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TRIM21 is involved in the tumorigenesis of several types of tumors[12, 13, 19, 20]. In the present study, we found overexpression of HA-TRIM21 significantly increased U2-OS and Saos-2 osteosarcoma cell viability using MTT assays (Figure 1A-B). Meanwhile, overexpression of HA-TRIM21 protected U2-OS and Saos-2 cells from cytotoxicity in response to several stresses, including exposure to CDDP (30 μg/mL), TRAIL (50 ng/mL), and serum starvation for 24 hours (Figure 1A-B). By contrast, TRIM21 knockdown with siRNA significantly decreased U2-OS and Saos-2 cell viability (Figure 1C-D), as well as amplified cytotoxicity from the aforementioned stresses (Figure 1C-D). These results suggest TRIM21 was a positive regulator of U2-OS and Saos-2 cell proliferation and increased U2-OS and Saos-2 cell tolerance to different stresses.
Figure 1. The effects of TRIM21 on osteosarcoma cell proliferation. (A) U2-OS and (B) Saos-2 cells were transfected with HA-TRIM21 or HA-vector plasmid in 6-well plates and incubated overnight. The cells were then resuspended in McCoy's 5A medium containing fetal bovine serum and seeded into 96-well plates. The cells were then treated with CDDP (30 μg/mL), TRAIL (50 ng/mL), or serum starvation for 24 hours as indicated. MTT assays were performed to assess cellular viability through monitoring the absorbance of formazan crystals at 570 nm. (C) U2-OS and (D) Saos-2 cells were transfected with control (si-NC) or TRIM21-targeting siRNA in 6-well plates overnight. The cells were treated as described in A and B. The absorbance of the untreated control group was set to 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
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Next, we identified TRIM21-interacting partners in U2-OS cells using co-IP coupled with LC-MS/MS analysis. To this end, U2-OS cells stably expressing H125-TRIM21 were employed, where the H125-TRIM21 expression system was recombinant lentivirus-based and regulated by a tetracycline (TET) response element (Figure 2A). After induction with TET (10 mg/mL) for 18 h, control U2-OS cells stably expressing the H125-Vector displayed strong green fluorescence (Figure 2B), whereas U2-OS cells stably expressing H125-TRIM21 produced large amounts of TRIM21 protein (Figure 2C).
Figure 2. Introduction of recombinant lentivirus vector expressing TRIM21 (H125-TRIM21) and co-IP assay prepared for LC-MS/MS analysis. (A) Schematic of the construction of recombinant lentivirus vector expressing TRIM21 (H125-TRIM21). (B) Expression of GFP by H125-Vector (H125-V)-transfected cells. U2-OS cells stably expressing H125-V were incubated with tetracycline (TET; 10 mg/mL) for 18 hours. The cells were then visualized using an inverted fluorescence microscope (Olympus, Japan) with a 20 × objective. (C) TRIM21 protein levels in U2-OS cells stably expressing H125-TRIM21. U2-OS cells stably expressing H125-TRIM21 were incubated with TET (10 mg/mL) for 18 hours and then analyzed by Western Blotting with TRIM21 and GAPDH antibodies. (D) Co-IP assay was used to identify the interacting partners of TRIM21. U2-OS cells stably expressing H125-V or H125-TRIM21 incubated with TET (10 mg/mL) for 18 hours were used in standard co-IP assay with control IgG or TRIM21 antibody. The resulting immune complexes containing IgG or TRIM21 antibody were subsequently subjected to in-solution digestion and analyzed by LC-MS/MS.
U2-OS cells stably expressing H125-TRIM21 were used in co-IP with TRIM21 antibody (Figure 2D). Proteins that co-precipitated with TRIM21 were identified by LC-MS/MS. The top three identified proteins are listed in Table 1. The top TRIM21-interacting partner was YWHAZ, which is overexpressed and acts as oncogene to promote proliferation in various tumors[21, 22]. However, there have been few reports on the role of YWHAZ in osteosarcoma. In the present study, to investigate the mechanism of TRIM21 as a positive regulator of U2-OS and Saos-2 cell proliferation, YWHAZ protein was selected for subsequent experiments.
Accession Gene Names Protein Names Molecular Weight (kD) Total Number of Peptides Assigned Sequence Coverage (%) Protein False Discovery Rate Confidence P63104 YWHAZ 14-3-3 protein zeta/delta 27.7 3 12.6531 High P26641 EEF1G Elongation factor 1-gamma 50.1 2 6.4073 High P17066 HSPA6 Heat-shock 70 kD protein 6 71.0 2 6.0653 High Table 1. Top Three TRIM21 Protein-interacting Partners Identified in this Study
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To confirm the interaction between TRIM21 and YWHAZ, co-IP of U2-OS cells transfected with HA-vector or HA-TRIM21 plasmid were performed. The interacting proteins bound by HA antibody were subsequently analyzed by immunoblot (IB) assay. As shown in Figure 3A, YWHAZ bound to TRIM21.
Figure 3. Interaction between TRIM21 and YWHAZ. (A) Co-IP assay was used to validate the interaction between TRIM21 and YWHAZ. U2-OS cells transfected with HA-vector or HA-TRIM21 plasmid were employed to carry out Co-IP assay with HA antibody. The immune complexes were analyzed by immunoblotting with TRIM21 and YWHAZ antibodies. (B) BIFC assay. U2-OS cells were co-transfected with pHA-VC155 (VC-V), pMyc-VN155 (VN-V), VC-TRIM21, or VN-YWHAZ as indicated in B. After 24 hours, the fluorescence was visualized using an inverted fluorescence microscope with a 20 × objective. (C) Confocal microscopy assay. TRIM21 and YWHAZ were immunostained and then visualized using a laser scanning confocal microscope. DAPI staining was used to assess the morphology of cell nuclei. In the merged panel, yellow represents co-localization of two proteins. Scale bar: 10 μm.
BiFC was then employed to further confirm the interaction between TRIM21 and YWHAZ. BiFC is a method used to observe protein-protein interactions in live cells[33]. BiFC vector plasmids, including pHA-VC155 and pMyc-VN155, were used to construct VC-TRIM21 and VN-YWHAZ, respectively. Interactions between TRIM21 and YWHAZ would promote linking of the complementary non-fluorescent fragments VC and VN, which produces Venus fluorescence. As shown in Figure 3B, 24 hours after transfection of U2-OS cells with VC-TRIM21 and VN-YWHAZ, fluorescence was only detected in cells co-transfected with VC-TRIM21 and VN-YWHAZ plasmids, suggesting binding of YWHAZ to TRIM21. Co-localization of TRIM21 with YWHAZ was then examined using confocal microscopy. As shown in Figure 3C, both TRIM21 and YWHAZ were mainly located in the cytoplasm within scattered granules, where co-localization of TRIM21 with YWHAZ is indicated in yellow in the merged panels. Taken together, these results suggest TRIM21 interacted with YWHAZ and these proteins co-localized in the cytoplasm.
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TRIM21 is an E3 ligase; therefore, we next investigated whether YWHAZ expression is regulated by TRIM21. Knockdown of TRIM21 with its siRNA resulted in a marked increase in YWHAZ levels (Figure 4A), whereas overexpression of TRIM21 after tetracycline induction resulted in a large decrease in YWHAZ levels (Figure 4B). Accordingly, the addition of proteasome inhibitor MG132 abrogated this decrease (Figure 4B). These results hint at the possibility TRIM21 regulated YWHAZ levels through a proteasome pathway.
Figure 4. RING domain of TRIM21 required for negative regulation of YWHAZ expression by TRIM21. (A) Knockdown of TRIM21 led to upregulation of YWHAZ expression. U2-OS cells were transfected with control (si-NC) or TRIM21-targeting short interfering RNA and then immunoblotted with the indicated antibodies. (B) Downregulation of YWHAZ induced by TRIM21 overexpression was inhibited by the proteasome inhibitor MG132. U2-OS cells stably expressing H125-V or H125-TRIM21 after induction with TET (10 mg/mL) for 18 hours were left untreated or treated with MG132 and then immunoblotted with the indicated antibodies. (C) Diagram displaying full-length TRIM21 and the TRIM21-ΔRING mutant. (D) Effect of the TRIM21 RING domain on YWHAZ expression. U2-OS cells were transfected with control HA-V, HA-TRIM21, or HA-TRIM21-ΔRING and then immunoblotted with the indicated antibodies.
The RING-finger domain of the TRIM protein contains conserved cysteine and histidine residues in a 'cross-brace' arrangement. The RING domain is necessary for recruiting ubiquitin-conjugating enzymes (E2 Ub) and possesses ubiquitin E3 ligase activity[35]. We then determined whether this RING domain takes part in TRIM21 regulation of YWHAZ. To this end, we constructed the HA-TRIM21-ΔRING mutant as shown in Figure 4C. Unlike the plasmid expressing full-length HA-TRIM21, the HA-TRIM21-ΔRING mutant failed to reduce YWHAZ expression (Figure 4D), indicating the RING domain of TRIM21 was required for TRIM21-mediated negative regulation of YWHAZ.
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Subsequently, we tested whether YWHAZ is involved in TRIM21 regulation of osteosarcoma cell proliferation. Based on our results showing that TRIM21 negatively regulated the expression level of YWHAZ, we investigated whether YWHAZ overexpression could antagonize the effects of TRIM21 on osteosarcoma cell proliferation. As shown in Figure 5A, overexpression of either HA-TRIM21 or Flag-YWHAZ alone, but not the HA-TRIM21-ΔRING mutant, significantly increased U2-OS cell viability. However, overexpression of Flag-YWHAZ failed to antagonize the effects of TRIM21. Moreover, co-expression of HA-TRIM21 and Flag-YWHAZ notably increased cell viability. Intriguingly, similar results were also obtained for cells co-expressing HA-TRIM21-ΔRING and Flag-YWHAZ (Figure 5A, upper panel). The protein levels in these cohorts are shown in the lower panel of Figure 5A. In agreement with the results in Figure 4, overexpression of full-length HA-TRIM21, but not the HA-TRIM21-ΔRING mutant, reduced Flag-YWHAZ levels. However, overexpression of Flag-YWHAZ had no effect on TRIM21 expression (Figure 5B).
Figure 5. Effects of YWHAZ overexpression on TRIM21 overexpression-induced osteosarcoma cell proliferation. (A) U2-OS cells were co-transfected with HA-TRIM21 or HA-TRIM21-ΔRING and Flag-YWHAZ as indicated and evaluated by MTT assays (upper panel) and Western Blotting (lower panel). Results for control cells co-transfected with HA-vector and Flag-vector were set at 100%. The asterisk indicates a statistically significant difference between the indicated cells and control cells. *: P < 0.05; **: P < 0.01; ***: P < 0.001. ###: P < 0.001. (B) U2-OS cells were transfected with Flag-vector or Flag-YWHAZ as indicated and performed standard IB assay with the TRIM21 and Flag antibodies. (C) U2-OS cells were co-transfected with HA-TRIM21 combined with Flag-YWHAZ as indicated and performed standard IB assay with antibodies against HA, Flag, or PCNA.
In addition, PCNA (proliferating cell nuclear antigen), a marker of cell proliferation[36], was used to evaluated the proliferation of U2-OS cells. As shown in Figure 5C, PCNA expression was upregulated in U2-OS cells overexpressing either HA-TRIM21 or Flag-YWHAZ alone compared to the control group. Furthermore, co-expression of both HA-TRIM21 and Flag-YWHAZ caused an obvious re-regulation of PCNA (Figure 5B), which is similar to the trend in cell proliferation observed in Figure 5A. Taken together, our results suggest that YWHAZ was not involved in TRIM21 regulation of osteosarcoma cell proliferation.