoptosis of peripheral T cells by an unknown mechanism. Because Gimap5 localizes to the endoplasmic reticulum (ER), we hypothesized that absence of functional Gimap5 protein initiates T cell death through disruptions in ER homeostasis. We observed increases in ER stress-associated chaperones in T cells but not thymocytes or B cells from Gimap5^−/− BBDP rats. We then discovered that ER stress-induced apoptotic signaling through C/EBP-homologous protein (CHOP) occurs in Gimap5^−/− T cells. Knockdown of CHOP by siRNA protected Gimap5^−/− T cells from ER stress-induced apoptosis, thereby identifying a role for this cellular pathway in the T cell lymphopenia of the BBDP rat. These findings indicate a direct relationship between Gimap5 and the maintenance of ER homeostasis in the survival of T cells.     

Development of Central Nervous System Autoimmunity Is Impaired in the Absence of Wiskott-Aldrich Syndrome Protein

Wiskott-Aldrich Syndrome protein (WASP) is a key regulator of the actin cytoskeleton in hematopoietic cells. Defective expression of WASP leads to multiple abnormalities in different hematopoietic cells. Despite severe impairment of T cell function, WAS patients exhibit a high prevalence of autoimmune disorders. We attempted to induce EAE, an animal model of organ-specific autoimmunity affecting the CNS that mimics human MS, in Was^−/− mice. We describe here that Was^−/− mice are markedly resistant against EAE, showing lower incidence and milder score, reduced CNS inflammation and demyelination as compared to WT mice. Microglia was only poorly activated in Was^−/− mice. Antigen-induced T-cell proliferation, Th-1 and -17 cytokine production and integrin-dependent adhesion were increased in Was^−/− mice. However, adoptive transfer of MOG-activated T cells from Was^−/− mice in WT mice failed to induce EAE. Was^−/− mice were resistant against EAE also when induced by adoptive transfer of MOG-activated T cells from WT mice. Was^+/− heterozygous mice developed an intermediate clinical phenotype between WT and Was^−/− mice, and they displayed a mixed population of WASP-positive and -negative T cells in the periphery but not in their CNS parenchyma, where the large majority of inflammatory cells expressed WASP. In conclusion, in absence of WASP, T-cell responses against a CNS autoantigen are increased, but the ability of autoreactive T cells to induce CNS autoimmunity is impaired, most probably because of an inefficient T-cell transmigration into the CNS and defective CNS resident microglial function.     

Rag2-deficient IL-1 Receptor Antagonist-deficient Mice Are a Novel Colitis Model in Which Innate Lymphoid Cell-derived IL-17 Is Involved in the Pathogenesis

Il1rn^−/− mice spontaneously develop arthritis and aortitis by an autoimmune mechanism and also develop dermatitis by an autoinflammatory mechanism. Here, we show that Rag2^−/−Il1rn^−/− mice develop spontaneous colitis with high mortality, making a contrast to the suppression of arthritis in these mice. Enhanced IL-17A expression in group 3 innate lymphoid cells (ILC3s) was observed in the colon of Rag2^−/−Il1rn^−/− mice. IL-17A-deficiency prolonged the survival of Rag2^−/−Il1rn^−/− mice, suggesting a pathogenic role of this cytokine in the development of intestinal inflammation. Although IL-17A-producing T cells were increased in Il1rn^−/− mice, these mice did not develop colitis, because CD4⁺Foxp3⁺ regulatory T cell population was also expanded. Thus, excess IL-1 signaling and IL-1-induced IL-17A from ILC3s cause colitis in Rag2^−/−Il1rn^−/− mice in which Treg cells are absent. These observations suggest that the balance between IL-17A-producing cells and Treg cells is important to keep the immune homeostasis of the colon.     

C/EBP Homologous Protein-induced Macrophage Apoptosis Protects Mice from Steatohepatitis

Nonalcoholic fatty liver disease is a heterogeneous disorder characterized by liver steatosis; inflammation and fibrosis are features of the progressive form nonalcoholic steatohepatitis. The endoplasmic reticulum stress response is postulated to play a role in the pathogenesis of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. In particular, C/EBP homologous protein (CHOP) is undetectable under normal conditions but is induced by cellular stress, including endoplasmic reticulum stress. Chop wild type (Chop^+/+) and knock-out (Chop^−/−) mice were used in these studies to elucidate the role of CHOP in the pathogenesis of fatty liver disease. Paradoxically, Chop^−/− mice developed greater liver injury, inflammation, and fibrosis than Chop^+/+ mice, with greater macrophage activation. Primary, bone marrow-derived, and peritoneal macrophages from Chop^+/+ and Chop^−/− were challenged with palmitic acid, an abundant saturated free fatty acid in plasma and liver lipids. Where palmitic acid treatment activated Chop^+/+ and Chop^−/− macrophages, Chop^−/− macrophages were resistant to its lipotoxicity. Chop^−/− mice were sensitized to liver injury in a second model of dietary steatohepatitis using the methionine-choline-deficient diet. Analysis of bone marrow chimeras between Chop^−/− and Chop^+/+ mice demonstrated that Chop in macrophages protects from liver injury and inflammation when fed the methionine-choline-deficient diet. We conclude that Chop deletion has a proinflammatory effect in fatty liver injury apparently due to decreased cell death of activated macrophages, resulting in their net accumulation in the liver. Thus, macrophage CHOP plays a key role in protecting the liver from steatohepatitis likely by limiting macrophage survival during lipotoxicity.     

ATF6alpha Promotes Astroglial Activation and Neuronal Survival in a Chronic Mouse Model of Parkinson’s Disease

Accumulating evidence suggests a crucial role for the unfolded protein response (UPR) in Parkinson’s disease (PD). In this study, we investigated the relevance of the UPR in a mouse model of chronic MPTP/probenecid (MPTP/P) injection, which causes severe and persistent degeneration of dopaminergic neurons. Enhanced activation of the UPR branches, including ATF6α and PERK/eIF2α/ATF4, was observed after MPTP/P injections into mice. Deletion of the ATF6α gene accelerated neuronal degeneration and ubiquitin accumulation relatively early in the MPTP/P injection course. Surprisingly, astroglial activation was strongly suppressed, and production of the brain-derived neurotrophic factor (BDNF) and anti-oxidative genes, such as heme oxygenase-1 (HO-1) and xCT, in astrocytes were reduced in ATF6α −/− mice after MPTP/P injections. Decreased BDNF expression in ATF6α −/− mice was associated with decreased expression of GRP78, an ATF6α-dependent molecular chaperone in the ER. Decreased HO-1 and xCT levels were associated with decreased expression of the ATF4-dependent pro-apoptotic gene CHOP. Consistent with these results, administration of the UPR-activating reagent tangeretin (5,6,7,8,4′-pentamethoxyflavone; IN19) into mice enhanced the expression of UPR-target genes in both dopaminergic neurons and astrocytes, and promoted neuronal survival after MPTP/P injections. These results suggest that the UPR is activated in a mouse model of chronic MPTP/P injection, and contributes to the survival of nigrostriatal dopaminergic neurons, in part, through activated astrocytes.     

γδ T Cells Are Required for Pulmonary IL-17A Expression after Ozone Exposure in Mice: Role of TNFα

Ozone is an air pollutant that causes pulmonary symptoms. In mice, ozone exposure causes pulmonary injury and increases bronchoalveolar lavage macrophages and neutrophils. We have shown that IL-17A is important in the recruitment of neutrophils after subacute ozone exposure (0.3 ppm for 24–72 h). We hypothesized that γδ T cells are the main producers of IL-17A after subacute ozone. To explore this hypothesis we exposed wildtype mice and mice deficient in γδ T cells (TCRδ^−/−) to ozone or room air. Ozone-induced increases in BAL macrophages and neutrophils were attenuated in TCRδ^−/− mice. Ozone increased the number of γδ T cells in the lungs and increased pulmonary Il17a mRNA expression and the number of IL-17A⁺ CD45⁺ cells in the lungs and these effects were abolished in TCRδ^−/− mice. Ozone-induced increases in factors downstream of IL-17A signaling, including G-CSF, IL-6, IP-10 and KC were also decreased in TCRδ^−/− versus wildtype mice. Neutralization of IL-17A during ozone exposure in wildtype mice mimicked the effects of γδ T cell deficiency. TNFR2 deficiency and etanercept, a TNFα antagonist, also reduced ozone-induced increases in Il17a mRNA, IL-17A⁺ CD45⁺ cells and BAL G-CSF as well as BAL neutrophils. TNFR2 deficient mice also had decreased ozone-induced increases in Ccl20, a chemoattractant for IL-17A⁺ γδ T cells. Il17a mRNA and IL-17A⁺ γδ T cells were also lower in obese Cpe^fat versus lean WT mice exposed to subacute ozone, consistent with the reduced neutrophil recruitment observed in the obese mice. Taken together, our data indicate that pulmonary inflammation induced by subacute ozone requires γδ T cells and TNFα-dependent recruitment of IL-17A⁺ γδ T cells to the lung.     

C/EBP homologous protein drives pro-catabolic responses in chondrocytes

Introduction

Excess C/EBP homologous protein (CHOP) expression is one feature of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress. Here, we focused on CHOP expression and function in chondrocytes.

Methods

We studied human knee osteoarthritis (OA) cartilage, bovine chondrocytes cultured in alginate and subjected to sub-lethal biomechanical injury, and knee chondrocytes of human autopsy donors. We performed siRNA knockdown and transfection.

Results

UPR activation was increased in human knee OA cartilage in situ, and in biomechanically injured cultured chondrocytes in vitro. In normal human chondrocytes, CHOP “gain of function” sensitized chondrocytes to IL-1β induced nitric oxide (NO) and matrix metalloproteinase (MMP)-3 release without inducing these responses by itself. Excess CHOP expression, by itself, induced superoxide production and apoptosis. Conversely, siRNA knockdown of CHOP and the UPR-specific mediator X-box binding protein (XBP1) inhibited NO release by >80% (P <0.0005) in response to IL-1β, and blunted MMP-3 release, whereas there were only minimal effects of the UPR mediator GRP78 on these responses. The anti-inflammatory metabolic “super-regulator” AMP kinase (AMPK) is known to limit UPR activation in vascular muscle cells. Here, CHOP supported the capacity of IL-1β to suppress AMPK activity in chondrocytes. We also observed that inhibition of AMPK activity promoted an increase in chondrocyte CHOP expression. Conversely, pharmacologic activation of AMPK by 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) blunted chondrocyte CHOP expression in response to biomechanical injury.

Conclusions

Biomechanical injury and IL-1 signaling stimulate UPR activation in chondrocytes. CHOP mediates chondrocyte catabolic and apoptotic responses to IL-1β, and does so partly by inhibiting AMPK activity. Conversely, development of excess CHOP activity is limited by AMPK activity in chondrocytes. Our findings suggest a mechanism for potential chondroprotection by AICAR and other AMPK activators. The work is of translational relevance for OA, since several drugs that activate AMPK are already in the clinic for arthritis (for example, allosteric AMPK activators sodium salicylate and high dose aspirin, and methotrexate, which activates AMPK by generating AICAR).     

Role of ERO1-α–mediated stimulation of inositol 1,4,5-triphosphate receptor activity in endoplasmic reticulum stress–induced apoptosis

Endoplasmic reticulum (ER) stress–induced apoptosis is involved in many diseases, but the mechanisms linking ER stress to apoptosis are incompletely understood. Based on roles for C/EPB homologous protein (CHOP) and ER calcium release in apoptosis, we hypothesized that apoptosis involves the activation of inositol 1,4,5-triphosphate (IP3) receptor (IP3R) via CHOP-induced ERO1-α (ER oxidase 1 α). In ER-stressed cells, ERO1-α is induced by CHOP, and small interfering RNA (siRNA) knockdown of ERO1-α suppresses apoptosis. IP3-induced calcium release (IICR) is increased during ER stress, and this response is blocked by siRNA-mediated silencing of ERO1-α or IP3R1 and by loss-of-function mutations in Ero1a or Chop. Reconstitution of ERO1-α in Chop^−/− macrophages restores ER stress–induced IICR and apoptosis. In vivo, macrophages from wild-type mice but not Chop^−/− mice have elevated IICR when the animals are challenged with the ER stressor tunicamycin. Macrophages from insulin-resistant ob/ob mice, another model of ER stress, also have elevated IICR. These data shed new light on how the CHOP pathway of apoptosis triggers calcium-dependent apoptosis through an ERO1-α–IP3R pathway.     

Role of the Adiponectin Binding Protein,T-Cadherin (cdh13), in Pulmonary Responses to Subacute Ozone

Adiponectin, an adipose derived hormone with pleiotropic functions, binds to several proteins, including T-cadherin. We have previously reported that adiponectin deficient (Adipo^−/−) mice have increased IL-17A-dependent neutrophil accumulation in their lungs after subacute exposure to ozone (0.3 ppm for 72 hrs). The purpose of this study was to determine whether this anti-inflammatory effect of adiponectin required adiponectin binding to T-cadherin. Wildtype, Adipo^−/−, T-cadherin deficient (T-cad^−/−), and bideficient (Adipo^−/−/T-cad^−/−) mice were exposed to subacute ozone or air. Compared to wildtype mice, ozone-induced increases in pulmonary IL-17A mRNA expression were augmented in T-cad^−/− and Adipo^−/− mice. Compared to T-cad^−/− mice, there was no further increase in IL-17A in Adipo^−/−/T-cad^−/− mice, indicating that adiponectin binding to T-cadherin is required for suppression of ozone-induced IL-17A expression. Similar results were obtained for pulmonary mRNA expression of saa3, an acute phase protein capable of inducing IL-17A expression. Comparison of lung histological sections across genotypes also indicated that adiponectin attenuation of ozone-induced inflammatory lesions at bronchiolar branch points required T-cadherin. BAL neutrophils and G-CSF were augmented in T-cad^−/− mice and further augmented in Adipo^−/−/T-cad^−/− mice. Taken together with previous observations indicating that augmentation of these moieties in ozone exposed Adipo^−/− mice is partially IL-17A dependent, the results indicate that effects of T-cadherin deficiency on BAL neutrophils and G-CSF are likely secondary to changes in IL-17A, but that adiponectin also acts via T-cadherin independent pathways. Our results indicate that T-cadherin is required for the ability of adiponectin to suppress some but not all aspects of ozone-induced pulmonary inflammation.     

Modulation of cellular signaling pathways in P23H rhodopsin photoreceptors

We previously reported activation of the unfolded protein response (UPR) in P23H rhodopsin (RHO) retinas with autosomal dominant retinitis pigmentosa (ADRP). Knowing that the UPR can trigger Ca^2 + release from the endoplasmic reticulum and regulate cellular signaling we examined the level of Ca^2 +-regulated proteins. We also looked for changes in the expression of Bcl2 family proteins, autophagy proteins and the mTOR/AKT pathways, as well as for the induction of mitochondria-associated apoptosis in the P23H RHO retina. Our data demonstrated that the elevation of calpain and caspase-12 activity was concomitantly observed with a decrease in the BCL2-XL/BAX ratio and an increase in mTor levels in the P23H-3 RHO retina suggesting a vulnerability of P23H RHO photoreceptors to apoptosis. The translocation of BAX to the mitochondria, as well as the release of cytochrome C and AIF into the cytosol supports this conclusion and indicates the involvement of mitochondria-induced apoptosis in the progression of ADRP. The level of autophagy proteins in general was found to be decreased in the P21–P30 P23H RHO retina. Injections of rapamycin, however, protected the P23H RHO rod photoreceptors from experiencing physiological decline. Despite this fact, the downregulation of mTOR did not alter the level of autophagy proteins. Our results imply that in addition to activation of the UPR during ADRP progression, photoreceptors also experience alterations in major proapoptotic pathways.     

PI3K p110γ Deletion Attenuates Murine Atherosclerosis by Reducing Macrophage Proliferation but Not Polarization or Apoptosis in Lesions

Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR^−/−p110γ^+/− and LDLR^−/−p110γ^−/− mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ^+/− and p110γ^−/− mice. Lack of p110γ in LDLR^−/− mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR^−/−p110γ^−/− mice were smaller than in LDLR^−/−p110γ^+/− controls, which coincided with decreased macrophage proliferation in LDLR^−/−p110γ^−/− mouse lesions. This proliferation defect was also observed in p110γ^−/− bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR^−/−p110γ^−/− mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR^−/− mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.