Induction of cellular Herp expression by Luman. (A) Scanned image of a representative microarray used in the study. 293 cells were either transfected with pcLuman or the vector pcDNA3.1. cDNA samples were labeled with Alexa Fluor 555 or 647 and hybridized to the 1.7K human cDNA microarray. (B) Induction of Herp mRNA expression by Luman. Cells were transfected with pcLuman, pcLuman(N), or the vector only. Treatment with the ER stressor Tm was used as a positive control. Total RNA was extracted and subjected to Northern blot analysis using a DNA probe specific for Herp. The relative intensities of the bands were normalized against 18S rRNA, shown at the bottom. (C) Overexpression of Luman triggers its proteolytic cleavage. HeLa cells in 35-mm dishes were transfected with 1 μg of 3F-Luman, 3F-Luman(N), or the parental vector pcDNA3.1. For the positive control, cells were treated with brefeldin A (1 μg/ml) in the presence of MG132 (5 μM) for 5 h. The affinity-purified FLAG monoclonal antibody M2 (Sigma) was used as the primary antibody in the Western blotting. β-Actin was used as a loading control. Bands with an asterisk on the left are proteolysis products of full-length Luman and are labeled Luman₄₀.

Luman induces transcription from the Herp promoter. (A) Schematic structure of full-length and N-terminal Luman as well as the Δ123-186 mutant lacking the basic region responsible for DNA binding. (B) Activation of a Herp promoter (−200/+98) reporter by Luman. 293 cells were transiently transfected with pGL3-Herp-Luciferase reporter together with the reference Renilla luciferase plasmid pRL-SV40 and the effector plasmids encoding Luman(N) and Luman(N)Δ123-186. The vector pcDNA3.1 and treatment with Tm were used as negative and positive controls, respectively. Luciferase values from three independent experiments were normalized to Renilla luciferase activity before being referenced to the control. Cell lysates from the luciferase assays in panel B were subjected to Western blot analysis using Luman antibody M13, shown at the bottom, with β-actin as a loading control. (C and D) Mapping of the Luman-responsive element in the Herp promoter. In both panels C and D, the pGL3-Herp-Luciferase or 10-bp scanning mutation reporter plasmids were cotransfected in 293 cells along with pcDNA3.1 or pcLuman(N). In all scanning mutations of the Herp promoter, nucleotides A, C, G, and T were substituted for C, A, T, and G, respectively. The vector pcDNA3.1 and treatment with Tm were used as negative and positive controls, respectively. The relative luciferase activity was determined by averaging triplicates in three independent experiments, shown with standard errors.

Luman binds and activates transcription from the second half-site of ERSE-II. (A) Dual luciferase assays were performed as described in the legend for Fig. 2. In all the half-site mutants of ERSE and ERSE-II, the nucleotides A, C, G, and T were substituted for C, A, T, and G, respectively. (B) In vitro binding of Luman to the second half-site of ERSE-II by EMSA. Mutant sequences of the two half-sites of ERSE-II are underlined. Equal amounts of purified GST (G) and GST-Luman (L) proteins were incubated with the indicated double-stranded probes labeled with ³²P and separated on a 4% nondenaturing polyacrylamide gel electrophoresis gel. (C) Direct binding of Luman to the Herp promoter as demonstrated by ChIP assay. Top, schematic diagram of the human Herp promoter, with positions of the primer pair used in this ChIP assay indicated. Bottom, 293 cells were transfected with plasmid pcDNA3.1 or pcFLAG-Luman and then cross-linked by formaldehyde. Chromatin was immunoprecipitated with the indicated antibodies. Purified precipitates or input DNA was analyzed by PCR using primers specific for Herp (−193/−15) or the control OPR150 (−311/−28) and Smad6 (−186/+63) promoters. PCR products were subjected to gel electrophoresis and visualized by ethidium bromide staining.

Activation of Luman by ER stress. (A) Induction of Luman cleavage upon various ER stressor treatments. 293 cells were treated with 2 μg/ml Tm, 300 nM Tg, 1 mM DTT, 300 nM H₂O₂,or 1 μg/ml brefeldin A for 8 h in the presence of 5 μM MG132. Cells were lysed in sample buffer. Affinity-purified polyclonal antibody (Rb5660) against Luman(N) and a Herp antibody (17) were used as primary antibodies in Western blotting. β-Actin was used as a loading control. (B) Induction of Luman transcription by Tm, Tg, DTT, and H₂O₂. After the same ER stressor treatments as for panel A, Northern blot analysis was carried out using Luman cDNA as a probe. Equal loading of RNA was confirmed by staining of 18S rRNA. Relative levels of Luman mRNA (bottom) were calculated by normalization to 18S RNA. Note: the glycosylated form of full-length Luman (A, top band of the Luman doublet) is absent in the treated with Tm (an N-glycosylation inhibitor) or brefeldin A (ER-to-Golgi transport inhibitor), as reported previously (36).

Luman contributes to the induction of cellular Herp during the ER stress response. (A) Endogenous Luman knockdown by siRNA (Northern blotting). 293 cells were transfected with Luman Stealth siRNA 755 and its corresponding control siRNA (Invitrogen). RNA was extracted with TRIzol (Invitrogen) 24 h posttransfection and subjected to electrophoresis and Northern blot analysis using Luman cDNA as a probe. (B and C) siRNA knockdown of transfected GFP-Luman shown by Western blotting (B) and microscopy (C). At 48 h posttransfection of GFP-Luman and siRNA, 293 cells were lysed in sample buffer and subjected to Western blotting using Luman antibody M13 (B), or cells growing on coverslips were photographed under a Leica DMRA2 microscope using a 63× objective lens (C). (D) Repression of Herp expression through siRNA knockdown of Luman. 293 cells were transfected twice successively with Luman siRNA755 at a ∼24-h interval (left panel) or transfected with the Luman dominant negative mutant LumanΔ1-52. At 36 h posttransfection, cells were treated with Tg at 300 nM for 12 h and total RNA was extracted. Northern blot analysis was performed for Herp and Luman, while an RT-PCR specific for LumanΔ1-52 mutant or the actin control was used. *, a faint Herp band can been seen in the Tg-untreated sample under longer exposure. (E) Inhibition of uninduced Herp transcription by dominant negative mutant LumanΔ1-52. 293 cells were transfected with pcDNA3.1 treated or untreated with 2 μg/ml tunicamycin (Tm) for 8 h, pcLuman, or pcLuman Δ1-52. Herp mRNA was detected by Northern blot analysis. Normalized transcript levels are shown at the bottom in the same order. (F) Semiquantitative analysis of cellular Luman, Herp, GRP78, GRP94 transcription, and the alternative splicing of XBP1 mRNA by RT-PCR.

Induction of Herp transcription by Luman is independent of the IRE1/XBP1 pathway. IRE1α^+/+ and IRE1α^−/− cells were transfected with pcLuman(N), pcLuman(N)Δ123-186, and the vector pcDNA3.1, along with the Herp reporter as shown. Dual luciferase assays were performed as described above.

Luman protects cells from ER stress-induced apoptotic cell death. (A) Luman represses the caspase 3 activity during ER stress. HeLa cells were transiently transfected with pcDNA3.1 or pcLuman(N). Caspase 3 activity was analyzed after cells were treated with Tm (2 μg/ml) for 48 h or staurosporine (St) for 24 h. The activity was normalized to the cells transfected with pcDNA3.1 with no treatment. The averages of the relative values from three independent experiments are shown with standard errors. The P value from Student's t test is shown for the pcLuman(N) with Tm treatment sample. (B) Luman increases cellular Herp expression in addition to ER stress stimulation. Cell lysates from the caspase 3 assay (shown in panel A) were subjected to Western blot analysis using affinity-purified Luman antibody (Rb5660) and a Herp antibody (17) as primary antibodies. β-Actin was used as a loading control. (C) Luman activates the Herp promoter in addition to Tm-induced ER stress. 293 cells were transiently transfected with the pGL3-Herp-Luciferase reporter together with the reference plasmid pRL-SV40 and pcLuman(N). The vector pcDNA3.1 was used as a negative control. Cells were treated with Tm for the indicated time before cellular lysates were harvested, and dual luciferase assays (top) and Western blotting (bottom) were conducted as described previously.

Summary of cis-acting elements and their regulatory factors in regulation of Herp gene expression during the mammalian UPR. The text at the bottom shows the cellular processes linked to the cis element and the genes in which the element has been found, in addition to Herp. Abbreviation: AA deprivation, amino acid deprivation.