Expression and subcellular localization of TLX and VHL in neuroblastoma cell lines upon hypoxia
We first examined by immunoblotting whether neuroblastoma cell lines derived from the developing peripheral nervous system might express TLX. TLX expression differs among the IMR-32, SK-N-BE2C, and SH-SY5Y neuroblastoma cell lines (). However, it is clearly higher compared with the human normal fibroblast AG1518 that expresses almost no TLX. It is well recognized that hypoxia up-regulates the expression of TLX in neuroprogenitors and retinal astrocytes during development (Chavali et al., 2011
; Uemura et al., 2006
). To test whether hypoxia induces TLX expression in neuroblastoma, IMR-32 and SH-SY5Y were cultured in 1.7% O2
, which efficiently induces neuroprogenitor proliferation (Chavali et al., 2011
), for 0, 4, 24, 48 h. The porcine aorta endothelial cell (PAEC) line was included because of its high hypoxia-sensitivity. The TLX expression increased in neuroblastoma cells and in PAEC following 4, 24, and 48 h of hypoxic treatment, along with glucose transporter-1 (Glut-1), which was used as a marker for hypoxia ().
Hypoxia increases TLX expression in neuroblastoma.
Since HIF-1α is the most well-studied hypoxia-induced transcription factor with multiple target genes, we compared its expression pattern with that of TLX in IMR-32 following 6 h of hypoxia (). Almost all cells stained positive for both TLX and HIF-1α, whereas none of these cells expressed HIF-1α in normoxia, even though a few cells expressed TLX quite strongly. In normal cells, HIF-αs are constantly degraded by pVHL. However, in neuroblastoma HIF-2α is often constitutively expressed, which is further upregulated upon hypoxia, but the up-regulation occurs much later than with HIF-1α (Holmquist-Mengelbier et al., 2006
). In order to find out whether TLX has any relation with VHL we next stained IMR-32 cultured in spheres for TLX and VHL (). TLX became up-regulated when neuroprogenitors were cultured in a defined medium stimulating neurosphere formation (Chavali et al., 2011
). In this condition, TLX was actually expressed stronger in the cells located close to the surface of the spheres while cells in the center are stained more for pVHL that was mostly in the cytoplasm. In fact, TLX colocalized with KI67, a marker for dividing cells, indicating that TLX is associated with proliferating cells in the spheres.
Subcellular localization of TLX.
In order to see the subcellular localization of pVHL and TLX in normoxia, proteins from IMR-32, SH-SY5Y, and SK-N-BE2C were fractionated into nuclear and cytoplasmic proteins and separately immunoblotted (supplementary material Fig. S1). In this condition, 3% (IMR-32), 14% (SK-N-BE2C), and 17% (SH-SY5Y) of the total VHL protein was detected in the nucleus. TLX expression was found in the nuclear fraction in both IMR-32 and SK-N-BE2C cells, whereas in SH-SY5Y a small fraction of TLX was found in the cytoplasm. We then examined whether VHL expression changes upon hypoxia with regard to its level and localization in relation to those of HIF-1α and -2α. Fractionated proteins from IMR-32, SH-SY5Y, and SK-N-BE2C were immunoblotted before and after 12 h hypoxia (). pVHL was mostly expressed in the cytoplasm in agreement with the immunofluorescence (). The pVHL in the nucleus decreased upon hypoxia, especially in IMR-32 and SK-N-BE2C. Adequate separation was confirmed by cytoplasmic β-tubulin and mostly nuclear BAF57 transcription factor. In hypoxia both HIF-1α and HIF-2α were accumulated only in the nucleus in contrast to pVHL that decreased. SK-N-BE2C cells contained HIF-2α in the nucleus and even slightly in the cytoplasm. These results suggest that in the nucleus the expression of VHL and TLX/HIF-α correlate negatively even though they may exist simultaneously in the nucleus. However, the mRNA levels of VHL and TLX as examined by qPCR remained unaltered in hypoxia (supplementary material Fig. S2). Thus, the protein expression of TLX seems to be stabilized upon hypoxia as in neuroprogenitors () (Chavali et al., 2011
Since pVHL acts as an ubiquitin ligase, we wanted to confirm that TLX is not a degradation target of pVHL. We thus performed a cyclohexamide (CHX) pulse-chase experiment in Cos-1 cells over-expressing exogenous Flag-TLX together with HA-VHL or vector (). Cells were harvested 48 h after transfection following treatment with CHX for various time periods. Coexpression of HA-VHL resulted in stabilization of TLX. This was also tested in VHL-deficient renal carcinoma 786-0 cells, where levels of pVHL and endogenous TLX were analyzed after transfection of VHL or its control plasmid in the presence of the proteasome inhibitor MG132 (supplementary material Fig. S3).
TLX binds VHL and stabilizes HIF-α, enhancing the VEGF-promoter activity
Next, we examined whether there is any interaction between TLX and pVHL, which might explain the stabilization of TLX and HIF-α. In order to determine TLX binding site to pVHL, Cos-1 cells were cotransfected with Flag-TLX and HA-full-length VHL30
, or the deletion mutant VHL30
Δ95-123, which failed to bind microtubules (Hergovich et al., 2006
). Twenty residues on the β-sheets of pVHL N67 to W117 are direct binding sites to HIF-αs (Min et al., 2002
) and among these amino acids a mutation of Y98, Y111, or Y117 made it incapable to bind HIF-αs (Ohh et al., 2000
). TLX was co-precipitated with the full-length pVHL30
but hardly with pVHL30Δ95-123 (). This suggests that the region 95-123 is important for binding to TLX.
Physical interaction of TLX and VHL.
We also examined whether TLX and pVHL can bind each other directly without any involvement of other proteins, and if this is the case, we wish to determine which region of TLX would bind pVHL. To this end we used GST-pull down of different GST-fused TLX deletion mutants and ThioHis-tagged full-length VHL and its deletion mutant as shown in . The result showed that deletion of the C-terminus (Δ187-385) of TLX abrogated VHL binding, while the N-terminal deletion construct (Δ1-187) was able to bind to pVHL. Even deletion of the C-terminal histone deacetylase (HDAC)5 interaction site (Δ341-385) could bind, indicating that the ligand-binding site is necessary for pVHL binding. However, pVHL30Δ95-123 bound very weakly to the full-length TLX. The N-terminal deleted construct (Δ1-187) and to a lesser extent the far-most C-terminal Δ341-385 construct could bind pVHL30Δ95-123, suggesting that exposing the ligand-binding region of TLX might increase its affinity to pVHL.
We further examined whether HIF-α and TLX could compete in binding to VHL involving the region containing residues 95-123. We thus treated cells with MG132 and performed coimmunoprecipitation with overexpressed Flag-HIF-1α in normoxia (), since the pVHL- binding sites of all HIF-αs are conserved. Cos-1 cells were transfected with Flag-HIF-1α together with HA-VHL30 and HA-TLX. When cells were transfected with increasing amounts of HA-TLX plasmid, the binding of pVHL to HIF-1α decreased. Furthermore, when we overexpressed increasing amounts of TLX in IMR-32 cells and the lysate was immunoprecipitated by endogenous HIF-2α, we saw that increased amounts of TLX diminished the amount of VHL to be coprecipitated with HIF-2α. These results suggest that increased amounts of TLX could prevent pVHL from binding to prolyl-hydroxylated HIF-α ().
Since TLX protein levels increased in the IMR-32 nucleus in hypoxia, we examined whether TLX could cooperate with HIF-α through binding to the TLX-binding motif (AAGTCA) close to the HRE element in the VEGF-promoter (). In the 5′ and 3′ of the HRE-sequence, 3 TLX-consensus-like sequences are present in the VEGF-promoter. We used the VHL-deficient 786 cells stably transfected with VHL and its control vector (786-VHL, 786-0) for the promoter-reporter assays. We transfected those cell lines with TLX, VHL, or both TLX and VHL, together with the VEGF promoter-luciferase vector in these cell lines and measured their respective promoter activities (). In 786-0, TLX activated the promoter-reporter 1.8-fold in normoxia in the presence of stabilized HIF-α caused by the loss of VHL. Thus, TLX activates the reporter not only by sequestering pVHL. As expected, VHL overexpression, which destabilizes HIF-α, decreased the activity. In 786-VHL, overexpression of TLX did not render any significant increase in the reporter activity. This may be due to depletion of HIF-α by abundant pVHL. We made a similar experiment in SH-SY5Y cells by using 2.2′BP that mimics a hypoxic condition. In normoxia, similarly to 786-0 cells, over-expression of TLX increased the promoter activity 2-fold, and excessive VHL decreased the activity to 30% of the control. In hypoxia, the activation due to TLX overexpression was marginal, although it counteracted the effect of VHL overexpression. These results suggest that TLX enhances the effect of HIF-α. Indeed, during hypoxia TLX protein levels increased, which was accompanied by a decrease of VHL protein levels and an apparent increase of VEGF protein levels after approximately 48 h. This supports the hypothesis that TLX protein might induce expression of VEGF. (). The effect of TLX is larger in normoxia, since TLX stabilizes HIF-α by sequestering VHL in accordance with VHL overexpression, which will remove this effect.
TLX activates but VHL represses the VEGF promoter.
TLX silencing suppresses cell growth, colony formation, and VEGF production of neuroblastoma cells
In order to investigate the biological roles of endogenous TLX in neuroblastoma, we silenced TLX in IMR-32 and SH-SY5Y cells. Several stable cell lines carrying TLX shRNA were made from both cell lines (supplementary material Fig. S4A). In agreement with the role of TLX in self-renewal of neural stem cells, the growth of silenced clones was clearly slower compared with the sh-controls (). Concordant with its potent proliferation-promoting activity, decrease of TLX expression led to differentiation of both of these neuroblastoma cell lines (supplementary material Fig. S4B,C). Since IMR-32 has N-Myc amplification and a more immature phenotype than SH-SY5Y, we examined in more detail the IMR-32 shTLX clones and compared them with the wild type and vector controls. When these clones were spread on agar plates, and the numbers of formed colonies were counted after 21 days, a significant reduction of colonies was found in shTLX clones as compared with the sh-control and wild-type cells ().
Effects of TLX silencing on cell growth and angiogenetic factors.
Next, we examined whether silencing of TLX affects the response of neuroblastoma to hypoxia. Silencing of TLX indeed decreased the expression of VEGF in normoxia (). In hypoxia, TLX gradually increased in controls, whereas in shTLX cells it remained low until 48 h when the silencing effect was diminished. In this experiment, 3% O2
was applied to prevent detachment of cells at later time points. The expression of HIF-2α is markedly reduced in shTLX cells. We found that VEGFA121
isoforms (Koch et al., 2011
) were expressed in shTLX control cells, and both isoforms were indeed diminished in shTLX. In addition, VHL was increased in shTLX cells, supporting the notion that expression of TLX correlates negatively with that of VHL but positively with HIF-2α. The increase of VHL might contribute to the differentiation of TLX-silenced IMR-32 cells.
So far, we found that TLX overexpression activated the VEGF-promoter in normoxia but, in hypoxia, the effect was somewhat mild, which could be explained by the endogenous increase of TLX. In order to see whether TLX actually binds the VEGF-promoter chromatin, ChIP was performed in IMR-32-derived cell clones, wild type, sh-control, and shTLX, on the VEGF-promoter containing the HRE-consensus site (). In normoxia, no TLX chromatin binding was detected in any of the cell lines, which agrees with the fact that TLX binds VHL and becomes sequestered. However, HIF-2α and RNA polymerase II (PolII) bound in both wild type and sh-control, while binding was greatly diminished in the shTLX clone. This suggests that in normoxia, TLX will indirectly activate HRE-regulated promoters through stabilizing HIF-2α. In hypoxia, both TLX and HIF-2α bind chromatin with an apparent difference from shTLX cells. However, the decrease of PolII binding was relatively small in the shTLX cells. VHL was not detected on the promoter chromatin at any time (not shown).
TLX and VHL interaction increases in early hypoxia, stabilizing HIF-α to induce angiogenesis
Having seen the binding of both HIF-2α and TLX in proximity to the HRE site of the promoter, we asked whether both TLX and HIF-2α exist in endogenous complexes with pVHL. Thus, endogenous pVHL was immunoprecipitated in both IMR-32 and SH-SY5Y, and the bound proteins were evaluated at 0, 4, and 24 h in 1.7% hypoxia (). In SH-SY5Y, the amount of precipitated pVHL increased to its maximum at 4 h in the hypoxic condition, whereas its expression in input protein decreased. In both cell lines, the same transient increase of TLX coimmunoprecipitated with pVHL at 4 h was seen, which might facilitate a faster stabilization of the remaining HIF-α. TLX expression in input protein continued to increase at 24 h, especially in IMR-32. Interestingly, HIF-2α, but not HIF-1α, was coimmunoprecipitated with pVHL, reaching maximum after 4 h in hypoxia and then to decrease at 24 h in SH-SY5Y. However, much lesser amount of HIF-2α was precipited in IMR-32, where only faint bands were detected for input of both HIF-αs. We also demonstrated that endogenous TLX could reciprocally coimmunoprecipitate VHL in wild type-, shTLX-, and the sh-control-IMR-32 cells (supplementary material Fig. S5).
TLX and VHL complex in vivo.
We next examined the expression of TLX, VHL, HIF-1α, HIF-2α in shTLX, sh-control, and wild type cells in normoxia and after 4 h of hypoxia (). As expected, shTLX cells expressed hardly any HIF-2α but HIF-1α remained almost unaffected. We then asked whether the decreased expression of TLX affects the amounts of HIF-αs in the VHL complex in normoxic and hypoxic conditions (). Binding of HIF-1α was unaltered in IMR-32 cells in both conditions, but HIF-2α was clearly decreased in shTLX cells in both normoxia and hypoxia, when compared with the sh-controls.
Silencing of HIF-2α and VHL affect the expression of TLX, HIF-2α, and VHL
In order to see whether decreased HIF-2α or VHL expression will affect TLX expression, we used siRNA oligos for HIF-2α, VHL, and TLX in normoxic and hypoxic conditions (). Since hypoxia affected the attachment of transiently siRNA-transfected IMR-32 cells, we used 2.2′BP to mimic hypoxic condition in SH-SY5Y. HIF-2α depletion led to a slight up-regulation of TLX in both conditions and an increased VHL level only in hypoxia. Depletion of VHL resulted in decreased TLX levels, as expected. In concordance with , TLX depletion increased VHL in normoxia and slightly so in hypoxia, but decreased HIF-2α in both normoxic and hypoxic conditions.
Effects of silencing HIF-2α and VHL.