Integration of the Unfolded Protein and Oxidative Stress Responses through SKN-1/Nrf

The Unfolded Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER stress, an underlying factor in various human diseases. During the UPR, numerous genes are activated that sustain and protect the ER. These responses are known to involve the canonical UPR transcription factors XBP1, ATF4, and ATF6. Here, we show in C. elegans that the conserved stress defense factor SKN-1/Nrf plays a central and essential role in the transcriptional UPR. While SKN-1/Nrf has a well-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes an overlapping but distinct response to ER stress. SKN-1/Nrf is regulated by the UPR, directly controls UPR signaling and transcription factor genes, binds to common downstream targets with XBP-1 and ATF-6, and is present at the ER. SKN-1/Nrf is also essential for resistance to ER stress, including reductive stress. Remarkably, SKN-1/Nrf-mediated responses to oxidative stress depend upon signaling from the ER. We conclude that SKN-1/Nrf plays a critical role in the UPR, but orchestrates a distinct oxidative stress response that is licensed by ER signaling. Regulatory integration through SKN-1/Nrf may coordinate ER and cytoplasmic homeostasis

Specific SKN-1/Nrf Stress Responses to Perturbations in Translation Elongation and Proteasome Activity

Peer reviewe

Assessing Potential Effects of Highway and Urban Runoff on Receiving Streams in Total Maximum Daily Load Watersheds in Oregon Using the Stochastic Empirical Loading and Dilution Model

The Stochastic Empirical Loading and Dilution Model (SELDM) was developed by the U.S. Geological Survey (USGS) in cooperation with the Federal Highway Administration to simulate stormwater quality. To assess the effects of runoff, SELDM uses a stochastic mass-balance approach to estimate combinations of pre-storm streamflow, stormflow, highway runoff, event mean concentrations (EMCs) and stormwater constituent loads from a site of interest. In addition, SELDM can be used to assess the effects of stormwater Best Management Practices (BMPs), which are designed to mitigate the adverse effects of runoff into a waterbody

SKN-1 directly regulates target genes during the UPR.

<p>(A–L) ER stress-induced SKN-1 recruitment and transcriptional activation was analyzed at the SKN-1-regulated genes <i>pcp-2</i> (A–D), <i>atf-5</i> (E–H), and <i>gst-4</i> (I–L). TM treatment leads to SKN-1 recruitment (A, E, I), accumulation of Pol II that is phosphorylated at CTD Ser 2 (P-Ser2) (B, F, J), decreased Histone H3 occupancy (C, G, K), and increased H3-AcK56 density (D, H, L) at the site of transcription. Maps mark qPCR amplicons relative to the predicted transcription start site, with exons marked as black boxes. % ChIP signal is relative to input, and normalized to the highest signal for each run <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701-GloverCutter1" target="_blank">[44]</a>. In (D, H, L), a ratio of acetyl histone to histone signal is presented. For ChIP experiments in this study error bars represent SEM, and * p≤.05, ** p≤.01, *** p≤.001, relative to <i>pL4440</i> Control calculated using one-sided student's t-test. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s002" target="_blank">Figure S2</a>.</p

Association of SKN-1 with the ER.

<p>(A, B) Interaction between endogenous SKN-1 and HSP-3/4, detected by IP/Western. Lysates were prepared from animals in which proteins had been crosslinked under ChIP conditions. (A) Monoclonal αSKN-1 IP blotted with αHsc3 (HSP-3/4). (B) αHsc3 (HSP-3/4) IP blotted with monoclonal αSKN-1. (C–E) Analyses of ER fractions prepared from whole worms. The fractionation scheme is described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s006" target="_blank">Fig. S6B</a>. (C) Detection of endogenous HSP-3/4 and the cytoplasmic marker GAPDH in ER and Mitochondrial fractions, and total worm lysate. Note the enrichment of the ER marker HSP-3/4 compared to GAPDH in the ER fraction. TM indicates lysates from animals that had been treated with TM. (D) Presence of endogenous SKN-1 in the ER fraction, detected by western and IP/western blotting. Note that TM treatment increased the levels of SKN-1 protein. (E) Association between endogenous SKN-1 and HSP-3/4 within the ER fraction, detected with polyclonal αSKN-1 and αBiP (HSP-3/4), by IP/Western that was performed without crosslinking. Fractionations and analyses were performed independently twice, with similar results. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s006" target="_blank">Figure S6</a>.</p

ER stress activates SKN-1 independently of oxidative stress.

<p>(A) Treatment with TM (16 hrs), thapsigargin (Thap, 2 hrs), or bortezomib (6 hrs) increased <i>skn-1</i> mRNA levels, as determined by qRT-PCR. RNAi knockdown of <i>hsp-4</i> or <i>atf-6</i> also increased <i>skn-1</i> mRNA levels. (B) Increased endogenous SKN-1 protein levels in response to TM-induced ER stress. SKN-1 was detected by Western blotting with the polyclonal antibody, with GAPDH serving as the loading control. (C) Induction of <i>skn-1</i> expression and SKN-1-regulated UPR target genes by reductive ER stress (DTT treatment for 2 hrs), assayed by qRT-PCR. (D) Induction of the UPR, <i>skn-1</i> expression, and SKN-1 target genes by <i>ero-1</i> RNAi, assayed by qRT-PCR. Different primer sets were used to distinguish among mRNAs that correspond to different <i>skn-1</i> isoforms. Error bars represent SEM, and * p≤.05, ** p≤.01, *** p≤.001, relative to <i>pL4440</i> Control calculated using student's t-test. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s004" target="_blank">Figure S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s010" target="_blank">Table S3</a>.</p

SKN-1 regulates diverse functions in response to ER stress.

<p>(A, B) ER stress induces <i>skn</i>-1-dependent activation of ER- or UPR-associated genes. qRT-PCR was performed after RNAi Control (<i>pL4440</i> in all panels) or <i>skn-1</i> RNAi, and Control or 5 µg/ml TM treatment. Known or predicted functions of these genes are described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s008" target="_blank">Table S1</a>. Genes are grouped in (A) or (B) according to the extent of TM-induced activation, and plotted on different scales. All analyses of TM-regulated gene expression involved a 16 hr TM treatment, based upon a time-course experiment (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s001" target="_blank">Figure S1B</a>) and published work in <i>C. elegans </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701-Harding1" target="_blank">[15]</a>. Shorter time courses were chosen for other ER stress treatments (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen-1003701-g004" target="_blank">Figure 4</a>, legend). (C) Upregulation of SKN-1-regulated oxidative stress defense genes in response to TM. Error bars represent SEM, * p≤.05, ** p≤.01, *** p≤.001, relative to <i>pL4440</i> Control. All qRT-PCR p-values were calculated as one or two-sided t-test as appropriate with n≥3. (D) Activation of the <i>gcs-1</i>::<i>GFP</i> transgene in the intestine, with GFP expression scored as High, Medium, or Low. *** p<.0001 chi² method. See Experimental Procedures for scoring method. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s001" target="_blank">Figure S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s008" target="_blank">Table S1</a>.</p

UPR factors required for ER stress-induced SKN-1 activation.

<p>(A) ER stress-induced activation of <i>skn-1</i> and its target genes requires core UPR factors. RNA levels were assayed by qRT-PCR after RNAi against core UPR genes or in core UPR factor mutants (indicated by ^M) after TM treatment. (B-E) IRE-1 is required for ER stress-induced SKN-1 accumulation and activity at SKN-1 target genes <i>gst-4</i> and <i>pcp-2</i>. Presence of SKN-1 and transcription markers was assayed by ChIP as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen-1003701-g002" target="_blank">Figure 2</a>, and <i>ire-1</i> was knocked down by RNAi. (F–H) Endogenous XBP-1 (F), ATF-6 (G), and SKN-1 (H) bind within the <i>skn-1</i> gene locus in response to TM-induced ER stress, with binding assayed by ChIP. Multiple start sites are noted within the <i>skn-1</i> locus. Error bars represent SEM, and * p≤.05, ** p≤.01, *** p≤.001 by student's t-test, relative to <i>pL4440</i> Control unless otherwise indicated. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003701#pgen.1003701.s005" target="_blank">Figure S5</a>.</p

The Hooskanaden Landslide: Historic and Recent Surge Behavior of an Active Earthflow on the Oregon Coast

This paper presents an analysis of the Hooskanaden Landslide, an earthflow, which experienced a dramatic surge event beginning on February 24, 2019, closing US Highway 101 near mile point 343.5 for nearly 2 weeks. This ~ 1 km long surge event resulted in horizontal displacements of up to 45 m and uplift of 6 m at the toe located on a gravel beach adjacent to the Pacific Ocean. The Hooskanaden Landslide, likely active since the eighteenth century, exhibits regular activity with a recurrence interval of major surge events of approximately every 20 years, transitioning from slow to relatively rapid velocities. During the 2019 event, maximum displacement rates of approximately 60 cm/h were observed, slowly decreasing to 15 cm/h for a sustained period of approximately 2 weeks before the eventual return to baseline conditions (\u3c 0.02 cm/h)

TOR Signaling and Rapamycin Influence Longevity by Regulating SKN-1/Nrf and DAF-16/FoxO

The TOR kinase, which is present in the functionally distinct complexes TORC1 and TORC2, is essential for growth but associated with disease and aging. Elucidation of how TOR influences life span will identify mechanisms of fundamental importance in aging and TOR functions. Here we show that when TORC1 is inhibited genetically in C. elegans, SKN-1/Nrf, and DAF-16/FoxO activate protective genes, and increase stress resistance and longevity. SKN-1 also upregulates TORC1 pathway gene expression in a feedback loop. Rapamycin triggers a similar protective response in C. elegans and mice, but increases worm life span dependent upon SKN-1 and not DAF-16, apparently by interfering with TORC2 along with TORC1. TORC1, TORC2, and insulin/IGF-1-like signaling regulate SKN-1 activity through different mechanisms. We conclude that modulation of SKN-1/Nrf and DAF-16/FoxO may be generally important in the effects of TOR signaling in vivo and that these transcription factors mediate an opposing relationship between growth signals and longevity.National Institutes of Health (U.S.) (Grant CA129105)Ellison Medical FoundationAmerican Federation for Aging ResearchStarr FoundationDavid H. Koch Institute for Integrative Cancer Research at MIT. Frontier Research ProgramNational Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (DRC Grant)National Institutes of Health (U.S.) (Ruth L. Kirschstein National Research Service Award) (F32 Postdoctoral Fellowship)American Diabetes Association (Fellowship