Limits...
Luman contributes to brefeldin A-induced prion protein gene expression by interacting with the ERSE26 element

View Article: PubMed Central - PubMed

ABSTRACT

The cellular prion protein (PrP) is essential for transmissible prion diseases, but its exact physiological function remains unclear. Better understanding the regulation of the human prion protein gene (PRNP) expression can provide insight into this elusive function. Spliced XBP1 (sXBP1) was recently shown to mediate endoplasmic reticulum (ER) stress-induced PRNP expression. In this manuscript, we identify Luman, a ubiquitous, non-canonical unfolded protein response (UPR), as a novel regulator of ER stress-induced PRNP expression. Luman activity was transcriptionally and proteolytically activated by the ER stressing drug brefeldin A (BFA) in human neurons, astrocytes, and breast cancer MCF-7 cells. Over-expression of active cleaved Luman (ΔLuman) increased PrP levels, while siRNA-mediated Luman silencing decreased BFA-induced PRNP expression. Site-directed mutagenesis and chromatin immunoprecipitation demonstrated that ΔLuman regulates PRNP expression by interacting with the ER stress response element 26 (ERSE26). Co-over-expression and siRNA-mediated silencing experiments showed that sXBP1 and ΔLuman both up-regulate ER stress-induced PRNP expression. Attempts to understand the function of PRNP up-regulation by Luman excluded a role in atorvastatin-induced neuritogenesis, ER-associated degradation, or proteasomal inhibition-induced cell death. Overall, these results refine our understanding of ER stress-induced PRNP expression and function.

No MeSH data available.


BFA-induced ER stress increases transcription and N-terminal cleavage of Luman.(a) Ethidium bromide stained agarose gel showing OASIS, BBF2H7, CREBH, AIbZIP, LUMAN, PRNP, HSPA5 and HPRT1 RT-PCR amplification products from MCF-7 cells (n = 4), neurons (n = 7) and astrocytes (n = 5) treated with DMSO (D, 0.1%), or 5 μg/mL of brefeldin A (BFA), thapsigargin (Th) or tunicamycin (TM) for 18 h. The fold increase relative to DMSO of band intensity normalized to HPRT1 is indicated. (b,c) Relative increase in LUMAN (b) or HSPA5 (c) mRNA levels assessed by qPCR 18 h after DMSO, BFA, Th or TM treatments (5 μg/mL). Data represent the mean ± SEM of three experiments, analysed using a one-way ANOVA followed by a Dunnett post-hoc test *p < 0.05 compared to DMSO. (d) Ethidium bromide stained agarose gel showing LUMAN and HPRT1 RT-PCR amplification products from MCF-7 cells treated with DMSO or BFA (5 μg/mL) for 18 h, in the presence of 1 μg/mL actinomycin D (Act. D) or 20 μg/mL cycloheximide (CHX)). (e) Western blot analysis of ΔLuman, BiP, and β-Actin protein levels from MCF-7 cells or primary human neuron and astrocytes treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (f) Ethidium bromide stained agarose gel showing MAP2, GFAP and HPRT1 RT-PCR amplification products from MCF-7 cells, neurons and astrocytes60. (g) Western blot analysis of HA-tagged ΔLuman, eGFP and β-Actin protein levels from HEK293T transfected with an N-terminal HA-tagged full length Luman treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (h) Time course assessment of Luman cleavage in HEK293T transfected with N-terminal HA-tagged full length Luman, and treated with BFA (5 μg/mL) or DMSO (Ctl, 0.1%). Vector designates HEK293T cells transfected with the pBud-eGFP vector. Full-length images of blots and gels are presented in Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5304227&req=5

f1: BFA-induced ER stress increases transcription and N-terminal cleavage of Luman.(a) Ethidium bromide stained agarose gel showing OASIS, BBF2H7, CREBH, AIbZIP, LUMAN, PRNP, HSPA5 and HPRT1 RT-PCR amplification products from MCF-7 cells (n = 4), neurons (n = 7) and astrocytes (n = 5) treated with DMSO (D, 0.1%), or 5 μg/mL of brefeldin A (BFA), thapsigargin (Th) or tunicamycin (TM) for 18 h. The fold increase relative to DMSO of band intensity normalized to HPRT1 is indicated. (b,c) Relative increase in LUMAN (b) or HSPA5 (c) mRNA levels assessed by qPCR 18 h after DMSO, BFA, Th or TM treatments (5 μg/mL). Data represent the mean ± SEM of three experiments, analysed using a one-way ANOVA followed by a Dunnett post-hoc test *p < 0.05 compared to DMSO. (d) Ethidium bromide stained agarose gel showing LUMAN and HPRT1 RT-PCR amplification products from MCF-7 cells treated with DMSO or BFA (5 μg/mL) for 18 h, in the presence of 1 μg/mL actinomycin D (Act. D) or 20 μg/mL cycloheximide (CHX)). (e) Western blot analysis of ΔLuman, BiP, and β-Actin protein levels from MCF-7 cells or primary human neuron and astrocytes treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (f) Ethidium bromide stained agarose gel showing MAP2, GFAP and HPRT1 RT-PCR amplification products from MCF-7 cells, neurons and astrocytes60. (g) Western blot analysis of HA-tagged ΔLuman, eGFP and β-Actin protein levels from HEK293T transfected with an N-terminal HA-tagged full length Luman treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (h) Time course assessment of Luman cleavage in HEK293T transfected with N-terminal HA-tagged full length Luman, and treated with BFA (5 μg/mL) or DMSO (Ctl, 0.1%). Vector designates HEK293T cells transfected with the pBud-eGFP vector. Full-length images of blots and gels are presented in Supplementary Information.

Mentions: To identify which members of the OASIS family could contribute to ER stress-induced PRNP expression, OASIS, BBF2H7, CREBH, AIbZIP, and LUMAN transcript levels were assessed by RT-PCR in breast carcinoma MCF-7 cells, human primary neurons and astrocytes treated with the ER stressing drugs BFA, Th or TM (Fig. 1a). As previously observed, all three ER stressors increased PRNP mRNA levels. OASIS mRNA levels were only increased by BFA in astrocytes. Levels of BBF2H7 and CREBH were undetectable, or very low, in the three cell types and seemed unaffected by ER stress, with the exception of a salient CREBH increase in astrocytes treated with BFA. AIbZIP was very weakly detected in neuronal preparations. TM increased AIbZIP levels in astrocytes, but reduced them in MCF-7 cells. LUMAN transcripts were detected in the three cell types, and BFA treatment clearly increased LUMAN mRNA levels in MCF-7 cells, neurons and astrocytes. However, Th and TM treatments only caused modest and inconsistent increases in LUMAN mRNA levels. To clarify these results, the induction of LUMAN mRNA by ER stress was assessed by quantitative PCR, and showed a significant LUMAN mRNA increase in MCF-7, neuronal and astrocytic cultures treated with BFA, but not with Th and TM (Fig. 1b). Amplification of BiP mRNA (HSPA5) by RT-PCR (Fig. 1a) and quantitative PCR (Fig. 1c) controlled for induction of ER stress by BFA, Th and TM treatments, and the housekeeping gene HPRT1 was used as a control for overall mRNA levels. To confirm that the induction of Luman mRNA levels by BFA was due to an increase in LUMAN transcription, MCF-7 cells were treated with BFA in the presence of the transcription inhibitor actinomycin D or of the translation inhibitor cycloheximide. BFA treatment increased LUMAN mRNA levels, and actinomycin D co-treatment attenuated LUMAN mRNA levels of BFA- and DMSO-treated cells (Fig. 1d). Cycloheximide treatment did not significantly influence BFA-induced LUMAN mRNA, but caused a small LUMAN mRNA increase in the DMSO control condition, as observed previously for other genes39. The housekeeping gene HPRT1 controlled for overall mRNA levels.


Luman contributes to brefeldin A-induced prion protein gene expression by interacting with the ERSE26 element
BFA-induced ER stress increases transcription and N-terminal cleavage of Luman.(a) Ethidium bromide stained agarose gel showing OASIS, BBF2H7, CREBH, AIbZIP, LUMAN, PRNP, HSPA5 and HPRT1 RT-PCR amplification products from MCF-7 cells (n = 4), neurons (n = 7) and astrocytes (n = 5) treated with DMSO (D, 0.1%), or 5 μg/mL of brefeldin A (BFA), thapsigargin (Th) or tunicamycin (TM) for 18 h. The fold increase relative to DMSO of band intensity normalized to HPRT1 is indicated. (b,c) Relative increase in LUMAN (b) or HSPA5 (c) mRNA levels assessed by qPCR 18 h after DMSO, BFA, Th or TM treatments (5 μg/mL). Data represent the mean ± SEM of three experiments, analysed using a one-way ANOVA followed by a Dunnett post-hoc test *p < 0.05 compared to DMSO. (d) Ethidium bromide stained agarose gel showing LUMAN and HPRT1 RT-PCR amplification products from MCF-7 cells treated with DMSO or BFA (5 μg/mL) for 18 h, in the presence of 1 μg/mL actinomycin D (Act. D) or 20 μg/mL cycloheximide (CHX)). (e) Western blot analysis of ΔLuman, BiP, and β-Actin protein levels from MCF-7 cells or primary human neuron and astrocytes treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (f) Ethidium bromide stained agarose gel showing MAP2, GFAP and HPRT1 RT-PCR amplification products from MCF-7 cells, neurons and astrocytes60. (g) Western blot analysis of HA-tagged ΔLuman, eGFP and β-Actin protein levels from HEK293T transfected with an N-terminal HA-tagged full length Luman treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (h) Time course assessment of Luman cleavage in HEK293T transfected with N-terminal HA-tagged full length Luman, and treated with BFA (5 μg/mL) or DMSO (Ctl, 0.1%). Vector designates HEK293T cells transfected with the pBud-eGFP vector. Full-length images of blots and gels are presented in Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5304227&req=5

f1: BFA-induced ER stress increases transcription and N-terminal cleavage of Luman.(a) Ethidium bromide stained agarose gel showing OASIS, BBF2H7, CREBH, AIbZIP, LUMAN, PRNP, HSPA5 and HPRT1 RT-PCR amplification products from MCF-7 cells (n = 4), neurons (n = 7) and astrocytes (n = 5) treated with DMSO (D, 0.1%), or 5 μg/mL of brefeldin A (BFA), thapsigargin (Th) or tunicamycin (TM) for 18 h. The fold increase relative to DMSO of band intensity normalized to HPRT1 is indicated. (b,c) Relative increase in LUMAN (b) or HSPA5 (c) mRNA levels assessed by qPCR 18 h after DMSO, BFA, Th or TM treatments (5 μg/mL). Data represent the mean ± SEM of three experiments, analysed using a one-way ANOVA followed by a Dunnett post-hoc test *p < 0.05 compared to DMSO. (d) Ethidium bromide stained agarose gel showing LUMAN and HPRT1 RT-PCR amplification products from MCF-7 cells treated with DMSO or BFA (5 μg/mL) for 18 h, in the presence of 1 μg/mL actinomycin D (Act. D) or 20 μg/mL cycloheximide (CHX)). (e) Western blot analysis of ΔLuman, BiP, and β-Actin protein levels from MCF-7 cells or primary human neuron and astrocytes treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (f) Ethidium bromide stained agarose gel showing MAP2, GFAP and HPRT1 RT-PCR amplification products from MCF-7 cells, neurons and astrocytes60. (g) Western blot analysis of HA-tagged ΔLuman, eGFP and β-Actin protein levels from HEK293T transfected with an N-terminal HA-tagged full length Luman treated with 0.1% DMSO or 5 μg/mL of BFA, Th or TM for 18 h. (h) Time course assessment of Luman cleavage in HEK293T transfected with N-terminal HA-tagged full length Luman, and treated with BFA (5 μg/mL) or DMSO (Ctl, 0.1%). Vector designates HEK293T cells transfected with the pBud-eGFP vector. Full-length images of blots and gels are presented in Supplementary Information.
Mentions: To identify which members of the OASIS family could contribute to ER stress-induced PRNP expression, OASIS, BBF2H7, CREBH, AIbZIP, and LUMAN transcript levels were assessed by RT-PCR in breast carcinoma MCF-7 cells, human primary neurons and astrocytes treated with the ER stressing drugs BFA, Th or TM (Fig. 1a). As previously observed, all three ER stressors increased PRNP mRNA levels. OASIS mRNA levels were only increased by BFA in astrocytes. Levels of BBF2H7 and CREBH were undetectable, or very low, in the three cell types and seemed unaffected by ER stress, with the exception of a salient CREBH increase in astrocytes treated with BFA. AIbZIP was very weakly detected in neuronal preparations. TM increased AIbZIP levels in astrocytes, but reduced them in MCF-7 cells. LUMAN transcripts were detected in the three cell types, and BFA treatment clearly increased LUMAN mRNA levels in MCF-7 cells, neurons and astrocytes. However, Th and TM treatments only caused modest and inconsistent increases in LUMAN mRNA levels. To clarify these results, the induction of LUMAN mRNA by ER stress was assessed by quantitative PCR, and showed a significant LUMAN mRNA increase in MCF-7, neuronal and astrocytic cultures treated with BFA, but not with Th and TM (Fig. 1b). Amplification of BiP mRNA (HSPA5) by RT-PCR (Fig. 1a) and quantitative PCR (Fig. 1c) controlled for induction of ER stress by BFA, Th and TM treatments, and the housekeeping gene HPRT1 was used as a control for overall mRNA levels. To confirm that the induction of Luman mRNA levels by BFA was due to an increase in LUMAN transcription, MCF-7 cells were treated with BFA in the presence of the transcription inhibitor actinomycin D or of the translation inhibitor cycloheximide. BFA treatment increased LUMAN mRNA levels, and actinomycin D co-treatment attenuated LUMAN mRNA levels of BFA- and DMSO-treated cells (Fig. 1d). Cycloheximide treatment did not significantly influence BFA-induced LUMAN mRNA, but caused a small LUMAN mRNA increase in the DMSO control condition, as observed previously for other genes39. The housekeeping gene HPRT1 controlled for overall mRNA levels.

View Article: PubMed Central - PubMed

ABSTRACT

The cellular prion protein (PrP) is essential for transmissible prion diseases, but its exact physiological function remains unclear. Better understanding the regulation of the human prion protein gene (PRNP) expression can provide insight into this elusive function. Spliced XBP1 (sXBP1) was recently shown to mediate endoplasmic reticulum (ER) stress-induced PRNP expression. In this manuscript, we identify Luman, a ubiquitous, non-canonical unfolded protein response (UPR), as a novel regulator of ER stress-induced PRNP expression. Luman activity was transcriptionally and proteolytically activated by the ER stressing drug brefeldin A (BFA) in human neurons, astrocytes, and breast cancer MCF-7 cells. Over-expression of active cleaved Luman (&Delta;Luman) increased PrP levels, while siRNA-mediated Luman silencing decreased BFA-induced PRNP expression. Site-directed mutagenesis and chromatin immunoprecipitation demonstrated that &Delta;Luman regulates PRNP expression by interacting with the ER stress response element 26 (ERSE26). Co-over-expression and siRNA-mediated silencing experiments showed that sXBP1 and &Delta;Luman both up-regulate ER stress-induced PRNP expression. Attempts to understand the function of PRNP up-regulation by Luman excluded a role in atorvastatin-induced neuritogenesis, ER-associated degradation, or proteasomal inhibition-induced cell death. Overall, these results refine our understanding of ER stress-induced PRNP expression and function.

No MeSH data available.