TMEM33: a new stress-inducible endoplasmic reticulum transmembrane protein and modulator of the unfolded protein response signaling
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Endoplasmic reticulum (ER) stress leads to activation of the unfolded protein response (UPR) signaling cascade and induction of an apoptotic cell death, autophagy, oncogenesis, metastasis, and/or resistance to cancer therapies. Mechanisms underlying regulation of ER transmembrane proteins PERK, IRE1α, and ATF6α/β, and how the balance of these activities determines outcome of the activated UPR, remain largely unclear. Here, we report a novel molecule transmembrane protein 33 (TMEM33) and its actions in UPR signaling. Immunoblotting and northern blot hybridization assays were used to determine the effects of ER stress on TMEM33 expression levels in various cell lines. Transient transfections, immunofluorescence, subcellular fractionation, immunoprecipitation, and immunoblotting were used to study the subcellular localization of TMEM33, the binding partners of TMEM33, and the expression of downstream effectors of PERK and IRE1α. Our data demonstrate that TMEM33 is a unique ER stress-inducible and ER transmembrane molecule, and a new binding partner of PERK. Exogenous expression of TMEM33 led to increased expression of p-eIF2α and p-IRE1α and their known downstream effectors, ATF4-CHOP and XBP1-S, respectively, in breast cancer cells. TMEM33 overexpression also correlated with increased expression of apoptotic signals including cleaved caspase-7 and cleaved PARP, and an autophagosome protein LC3II, and reduced expression of the autophagy marker p62. TMEM33 is a novel regulator of the PERK-eIE2α-ATF4 and IRE1-XBP1 axes of the UPR signaling. Therefore, TMEM33 may function as a determinant of the ER stress-responsive events in cancer cells.
KeywordsTMEM33 Endoplasmic reticulum stress and unfolded protein response PERK IRE1α Caspase-7 Autophagy Breast cancer
Activating transcription factor 4
Activating transcription factor 6
Cytosolic domain of ATF6
C/EBP(CCAAT/enhancer-binding protein) homologous protein
Eukaryotic translation initiation factor 2α
Glucose-regulated protein 78
Inositol-requiring enzyme 1α
Microtubule-associated protein 1 light chain 3
Protein kinase RNA-like ER kinase
Transmembrane protein 33
Unfolded protein response
X-box binding protein 1
Active (spliced) XBP1
The endoplasmic reticulum (ER) is involved in several fundamental cellular processes including synthesis and sorting of secretory and membrane proteins, detoxification, and intracellular calcium homeostasis . Correct folding of proteins in the ER lumen is regulated by folding and oxidizing enzymes in the presence of chaperones and glycosylating enzymes dependent on ATP and high Ca2+ levels. Misfolded proteins are exported to the cytoplasm for proteosomal degradation by a process known as the ER-associated degradation (ERAD) . ER stress, as defined by the accumulation of misfolded or unfolded proteins above the threshold levels in the ER lumen, leads to activation of an ER-to-nucleus unfolded protein response (UPR) signaling cascade. The ER transmembrane proteins protein kinase RNA-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α) and activating transcription factor 6 (ATF6α/β) sense ER stress, each then regulating one of the three distinct axes of the UPR signaling cascade . ER stress-activated PERK phosphorylates serine 51 of eukaryotic translation initiation factor 2α (eIF2α), followed by the suppression of protein synthesis and a selective increase in ATF4 activity. Downstream effectors of PERK signaling include both pro-survival factors such as the transcription factor NRF2 and microRNA miR-211, which are associated with adaptation response, and pro-apoptotic factors including CHOP that can induce cell death [4, 5]. Stress-induced phosphorylation and activation of IRE1α results in activation of the transcription factor X-box binding protein 1 (XBP1) and increased expression of ER chaperones such as GRP78/BiP which, in turn, increase protein folding capacity in the ER lumen. Activated IRE1α also reduces protein load in the ER lumen by cleaving mRNAs encoding secretory and membrane proteins through regulated IRE1α-dependent decay (RIDD). In the ATF6α/β arm of the UPR signaling, ATF6α/β is transported to the golgi where it undergoes cleavage; cleaved ATF6 (ATF6f/ATF6c) functions as a transcription factor for several ER chaperones.
Mechanisms underlying regulation of PERK, IRE1α, and ATF6α/β, and how the balance of these activities determine outcome of the activated UPR, remain largely unclear. Indeed, depending on the acute or chronic ER stress and cellular context, activation of the UPR may lead to apoptotic cell death, senescence, autophagy, oncogenesis, metastasis, and/or resistance to chemotherapeutics and endocrines [6, 7, 8, 9, 10, 11, 12, 13, 14]. Identification of new molecules regulating UPR signals may advance understanding of the mechanistic and functional significance of UPR in cancer biology and therapy.
Here, we report characterization of transmembrane protein 33, TMEM33 (also known as SHINC-3) as a novel ER stress-inducible and ER transmembrane molecule and regulator of two main drivers of the UPR: PERK and IRE1α. Our data show that TMEM33 is a new binding partner of PERK. TMEM33 overexpression led to increased expression levels of both p-eIF2α and p-IRE1α and of their respective downstream effectors, ATF4 and XBP1-S in breast cancer cells. TMEM33 overexpression also led to increased expression of CHOP, cleaved caspase-7, and the autophagosome marker LC3II in these cells. Collectively, this work provides new mechanistic insights into the regulation of PERK and IRE1α signaling pathways via TMEM33 in cancer cells.
Materials and methods
Antibodies, reagents, and chemicals
Rabbit polyclonal antibody was custom generated against a TMEM33-specific peptide, KKVLDARGSNSLPLLR (amino acids 127–143; Covance Research Products Inc., Denver, PA). Polyclonal anti-GAPDH antibody (2275-PC-1) was purchased from Trevigen (Gaithersburg, MD, USA). Monoclonal anti-α-tubulin antibody (TU-02), monoclonal anti-Myc antibody (9E10), monoclonal horseradish peroxidase-conjugated anti-cMyc antibody (9E10HRP), polyclonal anti-PERK antibody (H-300), polyclonal anti-GRP78/BiP antibody (C-20), polyclonal anti-IRE1 antibody (H-190), polyclonal anti-Calnexin antibody (C-20), monoclonal anti-PARP antibody (F-2), polyclonal anti-ATF4 antibody (H-290), polyclonal anti-ATF-6α antibody (H-280), monoclonal anti-Cyclin D1 antibody (sc-20044); polyclonal anti-β-actin antibody (sc-1616) and Protein A/G PLUS-Agarose immunoprecipitation reagent were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal anti-COX4 antibody (12C4) was obtained from Molecular Probes (Carlsbad, CA, USA). Monoclonal anti-Myc antibody (2276), monoclonal anti-PERK antibody (5683), polyclonal anti-phospho-eIF2α (Ser51) antibody (9721), polyclonal anti-eIF2α antibody (9722), monoclonal anti-phospho-eIF2α antibody (3597), monoclonal anti-ATF4 antibody (11815), monoclonal anti-LC3II antibody (12741), monoclonal anti-CHOP antibody (2895), polyclonal anti-cleaved-caspase-7 antibody (9491), polyclonal anti-caspase-7 antibody, and polyclonal anti-cleaved PARP antibody were all purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Additional antibodies and reagents used were as follows: monoclonal anti-ATF6 antibody (IMG-273, Imagnex); polyclonal anti-XBP1 antibody (GWB-BACB31, Genway); polyclonal anti-phospho-IRE1α antibody (PA1-16927, Thermoscientific); monoclonal anti-p62 antibody (610832, BD-Bioscience); FITC-conjugated monoclonal anti-Calnexin antibody (BD Transduction Laboratories); horseradish peroxidase-conjugated mouse and rabbit secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ, USA); Lipofectamine 2000, Lipofectamine LTX, and Lipofectin (Invitrogen Life Technologies, Carlsbad, CA, USA); Fu Gene HD (Roche); proteinase inhibitor cocktail tablets (Roche Diagnostics, Indianapolis, IN, USA); ECL Plus Western blotting detection system (Amersham Biosciences); Coomassie protein assay reagent and Surfact-Amps NP-40 (Pierce Biotechnology, Inc., Rockford, IL, USA); Tween 20 (Bio-Rad Laboratories, Inc., Hercules,CA, USA); Re-Blot plus mild antibody stripping solution (Chemicon International, Inc., Temecula, CA, USA); Restore Western blot stripping buffer (21059, ThermoScientific), and thapsigargin, tunicamycin from Streptomyces sp., dimethyl sulphoxide Hybri-Max Sterile filtered (DMSO), and etoposide (Sigma-Aldrich, St. Louis, MO, USA).
Cell lines and cultures
MCF-7 human breast cancer cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and Tissue Culture Shared Resource of the Georgetown Lombardi Comprehensive Cancer Center. HEK293T human embryonic kidney cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Human prostate cancer cells (PC-3 and DU-145), breast cancer cells (MDA-MB231), HeLa, and COS-1 cells were obtained from the Tissue Culture Shared Resource of the Georgetown Lombardi Comprehensive Cancer Center. All cell lines were grown as monolayers in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen Life Technologies, Carlsbad, CA, USA) supplemented with 5 or 10 % heat-inactivated fetal bovine serum. Endocrine-sensitive (LCC1) and endocrine-resistant breast cancer cells (LCC9), and antiestrogen-resistant MCF-7 cells (MCF7RR) were obtained and established as reported earlier [15, 16, 17]. LCC1, LCC9, and MCF7RR cells were maintained in IMEM without phenol red and supplemented with 5 % charcoal-stripped calf serum (CCS). All cell cultures were maintained at 37 °C under 95 % relative humidity and 95 % O2:5 % CO2 atmosphere.
Construction of Myc-TMEM33 expression vector
TMEM33 cDNA (741 bp) was amplified by RT-PCR using total mRNA from human testes (Ambion, Foster City, CA) and cloned into the pCR2.1 vector (Invitrogen). N-terminal Myc-tagged TMEM33 ORF (771 bp) was amplified by PCR using TMEM33 in pCR2.1 as template. The forward primer sequence containing the translation initiation codon, the Myc epitope (underlined), and Bgl II primer (bold) was 5′-GAGATCTGCCATGGAGCAGAAACTCATCTCTGAAGAGGACCTGATGGCAGATACGACCCCGAAC-3′, and the reverse primer sequence containing the MulI primer (bold) was 5′-GACGCGTCTATGGAACTGTTGGTGCC -3′ as described earlier . The PCR conditions were as follows: 95 °C for 4 min; 40 cycles of denaturation at 94 °C for 30 s; annealing at 65 °C for 1 min; extension at 72 °C for 1 min; and a final extension at 72 °C for 5 min. The amplified product was subjected to electrophoresis in 1 % agarose gels and cloned into the pCR3.1 expression vector. TMEM33 cDNA sequence was verified by automated DNA sequencing of both strands using vector-based forward and reverse primers as detailed earlier [18, 19].
Transient cDNA transfections
COS-1, HEK-293T, and PC-3 prostate cancer cells were transiently transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). HeLa cells were transiently transfected using FuGene HD (Roche), and MCF-7 and MDA-MB231 breast cancer cells were transiently transfected using Lipofectamine LTX (Invitrogen) as described in Supplementary Materials and methods.
Immunofluorescence and immunostaining
COS-1 cells were grown overnight on coverslips placed in a six well plate, one coverslip/well. Approximately, 3 × 104 cells were seeded/well. Next day, cells were transfected with 1 µg of Myc-TMEM33 or empty vector using Lipofectamine 2000. Forty eight hours post-transfection, the medium was removed and cells were immediately fixed in 3.7 % paraformaldehyde, followed by immunofluorescence and immunostaining using various antibodies as described in Supplementary Materials and methods.
Approximately, 5 × 106 MCF-7 cells were seeded per 150 mm tissue culture dish. Next day, the cells were collected by trypsinization and washed once with ice-cold phosphate-buffered saline (PBS). The cytosolic, mitochondrial (heavy membrane), microsomal (light membrane), and nuclear fractions were isolated as described in Supplementary Materials and methods.
Immunoprecipitation and immunoblotting
The whole cell lysate (approximately 2 mg protein) was incubated with 25 μL of agarose-conjugated anti-Myc antibody on a rotator at 4 °C overnight. The antibody-conjugated agarose beads were washed 1x in cell lysis buffer and used for immunoblotting as reported earlier  and detailed in Supplementary Materials and methods.
Thapsigargin and tunicamycin treatments
Stock solutions of thapsigargin (TG, 2 mM) and tunicamycin (TU, 2 mg/mL) were made in DMSO and stored at −20 °C. Cells from approximately 80 % confluent monolayers were used. The culture medium was removed and fresh DMEM containing 10 % FBS and the desired final concentration of TG or TU was added to the cells and incubation continued for various periods, followed by cell lysis and Western blotting as described in Supplementary Materials and methods.
TMEM33 is a novel endoplasmic reticulum transmembrane protein
Subcellular localization of the endogenous TMEM33 protein was examined by cell fractionation and immunoblotting using a custom-generated rabbit polyclonal antibody against a TMEM33-specific epitope (aa 127–143). The anti-TMEM33 antibody recognized an approximately 28 kDa protein in human prostate cancer cells (PC-3 and DU-145); pre-immune serum had no immune reactivity at the corresponding location (Supplementary Fig. 2a). The anti-TMEM33 antibody was further validated by sequential immunoblotting of cell lysates from COS-1 Myc-TMEM33 transfectants with anti-Myc and anti-TMEM33 antibodies. These two antibodies recognized an overlapping band at ~28 kDa in COS-1 transfectants (Supplementary Fig. 2b). Using the custom-generated anti-TMEM33 antibody, human endogenous TMEM33 (~28 kDa) was detected in several human cancer cell lines including A549, Aspc-1, Colo-357, MDA-MB435, MCF-7, and HeLa cells (data not shown). In the combined cell fractionation and immunoblotting assay, Calnexin was detected in both the heavy membrane pellet (HM) and the ER-containing microsomal fractions (MS) of MCF-7 cells; heavy membrane pellets contain mitochondrial, lysosomal, and ER-resident proteins. Similar to Calnexin, endogenous TMEM33 expression was seen in both the HM and MS fractions of MCF-7 cells but not in the nuclear or cytosolic fractions (Fig. 3c). Together with the predicted 2D topology of TMEM33 [26, 27] (Fig. 3d) these data establish TMEM33 as a novel ER transmembrane resident protein.
TMEM33 is an ER stress-inducible molecule
TMEM33 overexpression is associated with enhanced stimulation of the ER stress-responsive PERK/p-eIF2α/ATF4 signaling pathway
TMEM33 overexpression and increased expression of ER stress-induced cell death signals
TMEM33 overexpression and constitutive activation of the PERK-p-elF1α-ATF4 and IRE1α-XBP1 axes of the UPR signaling and autophagy in breast cancer cells
To address the consequences of TMEM33 overexpression in human cancer cells, MCF-7 breast cancer cells were transiently transfected with either the Myc-TMEM33 or pCR3.1 vector, followed by immunoblotting of the cell lysates with antibodies against various components of the UPR signaling pathways. Figure 7 shows that exogenous expression of TMEM33 was sufficient to increase basal levels of p-eIF2α, ATF4, CHOP, cleaved caspase 7, p-IRE1α, and XBP1-S but not cleaved ATF6. In addition, cyclin D1 expression, a marker of cell cycle progression and oncogenesis, was decreased in Myc-TMEM33 transfectant MCF-7 cells. The latter observations seem to be inconsistent with data showing high TMEM33 expression in several tumor tissues including breast cancer (Supplementary Fig. 5). Moreover, TMEM33 is reported to be amplified in approximately 10 % of breast cancer patient xenografts [28, 29, 30]. During autophagy, microtubule-associated protein-1 light chain 3 (LC3I) is converted to its membrane-bound form (LC3II). We have previously reported that overexpression of XBP1 can regulate autophagy through its control of BCL2 [31, 32, 33]. Increased expression of LC3II and decreased expression of p62/sequestosome 1 (SQSTM1), ubiquitin-binding protein and promoter of apoptosis, was detected in Myc-TMEM33 transfected MCF-7 cells relative to controls (Fig. 7). Similar observations were made in hormone refractory MDA-MB-231 breast cancer cells (Supplementary Fig. 6). Hence, TMEM33 overexpression is associated with constitutive activation of PERK-p-elF1α-ATF4 and IRE1α-XBP1-S signaling and autophagy in breast cancer cells.
How TMEM33 expression is regulated remains unknown. The proximal promoter region of the TMEM33 gene shows putative binding site for NRF-2, a prosurvival transcription factor downstream of the activated PERK (Supplementary Fig. 1c). It is tempting to speculate that an as yet unknown combination of feedback and cross-talk exists. For example, where ER stress activates PERK and increases TMEM33 expression via NRF-2, and increased TMEM33 overexpression, depending on the cell type, promotes PERK and IRE-1α activated signaling. TMEM33 may also interact with other proteins. At least one phosphorylation site (Thr 65) and two ubiquitination sites (Lys 148 and Lys 221) are predicted within the open reading frame of TMEM33 (Supplementary Fig. 1b). TMEM33 may be a member of the family of ubiquitin-modified proteins [38, 39, 40]. The STRING database  also predicts potential interaction of TMEM33 with ubiquitin C (UBC), and ubiquitin specific peptidase 19 (USP19), associated with ubiquitin-dependent proteolysis (Supplementary Fig. 8). TMEM33 may also interact with the retention in endoplasmic reticulum 1 protein (RER1), which is involved in the retrieval of ER membrane proteins from the early golgi compartment (Supplementary Fig. 8).
Present report supports the hypothesis that TMEM33 may function as a multi-faceted molecule in cancer cells. While our data suggest that TMEM33 overexpression correlates with enhanced expression of apoptotic signals (CHOP and cleaved caspase7), constitutive expression of TMEM33 was found to be high in a limited number of endocrine-resistant breast cancer cells and in early recurrent breast cancer specimens tested. The latter observations are consistent with the roles of PERK and ATF4 in tumor cell survival . Moreover, autophagy may increase survival of tumor cells [8, 9]. We observed increased expression of autophagosome marker LC3II and downregulation of p62/sequestosome 1 (SQSTM1) in breast cancer cells overexpressing TMEM33, suggesting induction and completion of autophagy. In conclusion, TMEM33 offers a new regulatory mechanism of the UPR and may serve as a determinant of the outcome of activated UPR signaling cascade.
We thank Dr. Sona Vasudevan for analysis of TMEM33 profiles in The Cancer Genome Atlas (TCGA) and cBioPortal databases. This work was funded by NIH Grants CA68322, CA74175, CA149147, and CA184902 and NeoPharm, Inc. Several cell lines were obtained from the Tissue Culture Shared Resource of the Georgetown Lombardi Cancer Center. The RNA array expression profiling was performed at the Genomics and Epigenomics Shared Resource and the immunofluorescence imaging was performed at the Microscopy & Imaging Shared Resource of the Georgetown Lombardi Cancer Center. All shared resources were supported by the NIH Grant P30-CA51008.
Compliance with ethical standards
Conflict of interest
TMEM33 (alias SHINC-3) is a Georgetown University patented technology, “Gene SHINC-3 and Diagnostic and Therapeutic Uses Thereof,” US Patent # 7244565. IS and UK are co-inventors of this technology. RH, LJ and RC declare that they have no conflict of interest.
The authors declare that all experiments reported in this manuscript were performed in compliance with all current laws and regulations of the United States of America.
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