Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

Abstract

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR.

Fig. 1 –

Characterization of Armet. A. The endogenous and transfected non-tagged Armet have the same size. Increasing amounts of plasmid encoding non-tagged Armet were transfected into HEK293 cells. Transfected cells were processed for IB for Armet using affinity-purified rabbit anti-Armet. B. Anti-Armet antibody specifically recognizes Armet-6xHis but not Armet-L1–6xHis. pCIneo-Armet-6xHis, pCIneo-Armet-L1–6xHis or pCIneo as control were transfected in HEK293 cells. Twenty-four hours post-transfection cells were processed for IP with anti 6xHis antibody and the precipitates were further analyzed by IB. The input represent 5% of the total proteins used for IP. Arrowhead indicates modified Armet-L1. Laddering bands in lane 4 (upper panel) are non-specific staining. C. Effects of Brefeldin A and tunicamycin on levels and migration of transfected Armet and Armet-L1. HEK293 cells transfected with Armet or Armet-L1 were treated with Tunicamycin (2.5 μg/ml) or Brefeldin A (5 μg/ml.) for 5 h before processed for IB. Arrowhead indicates glycosylated Armet-L1.

Fig. 2 –

Induction of Armet expression by chemical ER stress inducers in cells and by ischemia in rat brain. A. Armet is upregulated by different ER stress inducers in different cell lines. U2OS, HEK293 and SHSY-5Y were treated with tunicamycin (Tun, 2.5 μg/ml), thapsigargin (Tha, 500 nM), or proteasome inhibitor lactacystin (Lac, 10 μM) for 7 h. B and C. Armet is upregulated by ER stress in a time-dependent manner. D. Armet but not Armet-L1 is upregulated by tunicamycin. U2OS cells were treated with Tunicamycin for 0, 1, 2, 5 and 7 h. Total RNA was extracted and RT-PCR was performed. E. Anti-Armet antibody is specific for Armet in immunostaining. U2OS cells were transfected with pCIneo-Armet-FLAG. Twenty-four hours after transfection cells were fixed and stained with rabbit polyclonal anti-Armet and mouse monoclonal anti-FLAG antibodies in conjunction with Alexa Fluor-488 (green) and Alexa fluor-594 (red). F and G. Induction of Armet by experimental cerebral ischemia. Armet was revealed by immunohistochemical staining by ABC method of ischemic cerebral cortex (G) or contralateral side (F, negative control). Nuclei were counterstained by hematoxylin.

Fig. 3 –

Armet is localized in the ER and Golgi. A. Armet is associated with the microsomal fraction. HEK293 cells were transfected with pCIneo-Armet-FLAG. Twenty-four hours post-transfection cells were fractionated into microsomes (m) and cytosol (c) and analyzed by IB for the indicated proteins. B. Armet is localized in the lumen of the ER. Microsomes isolated from HeLa cells were subjected to digestion by increasing concentrations of proteinase K for 30 min on ice. Following the digestion, microsomes and supernatants were separated by centrifugation and analyzed by IB. C. Armet is cofractionated with the ER marker calnexin and partially with the Golgi marker Golgin-97. The post-nuclear fraction of HeLa cell homogenates were subjected to density gradient fractionation as described in Materials and Methods. An equal volume from each fraction was analyzed by IB for the indicated proteins. D, E, F, and G. Armet is localized in the ER and Golgi. D. Armet colocalizes with Hrd1, an ER resident ubiquitin ligase. HeLa cells were transfected with plasmid encoding Hrd1. Transfected cells were then processed for double immunofluorescent staining of Armet (green) and Hrd1 (red). E. Colocalization of Armet (green) and Golgin-97 in the juxta-nuclear region of HeLa cells. F and G. Treatment with BFA but not tunicamycin causes accumulation of Armet in the periphery of HeLa cells. HeLa cells were treated with BFA (C) or tunicamycin (D) for 5 h before processing for staining for endogenous Armet and Golgin-97 as a Golgi marker. H. Armet is not colocalized with mitochondrial marker. Live HeLa cells were labeled with red mitotracker for 15 min and then fixed and stained with anti-Armet antibody and Alexa Fluor-488.

Fig. 4 –

Armet and Armet-L1 are secreted proteins. A. Armet is a secreted protein. HEK293 cells were transfected with pCIneo-Armet-6xHis. Twenty-four hours after transfection cells were treated overnight with tunicamycin (2.5 μg/ml), Brefeldin A or grown in glucose-free medium. Then, the conditioned medium was harvested and precipitated using nickel-agarose beads. Armet-6xHis in precipitates was determined by IB using anti-6xHis antibody. Arrowhead indicates a non-specific band pulled down by nickel-beads, because it also presents in non-transfected control (lane 6). B. Endogenous Armet is a secreted protein. Conditioned medium was collected from HeLa cells after 6 h. Medium was concentrated using Amicon-4 Ultra centrifugal filter device. Equal volume of control and conditioned medium was analyzed by IB with anti-Armet antibody.C. Armet-L1 is a secreted protein. Experiment was done similarly to a. Intracell = Intracellular.

Fig. 5 –

Armet knockdown increases cell proliferation. A. siRNA efficiently silences Armet protein expression. Control or two siRNA oligos (siRNA1 and siRNA2) targeting Armet were transfected into HeLa cells. Transfected cells were harvested at days 2 and 3 post-transfection and Armet expression was determined by IB. Actin was blotted as loading control. B. Armet knockdown increases cell proliferation. HeLa cells were transfected with siRNA1, siRNA2 or control oligos. Twenty-four hours after transfection, equal numbers of transfected cells from each group were seeded in culture. After an additional 2 days in culture, cell numbers were counted. The results were expressed as mean ±SD, n=3.

Fig. 6 –

Transient knockdown of Armet decreases cell size, changes cell morphology, and renders cells more susceptible to ER stress-induced cell death. A. Armet knockdown cells are more sensitive to ER stress-induced cell death. Armet silencing was done as described in Fig. 5. At day 3 cells were treated for 24 h with tunicamycin or thapsigargin and the images were taken under phase contrast microscope. A and B. Armet knockdown changes cell morphology from polygon-like to flattened and round shape. At day 3 of Armet knockdown, 200 cells from each group were counted and separated into polygon-shaped and flattened-round morphology. The relative percentages of these two morphologies were expressed in the graph. C. Armet knockdown decreases cell size. At day 3 of Armet knockdown, cells were processed for flow cytometry as described in the Materials and Methods. The X-axis indicates relative cell size. Cell size distribution curve for the control is shaded in gray and the Armet-targeting siRNA curve is not shaded.

Fig. 7 –

Armet knockdown causes UPR that is aggravated by ER stress inducers. Armet knockdown causes UPR (lane 1 vs. 4 and 7) that is enhanced by the treatment with tunicamycin (lanes 5, 8) and thapsigargin (lanes 6, 9). Armet was knocked down in HeLa cells using siRNA1 and 2 as described in Fig. 6. At day 3 cells were treated with tunicamycin (2.5 μg/ml) or thapsigargin (500 nM) for 24 h. The indicated proteins were examined by IB.

Fig. 8 –

Overexpression of Armet inhibits cell proliferation and protects cells from ER stress-induced death. A. Armet protein overexpression. Control or two overexpression clones that stably express Armet were established in U2OS cells. Armet expression was determined by IB. GAPDH was blotted as loading control. B. Armet overexpression inhibits cell proliferation. Control and two clones (#2 and 11) that overexpress Armet were established by lentiviral vector-mediated transfection. C. Armet-overexpressing cells are resistant to cell death. Control and clone 2 and 11 cells were cultured under glucose-free condition (upper panel) or treated with tunicamycin (lower panel) for 17 h.