Hsp90 modulates PPARγ activity in a mouse model of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a highly prevalent complication of obesity, yet cellular mechanisms that lead to its development are not well defined. Previously, we have documented hepatic steatosis in mice carrying a mutation in the Sec61a1 gene. Here we examined the mechanism behind NAFLD in Sec61a1 mutant mice. Livers of mutant mice exhibited upregulation of Pparg and its target genes Cd36, Cidec, and Lpl, correlating with increased uptake of fatty acid. Interestingly, these mice also displayed activation of the heat shock response (HSR), with elevated levels of heat shock protein (Hsp) 70, Hsp90, and heat shock factor 1. In cell lines, inhibition of Hsp90 function reduced Pparγ signaling and protein levels. Conversely, overexpression of Hsp90 increased Pparγ signaling and protein levels by reducing degradation. This may occur via a physical interaction as Hsp90 and Pparγ coimmunoprecipitated in vivo. Furthermore, inhibition of Hsp90 in Sec61a1 mutant hepatocytes also reduced Pparγ protein levels and signaling. Finally, overexpression of Hsp90 in liver cell lines increased neutral lipid accumulation, and this accumulation was blocked by Hsp90 inhibition. Our results show that the HSR and Hsp90 play an important role in the development of NAFLD, opening new avenues for the prevention and treatment of this highly prevalent disease.

Defective ER associated degradation of a model luminal substrate in yeast carrying a mutation in the 4th ER luminal loop of Sec61p.

The major constituent of the eukaryotic ER protein-translocation channel (Sec61p in yeast, Sec61α in higher eukaryotes) shows a high degree of evolutionary conservation from yeast to humans. The vast majority of eukaryotic species have a conserved di-tyrosine in the 4th ER luminal loop. Previously, we discovered through a screen of ethylnitrosourea- (ENU-) mutagenized mice that substitution of the latter of these tyrosines with histidine (Y344H) of the murine Sec61α protein results in diabetes and hepatic steatosis in mice that is a result of ER stress. To further characterize the mechanism behind ER stress in these mice we made the homologous mutation in yeast Sec61p (Y345H). We found that this mutation increased sensitivity of yeast to ER stressing agents and to reduction of Inositol Requiring Enzyme 1 (IRE1) activity. Furthermore, we found that, while this mutation did not affect translocation, it did delay degradation of the model ER-associated degradation (ERAD) substrate CPY(∗). Replacing both ER luminal tyrosines with alanines resulted in a destabilization of the Sec61 protein that was rescued by over expression of Sss1p. This double mutant still lacked a noticeable translocation defect after stabilization by Sss1p, but exhibited a similar defect in CPY(∗) degradation.

Multi-week prediction of livestock chill conditions associated with the northwest Queensland floods of February 2019.

The compound extreme weather event that impacted northern Queensland in February 2019 featured record-breaking rainfall, persistent high wind gusts and relatively cold day-time temperatures. This caused livestock losses numbering around 500,000 in the northwest Queensland Gulf region. In this study, we examine the livestock chill conditions associated with this week-long compound weather event and its potential for prediction from eleven world-leading sub-seasonal to seasonal (S2S) forecast systems. The livestock chill index combines daily rainfall, wind and surface temperature data. Averaged over the event week, the potential heat loss of livestock was in the moderate to high category, with severe conditions on the day of peak rainfall (5 February). Using calibrated forecasts from the Bureau of Meteorology's S2S forecast system, ACCESS-S1, a 1-week lead prediction showed a 20-30% probability of extreme livestock chill conditions over the northwest Queensland Gulf region, however the highest probabilities were located to the west of where the greatest livestock impacts were observed. Of the remaining ten S2S systems, around half predicted a more than 20% chance of extreme conditions, more than twice the climatological probability. It appears that the prediction accuracy arose from the skilful forecasts of extreme rainfall, as opposed to cold day-time temperature and strong wind forecasts. Despite a clear association between the observed extreme weather conditions and an active Madden-Julian Oscillation (MJO) event stalling in the western Pacific, the majority of 1-week lead S2S forecasts showed little indication of a slow-down in the MJO. As the livestock chill index was developed for southern Australian sheep, it may not be the best metric to represent the effects of exposure on tropical cattle breeds. Hence, this study draws attention to the need for tailored diagnostics that better represent the cold effects of summer tropical cyclones and tropical depressions on northern Australian livestock.

ER stress drives Lipocalin 2 upregulation in prostate cancer cells in an NF-κB-dependent manner.

BACKGROUND: Tumor cells adapt to endoplasmic reticulum (ER) stress through a set of conserved intracellular pathways, as part of a process termed the unfolded protein response (UPR). The expression of UPR genes/proteins correlates with increasing progression and poor clinical outcome of several tumor types, including prostate cancer. UPR signaling can activate NF-κB, a master regulator of transcription of pro-inflammatory, tumorigenic cytokines. Previous studies have shown that Lipocalin 2 (Lcn2) is upregulated in several epithelial cancers, including prostate cancer, and recently Lcn2 was implicated as a key mediator of breast cancer progression. Here, we hypothesize that the tumor cell UPR regulates Lcn2 production. METHODS: We interrogated Lcn2 regulation in murine and human prostate cancer cells undergoing pharmacological and physiological ER stress, and tested UPR and NF-κB dependence by using pharmacological inhibitors of these signaling pathways. RESULTS: Induction of ER stress using thapsigargin (Tg), a canonical pharmacologic ER stress inducer, or via glucose deprivation, a physiologic ER stressor present in the tumor microenvironment, upregulates LCN2 production in murine and human prostate cancer cells. Inhibition of the UPR using 4-phenylbutyric acid (PBA) dramatically decreases Lcn2 transcription and translation. Inhibition of NF-κB in prostate cancer cells undergoing Tg-mediated ER stress by BAY 11-7082 abrogates Lcn2 upregulation. CONCLUSIONS: We conclude that the UPR activates Lcn2 production in prostate cancer cells in an NF-κB-dependent manner. Our results imply that the observed upregulation of Lipocalin 2 in various types of cancer cells may be the direct consequence of concomitant UPR activation, and that the ER stress/Lipocalin 2 axis is a potential new target for intervention in cancer progression.

Why Australia was not wet during spring 2020 despite La Niña.

The austral spring climate of 2020 was characterised by the occurrence of La Niña, which is the most predictable climate driver of Australian springtime rainfall. Consistent with this La Niña, the Bureau of Meteorology's dynamical sub-seasonal to seasonal forecast system, ACCESS-S1, made highly confident predictions of wetter-than-normal conditions over central and eastern Australia for spring when initialised in July 2020 and thereafter. However, many areas of Australia received near average to severely below average rainfall, particularly during November. Possible causes of the deviation of rainfall from its historical response to La Niña and causes of the forecast error are explored with observational and reanalysis data for the period 1979-2020 and real-time forecasts of ACCESS-S1 initialised in July to November 2020. Several compounding factors were identified as key contributors to the drier-than-anticipated spring conditions. Although the ocean surface to the north of Australia was warmer than normal, which would have acted to promote rainfall over northern Australia, it was not as warm as expected from its historical relationship with La Niña and its long-term warming trend. Moreover, a negative phase of the Indian Ocean Dipole mode, which typically acts to increase spring rainfall in southern Australia, decayed earlier than normal in October. Finally, the Madden-Julian Oscillation activity over the equatorial Indian Ocean acted to suppress rainfall across northern and eastern Australia during November. While ACCESS-S1 accurately predicted the strength of La Niña over the Niño3.4 region, it over-predicted the ocean warming to the north of Australia and under-predicted the strength of the November MJO event, leading to an over-prediction of the Australian spring rainfall and especially the November-mean rainfall.

TLR9-independent activation of B lymphocytes by bacterial DNA.

The intracellular Toll-like receptor 9 (TLR9) is unique in its ability to recognize single-stranded DNA unmethylated at CpG motifs. Work from this laboratory showed that plasmid DNA is spontaneously internalized in B lymphocytes. This event is followed by the upregulation of costimulatory molecules and the acquisition of antigen presenting function by these cells. However, it is not known whether this phenomenon depends on TLR9. Because of the relevant role played by DNA-based drugs in immunotherapy and vaccination, and the central role of TLR9 signaling by CpG motifs, we decided to investigate whether signaling through TLR9 is a prerequisite for spontaneous transgenesis of lymphocytes. Here we found that transgene expression and upregulation of CD40 and CD86 costimulatory molecules was not inhibited by chloroquine treatment. Spontaneous transgenesis also occurred in B lymphocytes from TLR9-/- mice, and the injection of TLR9-/- transgenic B lymphocytes in C57Bl/6 mice induced both CD4 and CD8 T cell responses comparable to those induced by wild-type B lymphocytes. Collectively, these results suggest that plasmid DNA activates mammalian B lymphocytes through a TLR9 independent pathway.

Defective transport of the obesity mutant PC1/3 N222D contributes to loss of function.

Mutations in the PCSK1 gene encoding prohormone convertase 1/3 (PC1/3) are strongly associated with obesity in humans. The PC1/3(N222D) mutant mouse thus far represents the only mouse model that mimics the PC1/3 obesity phenotype in humans. The present investigation addresses the cell biology of the N222D mutation. Metabolic labeling experiments reveal a clear defect in the kinetics of insulin biosynthesis in islets from PC1/3(N222D) mutant mice, resulting in an increase in both proinsulin and its processing intermediates, predominantly lacking cleavage at the Arg-Arg site. Although the mutant PC1/3 zymogen is correctly processed to the 87-kDa form, pulse-chase immunoprecipitation experiments, labeling, and immunohistochemical experiments using uncleavable variants all demonstrate that the PC1/3-N222D protein is largely mislocalized compared with similar wild-type (WT) constructs, being predominantly retained in the endoplasmic reticulum. The PC1/3-N222D mutant also undergoes more efficient degradation via the ubiquitin-proteasome system than the WT enzyme. Lastly, the mutant PC1/3-N222D protein coimmunoprecipitates with WT PC1/3 and exerts a modest effect on intracellular retention of the WT enzyme. These profound alterations in the cell biology of PC1/3-N222D are likely to contribute to the defective insulin biosynthetic events observed in the mutant mice and may be relevant to the dramatic contributions of polymorphisms in this gene to human obesity.

A gain-of-function mutation in adenylate cyclase 3 protects mice from diet-induced obesity.

In a screen for genes that affect the metabolic response to high-fat diet (HFD), we selected one line of N-ethyl-N-nitrosourea (ENU)-mutagenized mice, Jll, with dominantly inherited resistance to diet-induced obesity (DIO). Mutant animals had dramatically reduced body weight and fat mass, and low basal insulin and glucose levels relative to unaffected controls. Both white adipose tissue (WAT) and brown adipose tissue (BAT) depots were smaller in mutant animals. Mutant animals fed a HFD gained only slightly more weight than animals fed regular chow, and were protected from hepatic lipid accumulation. The phenotype was genetically linked to a 5.7-Mb interval on chromosome 12, and sequencing of the entire interval identified a single coding mutation, predicted to cause a methionine-to-isoleucine substitution at position 279 of the Adcy3 protein (Adcy3M279I, henceforth referred to as Adcy3Jll). The mutant protein is hyperactive, possibly constitutively so, producing elevated levels of cyclic AMP in a cell-based assay. These mice demonstrate that increased Adcy3 activity robustly protect animals from diet-induced metabolic derangements.

A point mutation in Sec61alpha1 leads to diabetes and hepatosteatosis in mice.

OBJECTIVE: Type 2 diabetes is caused by both environmental and genetic factors. To better understand the genetic factors we used forward genetics to discover genes that have not previously been implicated in the development of hyperglycemia or diabetes. RESEARCH DESIGN AND METHODS: Offspring of ethylnitrosurea-mutagenized C57BL/6 mice were bred to homozygosity, maintained on high-fat diet, and screened for hyperglycemia. The phenotype in one diabetic family of mice was mapped among hybrid F2s with single nucleotide polymorphic markers, followed by candidate gene sequencing to identify the gene harboring the causative mutation. Subsequent analysis was done on wild-type, heterozygous, and homozygous mutant mice on a pure C57BL/6 background. RESULTS: Diabetes mapped to a point mutation in the Sec61a1 gene that encodes a His to Tyr substitution at amino acid 344 (Y344H). Metabolic profiling, histological examination, and electron microscopy revealed that hyperglycemia was a result of insulin insufficiency due to beta-cell apoptosis brought on by endoplasmic reticulum (ER) stress. Transgenic beta-cell-specific expression of Sec61a1 in mutant mice rescued diabetes, beta-cell apoptosis, and ER stress. In vitro experiments showed that Sec61alpha1 plays a critical role in the beta-cell response to glucose. CONCLUSIONS: Here we phenotypically characterize diabetes in mice with a novel point mutation in a basic component of the cell's ER protein translocation machinery, Sec61alpha1. Translocation by the mutant protein does not appear to be affected. Rather, ER homeostasis is perturbed leading to beta-cell death and diabetes.

KDEL-retained antigen in B lymphocytes induces a proinflammatory response: a possible role for endoplasmic reticulum stress in adaptive T cell immunity.

Generally, APCs activate CD4 T cells against peptides derived from exogenous Ag in the context of MHC II molecules. In this study, using transgenic B lymphocytes as model APCs, we demonstrate CD4 T cell priming in vivo against peptides derived from endogenously synthesized Ag targeted either to the cytosol or to the endoplasmic reticulum (ER). Surprisingly, priming by Ag containing the KDEL-retention motif yielded higher levels of two important proinflammatory cytokines, IFN-gamma and TNF-alpha, in responding CD4 T cells. Importantly, we found that KDEL-mediated retention of Ag up-regulates ER-stress responsive genes in primary B lymphocytes. We also found that thapsigargin treatment of A20 lymphoma cells up-regulates transcription of ER stress and proinflammatory genes along with IL-23p19. Induction of ER stress by thapsigargin also up-regulated IL-23p19 in primary B lymphocytes, macrophages, and bone marrow-derived dendritic cells. We conclude that perturbation of the secretory pathway and/or ER stress play an important role in modulating the gene program in professional APCs and in shaping CD4 T cell responses in vivo. These findings are relevant to a better understanding of the immune response after infection by viral and bacterial pathogens and the pathogenesis of certain autoimmune diseases.

In utero DNA immunisation. Immunity over tolerance in fetal life.

The function and plasticity of the developing immune system during embryonic life has been central to immunological thinking for half a century. A classical view is that antigen encountered during fetal life induces a state of acquired immunological tolerance. However, the ability to develop T cell immune responses during the perinatal period would be of great importance against intracellular pathogens. Recent experiments have challenged this notion and shown that neonatal tolerance can be circumvented by extrinsic immunological manipulations. Here, we used DNA immunization targeted at B lymphocytes to induce a CD4 T cell response that could be measured 2 weeks after birth. We conclude that T cell immunity can be programmed in utero by manipulating the parameters of the immune response in the fetal environment. Furthermore, our data suggest that under appropriate conditions the fetal immune system can be programmed to immunity.