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Inhibition of inducible nitric oxide synthase expression by a novel small molecule activator of the unfolded protein response.

Symons KT, Massari ME, Dozier SJ, Nguyen PM, Jenkins D, Herbert M, Gahman TC, Noble SA, Rozenkrants N, Zhang Y, Rao TS, Shiau AK, Hassig CA - Curr Chem Genomics (2008)

Bottom Line: Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR.Further, erstressin activates the transcription of multiple genes involved in the UPR.These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

No MeSH data available.


Related in: MedlinePlus

Erstressin (Compound 1) induces biochemical markers of ER stress and expression of genes involved in the UPR. Panel A. Erstressin increases mRNA levels of ER stress genes. Heat map of normalized microarray data from A172 cells simultaneously induced with pro-inflammatory cytokines and treated with indicated compound at 10 µM for 6 hours. A selection of UPR-related genes is shown. Data are shown as a ratio of expression with compound treatment versus vehicle. Red indicates up-regulation, green down-regulation, and black shows no difference between compound and the vehicle control. Panel B. Immunoblot of extracts from RAW 264.7 cells after 30 minutes treatment with compound shows elevated phosphorylation of serine 51 of eIF-2α when treated with erstressin (Es, 10 µM) or thapsigargin (Tg, 1 µM), but not with nostressin (Ns, 10 µM) or vehicle (Veh) alone. Panel C. XBP-1 rtPCR of RNA isolated form HeLa cells following treatment for 1 hour with compound shows the spliced variant (XBP-1s) present in cells treated with thapsigargin (Tg, 50 nM) and erstressin (Es, 20 µM), but not in cells treated with nostressin (Ns, 20 µM).
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Figure 3: Erstressin (Compound 1) induces biochemical markers of ER stress and expression of genes involved in the UPR. Panel A. Erstressin increases mRNA levels of ER stress genes. Heat map of normalized microarray data from A172 cells simultaneously induced with pro-inflammatory cytokines and treated with indicated compound at 10 µM for 6 hours. A selection of UPR-related genes is shown. Data are shown as a ratio of expression with compound treatment versus vehicle. Red indicates up-regulation, green down-regulation, and black shows no difference between compound and the vehicle control. Panel B. Immunoblot of extracts from RAW 264.7 cells after 30 minutes treatment with compound shows elevated phosphorylation of serine 51 of eIF-2α when treated with erstressin (Es, 10 µM) or thapsigargin (Tg, 1 µM), but not with nostressin (Ns, 10 µM) or vehicle (Veh) alone. Panel C. XBP-1 rtPCR of RNA isolated form HeLa cells following treatment for 1 hour with compound shows the spliced variant (XBP-1s) present in cells treated with thapsigargin (Tg, 50 nM) and erstressin (Es, 20 µM), but not in cells treated with nostressin (Ns, 20 µM).

Mentions: Interestingly, in addition to altering inflammatory gene expression, compound 1 also affected the mRNA levels of multiple genes involved in the UPR. The transcription factors XBP-1 (2.7-fold), ATF3 (3.2-fold), ATF4 (3.3-fold), and CHOP (22-fold) were significantly induced in compound 1-treated cells. Molecular chaperones ERdj4 (24-fold) and BiP (2.7-fold) and ER to Golgi transporters SEC23B (2-fold) and SEC31L1 (4-fold) were also significantly elevated by compound 1. Further, Herpud1/ Herp, a membrane-associated protein which functions in ER associated degradation was upregulated 9.7-fold (Fig. 3A). Changes in the expression of several genes identified in the expression profiling study were confirmed by independent qPCR analysis (Supplemental Figs. 2A and 2B). Similar gene expression signatures have been described for known ER stressors, including tunicamycin and thapsigargin [12, 13] and these effects were absent in cells treated with compound 2. These and other findings described below suggest that compound 1 may induce the UPR, and as a result, we have named this compound erstressin and its inactive analog compound 2, nostressin.


Inhibition of inducible nitric oxide synthase expression by a novel small molecule activator of the unfolded protein response.

Symons KT, Massari ME, Dozier SJ, Nguyen PM, Jenkins D, Herbert M, Gahman TC, Noble SA, Rozenkrants N, Zhang Y, Rao TS, Shiau AK, Hassig CA - Curr Chem Genomics (2008)

Erstressin (Compound 1) induces biochemical markers of ER stress and expression of genes involved in the UPR. Panel A. Erstressin increases mRNA levels of ER stress genes. Heat map of normalized microarray data from A172 cells simultaneously induced with pro-inflammatory cytokines and treated with indicated compound at 10 µM for 6 hours. A selection of UPR-related genes is shown. Data are shown as a ratio of expression with compound treatment versus vehicle. Red indicates up-regulation, green down-regulation, and black shows no difference between compound and the vehicle control. Panel B. Immunoblot of extracts from RAW 264.7 cells after 30 minutes treatment with compound shows elevated phosphorylation of serine 51 of eIF-2α when treated with erstressin (Es, 10 µM) or thapsigargin (Tg, 1 µM), but not with nostressin (Ns, 10 µM) or vehicle (Veh) alone. Panel C. XBP-1 rtPCR of RNA isolated form HeLa cells following treatment for 1 hour with compound shows the spliced variant (XBP-1s) present in cells treated with thapsigargin (Tg, 50 nM) and erstressin (Es, 20 µM), but not in cells treated with nostressin (Ns, 20 µM).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2803434&req=5

Figure 3: Erstressin (Compound 1) induces biochemical markers of ER stress and expression of genes involved in the UPR. Panel A. Erstressin increases mRNA levels of ER stress genes. Heat map of normalized microarray data from A172 cells simultaneously induced with pro-inflammatory cytokines and treated with indicated compound at 10 µM for 6 hours. A selection of UPR-related genes is shown. Data are shown as a ratio of expression with compound treatment versus vehicle. Red indicates up-regulation, green down-regulation, and black shows no difference between compound and the vehicle control. Panel B. Immunoblot of extracts from RAW 264.7 cells after 30 minutes treatment with compound shows elevated phosphorylation of serine 51 of eIF-2α when treated with erstressin (Es, 10 µM) or thapsigargin (Tg, 1 µM), but not with nostressin (Ns, 10 µM) or vehicle (Veh) alone. Panel C. XBP-1 rtPCR of RNA isolated form HeLa cells following treatment for 1 hour with compound shows the spliced variant (XBP-1s) present in cells treated with thapsigargin (Tg, 50 nM) and erstressin (Es, 20 µM), but not in cells treated with nostressin (Ns, 20 µM).
Mentions: Interestingly, in addition to altering inflammatory gene expression, compound 1 also affected the mRNA levels of multiple genes involved in the UPR. The transcription factors XBP-1 (2.7-fold), ATF3 (3.2-fold), ATF4 (3.3-fold), and CHOP (22-fold) were significantly induced in compound 1-treated cells. Molecular chaperones ERdj4 (24-fold) and BiP (2.7-fold) and ER to Golgi transporters SEC23B (2-fold) and SEC31L1 (4-fold) were also significantly elevated by compound 1. Further, Herpud1/ Herp, a membrane-associated protein which functions in ER associated degradation was upregulated 9.7-fold (Fig. 3A). Changes in the expression of several genes identified in the expression profiling study were confirmed by independent qPCR analysis (Supplemental Figs. 2A and 2B). Similar gene expression signatures have been described for known ER stressors, including tunicamycin and thapsigargin [12, 13] and these effects were absent in cells treated with compound 2. These and other findings described below suggest that compound 1 may induce the UPR, and as a result, we have named this compound erstressin and its inactive analog compound 2, nostressin.

Bottom Line: Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR.Further, erstressin activates the transcription of multiple genes involved in the UPR.These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA.

ABSTRACT
The transcription of inducible nitric oxide synthase (iNOS) is activated by a network of proinflammatory signaling pathways. Here we describe the identification of a small molecule that downregulates the expression of iNOS mRNA and protein in cytokine-activated cells and suppresses nitric oxide production in vivo. Mechanistic analysis suggests that this small molecule, erstressin, also activates the unfolded protein response (UPR), a signaling pathway triggered by endoplasmic reticulum stress. Erstressin induces rapid phosphorylation of eIF2alpha and the alternative splicing of XBP-1, hallmark initiating events of the UPR. Further, erstressin activates the transcription of multiple genes involved in the UPR. These data suggest an inverse relationship between UPR activation and iNOS mRNA and protein expression under proinflammatory conditions.

No MeSH data available.


Related in: MedlinePlus