D. Thomas Rutkowski

Endoplasmic Reticulum Stress Activates Cleavage of CREBH to Induce a Systemic Inflammatory Response

Regulated intramembrane proteolysis (RIP) of endoplasmic reticulum (ER) membrane-anchored transcription factors is known to maintain sterol homeostasis and to mediate the unfolded protein response (UPR). Here, we identified CREBH as a RIP-regulated liver-specific transcription factor that is cleaved upon ER stress and required to activate expression of acute phase response (APR) genes. Proinflammatory cytokines increase expression of ER membrane-anchored CREBH. In response to ER stress, CREBH is cleaved by site-1 and site-2 proteases to liberate an amino-terminal fragment that transits to the nucleus to activate transcription of the genes encoding serum amyloid P-component (SAP) and C-reactive protein (CRP). Proinflammatory cytokines and lipopolysaccharide activate the UPR and induce cleavage of CREBH in the liver in vivo. Together, our studies delineate a molecular mechanism for activation of an ER-localized transcription factor, CREBH, and reveal an unprecedented link by which ER stress initiates an acute inflammatory response.

Adaptation to ER Stress Is Mediated by Differential Stabilities of Pro-Survival and Pro-Apoptotic mRNAs and Proteins

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a signaling cascade known as the unfolded protein response (UPR). Although activation of the UPR is well described, there is little sense of how the response, which initiates both apoptotic and adaptive pathways, can selectively allow for adaptation. Here we describe the reconstitution of an adaptive ER stress response in a cell culture system. Monitoring the activation and maintenance of representative UPR gene expression pathways that facilitate either adaptation or apoptosis, we demonstrate that mild ER stress activates all UPR sensors. However, survival is favored during mild stress as a consequence of the intrinsic instabilities of mRNAs and proteins that promote apoptosis compared to those that facilitate protein folding and adaptation. As a consequence, the expression of apoptotic proteins is short-lived as cells adapt to stress. We provide evidence that the selective persistence of ER chaperone expression is also applicable to at least one instance of genetic ER stress. This work provides new insight into how a stress response pathway can be structured to allow cells to avert death as they adapt. It underscores the contribution of posttranscriptional and posttranslational mechanisms in influencing this outcome.

UPR Pathways Combine to Prevent Hepatic Steatosis Caused by ER Stress-Mediated Suppression of Transcriptional Master Regulators

The unfolded protein response (UPR) is linked to metabolic dysfunction, yet it is not known how endoplasmic reticulum (ER) disruption might influence metabolic pathways. Using a multilayered genetic approach, we find that mice with genetic ablations of either ER stress-sensing pathways (ATF6alpha, eIF2alpha, IRE1alpha) or of ER quality control (p58(IPK)) share a common dysregulated response to ER stress that includes the development of hepatic microvesicular steatosis. Rescue of ER protein processing capacity by the combined action of UPR pathways during stress prevents the suppression of a subset of metabolic transcription factors that regulate lipid homeostasis. This suppression occurs in part by unresolved ER stress perpetuating expression of the transcriptional repressor CHOP. As a consequence, metabolic gene expression networks are directly responsive to ER homeostasis. These results reveal an unanticipated direct link between ER homeostasis and the transcriptional regulation of metabolism, and suggest mechanisms by which ER stress might underlie fatty liver disease.

Regulation of basal cellular physiology by the homeostatic unfolded protein response

The extensive membrane network of the endoplasmic reticulum (ER) is physically juxtaposed to and functionally entwined with essentially all other cellular compartments. Therefore, the ER must sense diverse and constantly changing physiological inputs so it can adjust its numerous functions to maintain cellular homeostasis. A growing body of new work suggests that the unfolded protein response (UPR), traditionally charged with signaling protein misfolding stress from the ER, has been co-opted for the maintenance of basal cellular homeostasis. Thus, the UPR can be activated, and its output modulated, by signals far outside the realm of protein misfolding. These findings are revealing that the UPR causally contributes to disease not just by its role in protein folding but also through its broad influence on cellular physiology.

The unfolded protein response mediates adaptation to exercise in skeletal muscle through a PGC-1α/ATF6α complex.

Exercise has been shown to be effective for treating obesity and type 2 diabetes. However, the molecular mechanisms for adaptation to exercise training are not fully understood. Endoplasmic reticulum (ER) stress has been linked to metabolic dysfunction. Here we show that the unfolded protein response (UPR), an adaptive response pathway that maintains ER homeostasis upon luminal stress, is activated in skeletal muscle during exercise and adapts skeletal muscle to exercise training. The transcriptional coactivator PGC-1α, which regulates several exercise-associated aspects of skeletal muscle function, mediates the UPR in myotubes and skeletal muscle through coactivation of ATF6α. Efficient recovery from acute exercise is compromised in ATF6α(-/-) mice. Blocking ER-stress-related cell death via deletion of CHOP partially rescues the exercise intolerance phenotype in muscle-specific PGC-1α KO mice. These findings suggest that modulation of the UPR through PGC1α represents an alternative avenue to improve skeletal muscle function and achieve metabolic benefits.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110s, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially “compensated” to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Influenza A Viral Replication Is Blocked by Inhibition of the Inositol-requiring Enzyme 1 (IRE1) Stress Pathway

Background: The role of endoplasmic reticulum (ER) stress in influenza A viral infection is unknown. Results: Influenza A virus induces the IRE1 pathway of the ER stress response. Inhibition of IRE1 activity leads to decreased viral replication. Conclusion: IRE1 is a potential therapeutic target for influenza A virus. Significance: Targeting a host molecular mechanism is a novel therapeutic strategy that is less likely to be invalidated by viral mutagenesis. Known therapies for influenza A virus infection are complicated by the frequent emergence of resistance. A therapeutic strategy that may escape viral resistance is targeting host cellular mechanisms involved in viral replication and pathogenesis. The endoplasmic reticulum (ER) stress response, also known as the unfolded protein response (UPR), is a primitive, evolutionary conserved molecular signaling cascade that has been implicated in multiple biological phenomena including innate immunity and the pathogenesis of certain viral infections. We investigated the effect of influenza A viral infection on ER stress pathways in lung epithelial cells. Influenza A virus induced ER stress in a pathway-specific manner. We showed that the virus activates the IRE1 pathway with little or no concomitant activation of the PERK and the ATF6 pathways. When we examined the effects of modulating the ER stress response on the virus, we found that the molecular chaperone tauroursodeoxycholic acid (TUDCA) significantly inhibits influenza A viral replication. In addition, a specific inhibitor of the IRE1 pathway also blocked viral replication. Our findings constitute the first evidence that ER stress plays a role in the pathogenesis of influenza A viral infection. Decreasing viral replication by modulating the host ER stress response is a novel strategy that has important therapeutic implications.

Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome–translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.

The Stress-Regulated Transcription Factor CHOP Promotes Hepatic Inflammatory Gene Expression, Fibrosis, and Oncogenesis

Viral hepatitis, obesity, and alcoholism all represent major risk factors for hepatocellular carcinoma (HCC). Although these conditions also lead to integrated stress response (ISR) or unfolded protein response (UPR) activation, the extent to which these stress pathways influence the pathogenesis of HCC has not been tested. Here we provide multiple lines of evidence demonstrating that the ISR-regulated transcription factor CHOP promotes liver cancer. We show that CHOP expression is up-regulated in liver tumors in human HCC and two mouse models thereof. Chop-null mice are resistant to chemical hepatocarcinogenesis, and these mice exhibit attenuation of both apoptosis and cellular proliferation. Chop-null mice are also resistant to fibrosis, which is a key risk factor for HCC. Global gene expression profiling suggests that deletion of CHOP reduces the levels of basal inflammatory signaling in the liver. Our results are consistent with a model whereby CHOP contributes to hepatic carcinogenesis by promoting inflammation, fibrosis, cell death, and compensatory proliferation. They implicate CHOP as a common contributing factor in the development of HCC in a variety of chronic liver diseases.

Regulation of the transcriptome by ER stress: non-canonical mechanisms and physiological consequences

The mammalian unfolded protein response (UPR) is propagated by three ER-resident transmembrane proteins, each of which initiates a signaling cascade that ultimately culminates in production of a transcriptional activator. The UPR was originally characterized as a pathway for upregulating ER chaperones, and a comprehensive body of subsequent work has shown that protein synthesis, folding, oxidation, trafficking, and degradation are all transcriptionally enhanced by the UPR. However, the global reach of the UPR extends to genes involved in diverse physiological processes having seemingly little to do with ER protein folding, and this includes a substantial number of mRNAs that are suppressed by stress rather than stimulated. Through multiple non-canonical mechanisms emanating from each of the UPR pathways, the cell dynamically regulates transcription and mRNA degradation. Here we highlight these mechanisms and their increasingly appreciated impact on physiological processes.