WWOXf cells undergo nuclear bubbling or BCD at room temperature and below. WWOXf cells undergo nuclear bubbling and death in response to UV or UV/cold shock11,12. A panel of WWOXf cells used for experiments were TNF-sensitive L929S fibroblasts, human breast MCF7 cells, monkey kidney COS7 fibroblasts, prostate DU145 cancer cells, and squamous cell carcinoma SCC9 and SCC15 cells14. When TNF-sensitive L929S fibroblasts were exposed to UV irradiation (480 mJoule/cm2) and then cold shock at 4oC for indicated times, the cells underwent time-related bubbling (see arrows for bubbles; Fig. 1a).
The aforementioned cells were exposed to UV only, UV then cold shock (4oC for 5 min), or cold shock then UV. Post treatment, these cells were cultured at 4, 10, 22, and 37oC, respectively, for indicated times (Fig. 1b-g). Nuclear bubbling of MCF7 cells occurred most effectively at 22oC or room temperature (Fig. 1b-d; Supplementary Video 1), and bubbling blocked at 37oC by greater than 70%. For L929S cells, 4oC was the best temperature for UV-mediated nuclear bubbling (Fig. 1e-g). In stark contrast, WWOXd MDA-MB-231, MDA-MB-231(NH) and metastatic MDA-MB-231 cells (IV-2-3) were UV irradiated and then cold shocked, and underwent POD at all indicated temperatures (Fig. 1h-j; Supplementary Video 2). Triple-negative breast MDA-MB-231 do not express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), whereas MCF7 is ER positive16.
Similarly, COS7 cells underwent UV/cold shock-mediated nuclear bubbling, and the bubbling reduced at 37oC (Supplementary Fig. 1a). In response to UV only or UV and then cold shock, WWOXd TNF-resistant L929R fibroblasts underwent pop-out explosion at 10 and 22oC, and the explosion retarded at 37oC (Supplementary Fig. 1b). Again, POD ocurred in MDA-MB-231(NH) and MDA-MB-231 (IV-2-3) cells upon exposure to UV only (Supplementary Fig. 1c). Similar results were observed when MDA-MB-231 cells treated with cold shock and then UV (Supplementary Fig. 1c). Temperature changes failed to alter the extent of POD (Supplementary Fig. 1c).
Failure in trypan blue exclusion and mitochondrial respiration during BCD. When cells start undergoing nuclear bubbling, nuclear permeability is increased as indicated by the presence of DAPI, but not propidium iodide (PI), in the nuclei11. When the bubble enlarges to a certain extent, PI uptake in the nuclei occurs, indicating that the cell is dead11. When COS7 cells were exposed to UV and incubated at room temperature for 2 hour, the cells failed to exclude trypan blue (see red arrows; Supplementary Fig. 2a). Similar results were observed in UV/cold shock-treated COS7 cells (Supplementary Fig. 2b).
Rapid loss of miotchondrial function occurred in UV-treated COS7 cells, as determined by MTT assay (Supplementary Fig. 2c). Dramatically functional loss occurred if UV-irradiated cells were cultured overnight at 37oC (Supplementary Fig. 2d). Unlike apoptosis, BCD is an irreverible cell death event11,12. COS7 cells were UV irradiated and then cultured at 4, 22, or 37oC for various durations. Subculture of these cells for overnight incubation at 37oC resulted in cell death by >90% (Supplementary Fig. 2e). Similar results were observed when these UV-treated cells were direclty cultured overnight at 37oC (Supplementary Fig. 2f). Rapid suppression of mitochondrial function was observed when the UV-treated COS7 cells were cultured at 4oC for 1-4 hr, compared to other temperatures (Supplementary Fig. 2g).
Next, COS7 cells were treated with betulinic acid (10 µM) or staurosporine (1 µM) for 1 hour to initiate mitochondrial apoptosis, then exposed to UV and cold shock (4oC for 5 min), and incubated at room temperature for indicated times. Compared to UV/cold shock-treated cells, pre-induction of mitochondrial apoptosis did not block BCD (Supplementary Fig. 2h). Pre-inhibition of caspases by caspase inhibitor I (z-VAD-FMK) or caspase inhibitor X (specific for caspase 3, 7, and 8) failed to block BCD (Supplementary Fig. 2h). Internucleosomal DNA fragmentation was not detected in UV/cold shock-treated cells during incubation at RT (Supplementary Fig. 2i). When L929S, but not COS7, were exposed to UV and then culturing at 37oC for 8 hr, DNA fragmentation occurred (Supplementary Fig. 2j, k). As positive controls, staurosporine at 1 µM induced DNA fragmentation at 37oC but not at RT or 4oC (Supplementary Fig. 2j, k). Failure of UV-treated COS7 to undergo DNA fragmentation during incubation for 8 hr at 37oC was probably due to insufficient incubation time.
Inhibitors of necroptosis fail to block UV/cold shock-induced BCD in WWOXf cells and POD in WWOXd cells
When L929S cells were pretreated with a necroptosis inhibitor Nec-1 (30 µM) for 1 hr and then exposed to UV (480 mJoule/cm2 ) and cold shock for 5 min at 4o C, the treated cells exhibited bubbling in 1.5 hr (Supplementary Fig. 3; Supplementary Table I). Similar results were observed by using a protein synthesis inhibitor actinomycin D (ActD; Supplementary Table I).
Supplementary Table I shows the detailed characteristics of BCD and POD, including cell shrinkage, release of nucleolar contents, nuclear bubbling, and release of extracellular vesicles. Release of nucleolar content occurred just prior to bubbling from the nucleus (Supplementary Table I; Supplementary Video 1). ActD delayed the release of nucleolar content. Pretreatment of L929S cells with necroptosis inhibitors Nec-1 and GSK’872 for 30 min failed to abolish the nuclear bubbling (Supplementary Fig. 3a). Under similar conditions, L929R cells underwent POD without nuclear bubbling (Supplementary Fig. 3b). Also, Nec-1 had no effects (Supplementary Fig. 3b; Supplementary Table I). Oral squamous cell carcinoma SCC15 and SCC9 are WWOXf cell lines7,14. These cells underwent BCD in response to UV or UV/cold shock (Supplementary Table I). ActD and β-mercaptoethanol (10%) abolished UV/cold shock-mediated release of nucleolar content.
Antioxidant U74389G suppresses BCD in WWOXf cells at RT
UV/cold shock induces translocation of nitric oxide synthase 2 (NOS2) to the nucleus to generate nitric oxide (NO) for causing nuclear bubbling11,12. To further elucidate the nuclear bubbling, SCC15 cells were pretreated with EGTA (1 mM), proteasome inhibitor MG132 (30 µM), p53 activator Prima-1 (30 µM), CHK2/ATM inhibitor (30 µM), or antioxidant U74389G (30 µM) for 30 min. These cells were exposed to UV irradiation (480 mJoule/cm2) or UV/cold shock (5 min at 4oC). By time-lapse microscopy at room temperature, U74389G strongly suppressed the nuclear bubbling (Supplementary Fig. 4). EGTA, a calcium ion chelator, also retarded the occurrence of BCD (Supplementary Fig. 4). Antioxidant U74389G is an inhibitor of free radical production and lipid peroxidation and caspase 117,18.
Dramatic increases in cellular thickness or height during apoptosis, but not BCD, as determined by time-lapse holographic microscopy
Holographic microscopy allows visualization of three-dimensional changes of cells undergoing division, apoptosis and others19. When COS7 or L929S cells were exposed to UV and then cold shock at various durations at 4oC, cell bubbling were observed (Fig. 2a; Supplementary Videos 3, 4). The thickness or heights of cell bubbles were relatively low in COS7 and L929S cells during BCD (less than 2 µm; Fig. 2b, c; Supplementary Video 5). When L929S cells were treated with staurosporine and cultured at 37 oC, cells underwent apoptosis, which showed shrinkage and increased whole cell thickness reaching ~12 µm (Fig. 2d; Supplementary Video 6). In comparison, when UV/cold shock-treated L929S cells were cultured at 37oC, apoptosis occurred and the cell bubble heights reached ~15 µm (Supplementary Video 7). During cell division, L929S cells reached as tall as ~17 µm (Supplementary Videos 8, 9). Thus, during BCD, there were brief increases in cell volumes followed by reduction, and cell areas remained relatively stable (Fig. 2e). When cells underwent apoptosis, cell areas and volumes were reduced. However, cells became taller, which lasted for 5-8 hr (Fig. 2e).
UV induces WWOXf cells to rapidly lose mitochondrial membrane potential and meanwhile increase the production of nitric oxide (NO)
UV treatment leads WWOXf cells to undergo nuclear bubbling and calcium (Ca2+) influx and eventual death, whereas UV causes WWOXd cells to explode in 30 min with a poor efficiency in Ca2+ influx14. By using live cell dyes for time-lapse microscopy (Fig. 3), UV-treated L929S cells exhibited 1) rapid loss of mitochondrial membrane potential (using MitoTracker Red) (Fig. 3a, b, e, f), 2) reduction in cell survival from low retention of calcein dye (Fig. 3b), 3) rapid increase in the production of ROS (Dichlorofluorescein dye) (Fig. 3a) and NO (DAF-fm diacetate dye) (Fig. 3c), and 4) relatively low in stress fiber formation (Phalloidin green dye) (Fig. 3e). UV-mediated bubbling initiation occurred approximately in 30 min in L929S cells, whereas whole cell explosion occurred in L929R in less than 30 min. UV induced L929R cells to form stress fibers after whole cell explosion in less than 30 min, but not in L929S cells (Fig. 3e). Many other WWOXd cells were readily to form UV-induced stress fibers, whereas WWOXf cells failed to do so. No apparent changes were shown with intracellular utilization of ATP (BioTracker ATP-Red Live Cell Dye), incorporation of NTP (BioTracker Red NTP Transporter), and changes in cell stemness (BioTracker 529 Green Pluripotent Stem Cell Dye) in both L929S and L929R cells (Fig. 3d). NO levels were 2.5-fold higher in L929S than in L929R cells (Fig. 3c).
Calcein retention for measuring survival was better in WWOXf L929S (Fig. 3b) and lung WWOXf H441 cells (Fig. 3h), but not in WWOXd L929R (Fig. 3b) and lung H661 cells (Fig. 3h). UV-induced Ca2+ influx (Fluo-8 dye) was shown in WWOXf H441 (Fig. 3g) and NCI-H1299 cells (Fig. 3i), but not in WWOXd H661 (Fig. 3g) and PC9 cells (Fig. 3i). Next, compared to non-sphere areas in the 4T1 monolayers, 4T1 spheres had higher levels of ROS (Fig. 3j. left panel) and stemness (Fig. 3k), but not NO (Fig. 3j, right panel). A typical 4T1 sphere is shown (Fig. 3l). Finally, DAPI (nuclear dye) uptake occurred in a time-related manner with similar kinetics in all cells (Fig. 3).
p53, WWOX and p38, but not TβRI, participate in UV-mediated nuclear bubbling and calcium influx in WWOXf cells
UV or UV/cold shock initiates nuclear bubbling and calcium influx, which takes approximately 30 min in WWOXf cells (e.g. L929S) at room temperature14. We examined how both events occur in WWOXf HCT116 cells. In the control experiments (Fig. 4a1-3), HCT116 cells were cultured overnight and added a 100 µl aliquot of medium, PBS, or normal rabbit serum (1:250 dilution). UV caused the initiation of nuclear bubbling and calcium influx in approximately 30 min at room temperature (Fig. 4a1-3). Calcium influx reached maximally in about an hour, followed by reduction with time (Fig. 4a1-2). In contrast, normal rabbit serum sustained the increase in calcium influx with time (Fig. 4a3).
When HCT116 cells were pretreated with calcium chelator EGTA at 200 µM for 1 hr, calcium influx was not effectively blocked and the first bubbling time was prolonged to approximately 48 min (Fig. 4a4). To be effective, greater than 1 mM EGTA was needed to achieve 50% inhibition of calcium influx. As an inhibitor of NO synthase, Nω-nitro-L-arginine methyl ester hydrochloride (Nω-LAME) at 300 µM retarded the nuclear bubbling but had no effect on calcium influx (Fig. 4a5), suggesting that NO is needed for bubbling but not for calcium influx. To suppress >50% nuclear bubbling, 1 mM Nω-LAME is needed11.
We examined whether WWOX and its binding protein partners such as p53, type II TGFβ receptor (TβRII) and Hyal-2 participate in calcium influx and nuclear bubbling20−25. p53 wild type and isoforms such as p53β and p53γ have been implicated in cancer progression26,27. Firstly, we examined the role of p53 and isoforms (Fig. 4b1-4). When HCT116 cells were pretreated with an aliquot of antiserum against p53 (1 µg/ml) followed by exposure to UV, the antibody did not block nuclear bubbling but partially inhibited calcium influx (Fig. 4b1). In contrast, polyclonal p53 antibody (1 µg/ml) was potent in blocking both events (Fig. 4b2). In contrast, p53β or p53γ antibody retarded the nuclear bubbling but not calcium influx (Fig. 4b3-4). The observations suggest that wild type p53 participate in both nuclear bubbling and calcium influx, whereas p53β and p53γ are needed for nuclear bubbling.
Secondly, we investigated the role of TGFβ receptor (TβR) on both aforementioned events (Fig. 4c1-3). HCT116 cells are deficient in type II TGFβ receptor (TβRII)28. HCT116 cells were pretreated with a chemical inhibitor for TβRI for 30 min and then UV exposure. The treatment did not result in inhibition of calcium influx and nuclear bubbling (Fig. 4c1). Also, monoclonal antibody against TβRII did not alter UV-mediated responses in HCT116 cells (Fig. 4c2). The chemical inhibitor SD208 for TβRI had no apparent inhibitory effects (Fig. 4c3).
Thirdly, we examined the effect of WWOX protein on the UV-mediated cellular responses (Fig. 4d1-13). WWOX7-21 and WWOX7-11 peptides enhance ceritinib-mediated breast cancer cell death24,25. When HCT116 cells were pretreated with either WWOX7-21 or WWOX7-11 peptide (20 µM) for 30 min and then exposed to UV, there were no apparent changes in nuclear bubbling and calcium influx (Fig. 4d1-2). Similar results were observed using mutant peptides WWOX7-11(A7R) and WWOX7-11(G7R) (Fig. 4d3-4). Three antibodies against indicated different regions of WWOX at amino acid 7-21, 28-42 and 286-299, respectively, have been made20–22, 29. These antibodies retarded calcium influx but had no effect on nuclear bubbling (Fig. 4d5-7). Antibodies against WWOX phosphorylation sites, including pY33, pT12 and pS14-WWOX, strongly blocked calcium influx, whereas pY34 and pY287-WWOX antibodies were partially effective (Fig. 4d8-12). pS14-WWOX strongly retarded nuclear bubbling (Fig. 4d11). Antibody against isoform WWOX2 suppressed calcium influx but failed to block nuclear bubbling (Fig. 4d13). The observations suggest that when WWOX is Ser14 phosphorylated, it is potent in blocking nuclear bubbling and calcium influx.
Finally, antisera against Hyal-2 and pY216-Hyal-2 did not effectively block nuclear bubbling and calcium influx (Fig. 4e1-4). Membrane Hyal-2 acts as a cognate receptor for both TGF-β1 and hyaluronan to signal together with WWOX and Smad4 for exerting growth enhancement or suppression13,29. pY216-Hyal-2 antibody is potent in blocking cancer growth24. Intriguingly, p38 inhibitor SB203580 strongly suppressed both nuclear bubbling and calcium influx (Fig. 4f1).
Taken together, when HCT116 cells are exposed to UV, p53, WWOX and p38 participate in UV-mediated nuclear bubbling and calcium influx. In contrast, Hyal-2 and TβRI are not effective.
Ectopic WWOX restores calcium influx and nuclear bubbling in WWOXd cells in response to UV exposure
4T1 cancer cells were transiently overexpressed with an indicated WWOX construct using liposome-based Genefector and cultured for 48 hr. The cells were UV irradiated and then imaged by time-lapse microscopy at room temperature. The full-length WWOX restored calcium influx in 4T1 cells (Supplementary Fig. 5a; Supplementary Videos 10, 11). The first WW domain WW1 had a low activity in restoring calcium influx, and Y287F and Y61R mutants abolished the activity of endogenous WWOX (Supplementary Fig. 5a). 4T1 is a WWOXd cell line due to its failure in UV-mediated calcium influx14. At 200 µM, L-arginine failed to increase calcium influx and nuclear bubbling. L-arginine is needed for the synthesis of NO. In MDA-MB-231 cells, intracellular WWOX levels are fairly low16. Compared to other isoforms, WW1 was the most effective in restoring nuclear bubbling (or BCD) but failed to induce calcium influx (Supplementary Fig. 5b). Other indicated forms failed to restore calcium influx but enabled nuclear bubbling in MDA-MB-231 cells.
UV/cold shock induces global downregulation of cellular and housekeeping proteins
We examined whether UV/cold shock affects the protein expression in cells. COS7 cells were exposed to UV irradiation and subsequent incubation at 4℃ for indicated times. Dramatic and rapid downregulation of house-keeping proteins α-tubulin and β-actin was observed (Fig. 5a; >70% for α-tubulin and >30% for β-actin). Cortactin, a protein involved in cell shape and migration30, was downregulated rapidly in UV-irradiated COS7 cells (Fig. 5a; >90%). In response to UV/cold shock, the level of p53 was relatively unchanged (<10% reduction), whereas reduced p53 phosphorylation at S20 occurred in a time-related manner (Fig. 5a; >90% reduction in 2 hr).
Under similar conditions, L929S cells were exposed to UV and then cold shock, followed by incubation at specified temperatures and durations (Fig. 5b). UV/cold shock, or UV alone, drastically downregulated the expression of caspase 3, Bak and α-tubulin by greater than 90% in L929S cells at 4oC (Fig. 5b). Bcl-2, Bcl-x (Bcl-xL and Bcl-xS) and heat shock protein 70 (HSC70) were less affected (Fig. 5b; <10%). The reduction of caspase 3 may account for the failure of BCD-induced internucleosomal DNA fragmentation at room temperature or 4o C (Supplementary Fig. 2i-k). UV/cold shock-treated cells failed to migrate, which is mainly due to drastic loss of α-tubulin.
Relocation of α-tubulin and lamin B1 to the nucleus and bubble in UV/cold shock-treated L929S
By confocal microscopy, lamin B1 was shown to localize in the nuclear membrane, and α-tubulin in the cytoplasm of untreated L929S cells (Fig. 5c; top panel). When L929S cells were exposed to UV and cold shock, downregulation of α-tubulin was observed (Fig. 5c; bottom panel). Nuclear condensation occurred and lamin B1 was still in the nuclear membrane (Fig. 5c; bottom panel). Shown in the Fig. 5d (left panel) is a merge of confocal sections for nucleus (DAPI), lamin B1 and α-tubulin. UV/cold shock-treated L929S cells exhibited relocation of α-tubulin to the nuclear bubble (Fig. 5d, right panel; red arrows). In certain confocal sections, bubbles contained α-tubulin and lamin B1, and DAPI (Fig. 5d, right panel; white arrow). We have reported that the inner membrane of the bubble is from the nucleus, and the outer membrane from the cell membranea11,12. In stark contrast, α-tubulin did not relocate to the bubbles (yellow arrows) of COS7 cells during BCD (Fig. 5e; yellow arrows).
Accumulating evidence shows that α-tubulin is associated with apoptosis31,32. We examined whether α-tubulin undergoes morphological changes caused by UV exposure and temperature alterations. COS7 cells were exposed to UV (480 mJ/cm2), followed by culturing at 4 or 37℃ for 90 min. The UV/cold shock-treated cells underwent BCD (Supplementary Fig. 6). Cold shock shrank the cells and reduced the cell sizes (Supplementary Fig. 6). UV rapidly decreased the expression of α-tubulin, whereas cold shock had no effect (Supplementary Fig. 5a). UV/cold shock-treated cells had reduced levels of α-tubulin, and part of the α-tubulin relocated to the nucleus (Supplementary Fig. 6). At 37℃, the UV-treated cells underwent apoptosis, and α-tubulin did not relocate to the nuclei (Supplementary Fig. 6).
UV and/or cold shock suppress RNA transcription and shuts down mRNA processing machinery
L929S cells were exposed to UV and/or cold shock, followed by purification of whole cell total RNA20 and NanoDrop analysis for quality control (Fig. 6a). The concentration of total RNA was decreased by greater than 80% after UV irradiation. By reverse transcriptase-polymerase chain reaction (RT-PCR), mRNA expressions for p53, GAPDH, and β-actin were all dramatically decreased by greater than 95% after exposure to UV, cold shock, or UV/cold shock (Fig. 6a).
By gene chip analysis for mRNA expression profiling, we determined that downregulated genes are involved in mRNA processing and metabolic process and RNA splicing in L929S cells (Fig. 6b; Supplementary Fig. 7). Notably, upregulated genes included sensory perception of smell and chemical stimulus and G-protein coupled receptor protein signaling pathway (Fig. 6b-d). Specifically, microarray analysis showed that gene expression of glutathione peroxidase 2 (Gpx2) and eukaryotic translation initiation factor 2-alpha kinase 2 (Eif2ak2) were both increased after treated with UV/cold shock in L929S cells, respectively (Fig. 6c-d). By overlapping analysis, there are 12 upregulated genes and 4 downregulated genes (Fig. 6e). These regulated genes are: Gpx2 codes for glutathione peroxidase 2; Eif2ak2 for eukaryotic translation initiation factor 2-α kinase 2; Bpifa5 for BPI Fold Containing Family A Member 1; Ces3b for Carboxylesterase 3; Mir139 for MicroRNA 139; Olfr12 for Olfactory Receptor Family 5 Subfamily S Member 1 Pseudogene; Rps15 for Ribosomal Protein S15; Tcrb-J for T cell receptor β joining region; mt-Tm for Mitochondrially Encoded TRNA-Met (AUA/G). Gm10736, Gm11353, and Gm6613 do not code for proteins. Among the downregulated genes, Mbtps2 codes for Membrane Bound Transcription Factor Peptidase, and Sept7 for CDC10 Protein Homolog. Gm5446 and Gm5792 do not code for proteins.
UV and cold shock together rapidly suppress protein expression from skin to liver in the hairless mice
We investigated the effect of UV/cold shock in regulating protein expression in hairless mice33. Hairless mice were subjected to whole-body UV irradiation (960 mJ/cm2) and then subjected to temperature shock at -30o or 37o C for indicated times. Mice were then sacrificed. In the mouse skin, UV had little or no effect on the expression of β-actin, GPX1/234, PKR (protein kinase R; encoded by Eif2ak2 gene)35, α-tubulin, and TRAF211,12, but significantly upregulated the expression of WWOX (Fig. 7a-d; Supplementary Fig. 8a-l). Of particular interest is that UV upregulated WWOX phosphorylation at Ser14 (pS14-WWOX) by one-fold (Fig. 8k-l). pS14-WWOX enhances the progression of cancer and Alzheimer’s disease36−39. During cold shock of the animals at -30oC for 20 min, upregulation of the aforementioned proteins occurred (Fig. 7a-d; Supplementary Fig. 8a-l). However, the protein levels were significantly reduced when the mice were under cold shock for 40 min. When the mice were exposed to UV and then cold shock for 20 or 40 min, dramatic downregulation of the aforementioned proteins occurred. However, when UV-treated mice were kept at 37 C for 20 min, majority of protein expression remained largely unchanged, except that pS14-WWOX was significantly upregulated (Fig. 7a-d; Supplementary Fig. 8a-l).
In the mouse liver, UV rapidly suppressed the expression of β-actin (Fig. 7e-f), TRAF2 (Supplementary Fig. 9a), p53 and pS20-p53 (Supplementary Fig. 9f-g), and Bcl-x (Supplementary Fig. 9h). However, UV alone had no effect on the expression of GPX1/2 (Fig. 7g), PKR (Fig. 7h; Supplementary Fig. 9b), and WWOX and pS14-WWOX (Supplementary Fig. 9c, d) in the liver. In contrast, UV, cold shock, or UV/cold shock reduced the expression of pY33-WWOX in the liver (Supplementary Fig. 9e). When mice were exposed to cold shock (-30oC) for 30 min and then kept at 37o C for indicated times, significant downregulation of the aforementioned proteins were observed in the mouse liver (Fig. 7; Supplementary Fig. 9). pY33-WWOX was significantly increased in the liver post treatment with UV and 37o C for 15 min, followed by reduction in the next 15 min (Supplementary Fig. 9). Significant upregulation of pY33-WWOX in organs at 37o C is probably needed to exert apoptosis in vivo20–25, 40. Additionally, downregulation of p53, pS20-p53, and Bclx occurred when mice were subjected to UV and/or cold shock or exposure to 37o C (Supplementary Fig. 9f-h).
Collectively, when hairless mice were UV irradiated and cold-shocked, dramatic protein downregulation occurred in the skin. The signal was then transmitted to the liver to cause protein downregulation. The event may account for the pathologies found in patients with frostbite.
UV/cold shock induces nuclear accumulation of TRAF2/WWOX complex to regulate bubbling
We reported that antiapoptotic TRAF2 is dramatically downregulated during frostbite in human skin, and that TRAF2 counteracts with the function of WWOX and p53 in inducing BCD11,12. Here, we further elucidate the functional interactions between TRAF2 and WWOX during BCD. When L929S cells were exposed to UV (480 mJoule/cm2) and then cold shock at 4℃ for 15 min to 4 hr, expression of WWOX, TRAF2 and α-tubulin was downregulated by greater than 50% (Fig. 8a). Cold shock alone did not affect the expression of WWOX, TRAF2 and α-tubulin (Fig. 8a). pY33-WWOX levels were not reduced. Cold shock or UV/cold shock gradually reduced p53 expression (Fig. 8a).
An EGFP-TRAF2(124-233) construct was made for transient expression of the zinc finger domain in TRAF2. We established stable transfectants of L929S cells for expressing EGFP-TRAF2(124-233), ECFP-TRAF2, ECGP, or EGFP (Fig. 8b). EGFP-TRAF2(124-233) strongly suppressed UV/cold shock-induced BCD. The full-length ECFP-TRAF2 was not expressed and did not exhibit an inhibitory activity (Fig. 8b). Similar results were observed for the suppression of BCD when COS7 cells were stably transfected with EGFP-TRAF2(124-233) and L929S with ECFP-TRAF2 (Fig. 8c-d).
By Cytotrap yeast two-hybrid analysis13,20−22, we determined the positive binding of full-length WWOX with full-length TRAF2 (Fig. 8e). In positive controls, p53 physically bound WWOX and MafB underwent self-association (Fig. 8e). Additionally, we carried out Förster resonance energy transfer (FRET) microscopy13,29,40−42. There was an increased binding of EGFP-TRAF2 with DsRedM (monomeric DsRed)-WWOX in UV/cold shock-treated COS7 cells at 37oC (Fig. 8f). At low temperature (4oC), the EGFP-TRAF2/DsRedM-WWOX complex became dissociated in 60 min (Fig. 8f). By fluorescent immunostaining, UV/cold shock induced nuclear co-localization of endogenous WWOX with TRAF2 in COS7 cells (Fig. 8g). In the absence of WWOX, endogenous TRAF2 in Wwox−/− knockout MEF cells did not relocate to the nucleus in response to UV/cold shock (Fig. 8h), suggesting that TRAF2 requires WWOX to co-translocate to the nucleus.
By co-immunoprecipitation, UV/cold shock induced the dissociation of the endogenous WWOX/TRAF2 complex in 30 min in COS7 cells (Fig. 8i). TRADD, a TNF receptor adaptor protein43, physically bound TRAF2 without dissociation during the course of UV/cold shock (Fig. 8i). Dissociation of the TRAF2/WWOX complex correlates with WWOX dephosphorylation at Y287. pY287-WWOX is subjected to ubiquitination and proteasomal degradation44. In the absence of WWOX, UV/cold shock failed to induce translocation of cytosolic TRAF2 to the nucleus in the knockout Wwox−/− MEF cells (Fig. 8J). However, p53 underwent nuclear translocation (Fig. 8j). Overall, the zinc finger domain of TRAF2 blocked the function of WWOX in inducing BCD at room temperature. Both WWOX and TRAF2 form complex to relocate to the nucleus. Conceivably, dissociation of the complex at low temperature facilitates WWOX function in inducing NO production.