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Tumor suppressor protein VHL inhibits Hedgehog–Gli activation through suppression of Gli1 nuclear localization
FEBS Letters 587 (2013) 826–832
journal homepage: www.FEBSLetters.org
Tumor suppressor protein VHL inhibits Hedgehog–Gli activation through suppression of Gli1 nuclear localization Hyun Kook Cho a, So Young Kim a, Kook Hwan Kim a, Hyeong Hoe Kim b, JaeHun Cheong a,⇑ a b
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea Department of Experimental Medicine, Pusan National University Hospital, Busan 602-739, Republic of Korea
a r t i c l e
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Article history: Received 18 November 2012 Revised 19 January 2013 Accepted 23 January 2013 Available online 8 February 2013 Edited by Lukas Huber Keywords: VHL Gli Hedgehog signaling Tumor suppression
a b s t r a c t The transcription factor Gli1 acts in the last known step of the Hedgehog signaling, and deregulation of Gli1 is implicated in human cancers. VHL protein is widely expressed in both fetal and adult tissues and acts as a tumor suppressor. Here, we demonstrate the molecular mechanism through which VHL inhibits the Hedgehog–Gli pathway. VHL decreased Gli1-mediated promoter transactivation as well as the expression of Hedgehog/Gli pathway target genes. Nuclear translocation of cytosolic Gli1 protein was inhibited by VHL via protein–protein interaction. These results indicate that overexpression of VHL may antagonize Hedgehog–Gli activation at the post-translational level in Hedgehog pathway-induced cancers. Structured summary of protein interactions: VHL-30 physically interacts with GLI1 by anti tag coimmunoprecipitation (View Interaction: 1, 2) GLI1 and VHL colocalize by fluorescence microscopy (View interaction) VHL-19 physically interacts with GLI1 by anti tag coimmunoprecipitation (View Interaction: 1, 2) VHL physically interacts with GLI1 by pull down (View interaction) Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction The Hedgehog signaling pathway is one of the most fundamental signal transduction pathways in embryonic development and is highly conserved from flies up through humans [1]. The pathway is initiated by the binding of Hedgehog (Hh) ligand to PTCH1 receptor on the target cell surface, resulting in activation of SMO and transmission of the Hh signal through the protein complex [2]. Finally, activated glioma-associated oncogene homologue (Gli), a zinc finger transcription factor, translocates to nucleus whereupon it increases the expression of Gli target genes such as Gli1, PTCH1, Cyclin D1, Snail, BCL2, VEGF, and so on [3]. Gli1 and Gli2 mostly function as transcriptional activators while Gli3 functions as a repressor [4]. Recent findings have shown that Hh signaling is also important in the regulation of proliferation, survival, and growth of adult tissues [5]. Aberrant activation of the Hedgehog signaling pathway has been implicated in several human cancers, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, breast cancer, small-cell lung carcinoma, hepatocellular carcinoma, colon cancer, pancreatic cancer, prostate cancer, and digestive tract cancer [6–15]. Furthermore, various studies have shown that transcriptional activation of Gli1 and Gli2 is important for cell ⇑ Corresponding author. Fax: +82 51 513 9258. E-mail address: [email protected] (J. Cheong).
proliferation, cell cycle progression, and anti-apoptosis [14,16– 19]. Other studies have identified several negative regulators of Gli proteins. According to recent models, Gli1 activity is mainly regulated by nuclear-cytoplasmic shuttling [20–23]. Von Hippel–Lindau (VHL) disease is the result of inheriting a mutation in the VHL tumor-suppressor gene [24]. VHL mutations, including missense, non-sense, splice site, deletion, and insertion mutations, are associated with several types of tumors, which suggests that VHL protein exerts tumor suppressor activity in various cancers [25]. The VHL gene located on chromosome locus 3p25 is translated into two biological active proteins, pVHL30 and pVHL19. Internal translation is initiated at the 54th codon of VHL mRNA, thus generating pVHL19 [26]. Signs of VHL disease, including retinal angiomas, endolymphatic sac tumors, central nervous system hemangioblastomas, clear-cell renal cancers, and pancreatic neuroendocrine tumors, suggest that VHL protein has multiple functions [27]. Specifically, VHL protein interacts with several proteins and acts as a tumor suppressor [28]. The best characterized target of VHL protein is the a-subunit of HIF-1a and HIF-2a, which are hypoxia-inducible factor (HIF) family members [29]. Interaction of VHL protein with the HIF-1a subunit results in ubiquitylation and degradation of HIF-1a under normoxic conditions [30]. A recent study indicated that HIF-1a activates the Hedgehog pathway under hypoxic conditions [31]. This suggests that the tumor suppressor VHL may regulate the Hedgehog signaling
0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.01.050
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pathway through suppression of HIF-1a. However, a direct effect of VHL on Hedgehog/Gli pathway-related cancer development has not yet been confirmed. Therefore, we investigated whether or not VHL can inhibit the Hedgehog pathway. We found that VHL suppressed the Hedgehog/Gli pathway, leading to repression of Hedgehog target gene expression. Furthermore, the protein–protein interaction between VHL and Gli1 inhibited nuclear localization and transcriptional activity of Gli1. These results suggest that VHL may act as a tumor suppressor in some Hedgehog-related cancers. 2. Materials and methods 2.1. Plasmid constructs and reagents The Gli1-luc reporter, PTCH1-luc reporter, and BCL2-luc reporter constructs (containing the Gli1, PTCH1, and BCL2 promoters fused to luciferase reporter, respectively) were provided by Dr. Fritz Aberger (University of Salzburg, Salzburg, Austria). The pcDNA3/HA-hGli1, pcDNA3/GFP-hGli1, pcDNA3/GFP-Gli1/NESmut expression plasmids were constructed as previously described
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[23]. VHL constructs (pcDNA3/HA-VHL19, pcDNA3/HA-VHL30, pcDNA3/RFP-VHL30, and pcDNA3/GST-VHL30) were kindly provided by Dr. K. H. Kim. LMB (leptomycin B) and SAG was purchased from Sigma–Aldrich. The transfection reagents jetPEI and jetPRIME were purchased from Polyplus Transfection. 2.2. Cell culture Huh7, HEK-293, and SKBR3 cells were maintained in DMEM containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin (PS) (GIBCO BRL) at 37 °C in a humid atmosphere of 5% CO2, respectively. NIH3T3 cells were cultured in DMEM containing 10% fatal calf serum (FCS) and 1% (v/v) PS (GIBCO BRL) at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. SDS–PAGE and Western blotting Whole cell lysates (50 lg) were subjected to SDS–PAGE (8–12%) and transferred onto a PVDF membrane (Millipore) by semi-dry electroblotting. Membranes were then incubated with anti-actin
Fig. 1. VHL decreases Gli1 transcriptional activity. (A) Effect of VHL on Gli1 expression. Huh7 cells were transiently transfected with empty vector, VHL30, or VHL19 expression plasmids. After 12 h of transfection, the cells were treated with or without SAG (1 lM) dissolved in distilled water for 24 h. The cell lysates were analyzed for expression of Gli1, HA-VHL or Actin by Western blotting (top panels). Total RNAs were prepared from the cells, and then Gli1 or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (bottom panels). (B) Transcriptional repression of VHL on Gli1 expression. Huh7 cells were transfected with expression vectors encoding VHL30 or VHL19, along with Gli1 promoter luciferase reporter plasmid. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for luciferase activity. Luciferase activity was normalized for transfection efficiency based on corresponding b-galactosidase activity. Values are means ± S.D. (n = 3). ⁄⁄P < 0.01 compared with mock transfectants, ##P < 0.01 compared with SAG treated mock transfectants. (C) Effect of VHL-knockdown on Gli1 expression. Huh7 cells were transiently transfected with negative siRNA or siVHL. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for expression of Gli1, VHL or Actin by Western blotting (top panels). Total RNAs were prepared from the cells and then Gli1 or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (bottom panels). (D) Transcriptional activation of VHL-knockdown on Gli1 expression. Huh7 cells were transiently transfected with negative siRNA or siVHL, along with Gli1 promoter luciferase reporter plasmid. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for luciferase activity. Luciferase activity was normalized for transfection efficiency based on corresponding b-galactosidase activity. Values are means ± S.D. (n = 3). ⁄⁄P < 0.01, ⁄P < 0.05 compared with mock transfectants.
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antibody (A2066; Sigma), anti-HA antibody (1 867423; Roche), anti-Gli1 antibody (#2534; Cell Signaling), anti-patched antibody (sc-9016; Santa Cruz Biotechnology), anti-BCL2 antibody (sc-783; Santa Cruz Biotechnology), and anti-GFP antibody (sc-9996; Santa Cruz Biotechnology) in TBST (Tris-buffered saline containing 1% Tween-20) supplemented with 1% (w/v) non-fat dry milk. After washing three times with TBST, the blotted membranes were incubated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 30 min at room temperature. After washing three times with TBST, bands were detected using an ECL (enhanced chemiluminescence) system (Amersham Biosciences). 2.4. RNA isolation and RT-PCR Total RNAs from Huh7 or NIH3T3 cells were prepared using Trizol reagent (Invitrogen) according to the manufacturer’s recommendation. Total RNAs were converted into a single-stranded cDNA using MMLV (Moloney-murine-leukaemia virus) reverse transcriptase (RT) (Promega) with a random hexamer at 42 °C. An aliquot (1/20 vol) of the cDNA was then subjected to PCR amplification using gene-specific primers. 2.5. Transfection and luciferase assay Huh7 cells were seeded on a 24-well culture plate and transfected with reporter vector and b-galactosidase expression plasmid,
along with each indicated expression plasmid or VHL siRNA (50 GUC AUC UUC UGC AAU CGC AGU-30 , knockdown efficiency: 70%; Bioneer siRNA No. 1161840, knockdown efficiency: unchanged), using jetPEI or jetPRIME (Polyplus Transfection), respectively. The pcDNA3 plasmid DNA was then added to achieve the same total amount of plasmid DNA transfection. Luciferase activity was determined using an analytical luminometer according to the manufacturer’s instructions. Luciferase activity was then normalized for transfection efficiency based on corresponding b-galactosidase activity. All assays were performed at least in triplicate. 2.6. Co-immunoprecipitation and in vivo GST (gluthathione transferase) pull-down assay Cells were lysed in radio-immunoprecipitation assay buffer. Cell lysates were mixed with anti-HA antibody (1 867423; Roche) or anti-GFP antibody (sc-9996; Santa Cruz Biotechnology) at 4 °C for 12 h with gentle agitation. Immune complexes were then collected on protein G-Sepharose beads (Invitrogen) and incubated for 2 h in a cold-room. After washing three times extensively with RIPA buffer, the precipitates were boiled with an equal volume of 2 Laemmli sample buffer, separated by SDS–PAGE, and subjected to Western blot analysis. For GST pull-down assay, whole cell lysates were obtained by subsequent centrifugation at 13 000g for 20 min at 4 °C. Cell lysates were subjected to SDS–PAGE (6–12% gels) and transferred
Fig. 2. VHL suppresses the expression of Gli1 target genes. (A) Effect of VHL on of PTCH1 or BCL2 transcriptional activities. Huh7 cells were transfected with expression vectors encoding VHL30 or VHL19, along with PTCH1 or BCL2 promoter reporter plasmid. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for luciferase activity. Luciferase activity was normalized for transfection efficiency based on corresponding b-galactosidase activity. Values are means ± S.D. (n = 3). ⁄⁄P < 0.01, ⁄P < 0.05 compared with mock transfectants, ##P < 0.01, ##P < 0.05 compared with SAG treated mock transfectants. (B and C) Effect of VHL on PTCH1 or BCL2 expression. Huh7 cells were transiently transfected with empty vector, VHL30, or VHL19 expression plasmids. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. Total RNAs were prepared from the cells, and then PTCH1, BCL2, or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (B). The cell lysates were analyzed for expression of PTCH1, BCL2, HA-VHL, or Actin by Western blotting (C). (D) Effect of VHL-knockdown on the expression of Gli1 target genes. Huh7 cells were transiently transfected with negative siRNA or siVHL. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for expression of PTCH1, BCL2, VHL or Actin by Western blotting (top panels). Total RNAs were prepared from the cells and then PTCH1, BCL2, or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (bottom panels).
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onto a PVDF membrane (Millipore) by semi-dry electroblotting. Membranes were then incubated with anti-GST antibody (sc138; Santa Cruz Biotechnology) or anti-GFP antibody (sc-9996; Santa Cruz Biotechnology) in TBST (Tris-buffered saline containing 1% Tween-20) supplemented with 3% (w/v) non-fat dry milk. The bands were detected using an ECL (enhanced chemiluminescence) system (Amersham Biosciences). 2.7. Fluorescence microscopy in living cells Following transfection, cells were incubated for 24 h. Prior to imaging, cells were counterstained with Hoechst dye for 10 min at 37 °C in order to stain nuclei. Cells were visualized with a Zeiss Axiovert 200 M microscope. 2.8. Statistical analysis Statistical analyses were carried out by unpaired or paired t test as appropriate. All data are reported as the means ± S.D. P value of <0.05 was considered as significant. 3. Results 3.1. VHL decreases the expression of Gli1 gene To determine whether or not VHL regulates the expression of Hedgehog pathway-related target genes, the expression of Gli1 protein was investigated in Huh7 cells following transient transfection with VHL30 or VHL19 expression vector. The expression of Gli1 protein and mRNA was down-regulated by VHL30 or VHL19, and SAG (SMO agonist/Hedgehog signaling activator)-induced Gli expression was attenuated by VHL30 or VHL19 (Fig. 1A) [32]. To confirm whether or not the effect of VHL on Gli1 expression is dependent on the Gli1 promoter, luciferase assay was performed with Gli1 promoter linked to the luciferase gene. As
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shown in Fig. 1B, VHL30 or VHL19 significantly suppressed Gli1 and SAG-induced Gli1 promoter activity. Small interfering RNA (siRNA) is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. To confirm the effect of VHL on expression of Gli1, we used siRNA against VHL. As shown in Fig. 1C and D, Gli1 expression and promoter activity were increased by knock-down of the VHL gene. Further, SAG-induced Gli expression and promoter activity were enhanced by knockdown of the VHL gene. These results indicate that VHL proteins inhibit Gli1 expression at the transcriptional level. 3.2. VHL represses the expression of Hedgehog target genes To investigate whether VHL30 and VHL19 affect the expression of Hedgehog/Gli target genes, a luciferase reporter assay was performed. PTCH1 and BCL2 have been well characterized as markers of Hedgehog signaling activation [33]. As shown in Fig. 2A, both VHL30 and VHL19 decreased PTCH1 and BCL2 promoter activities under control and Hedgehog signaling activated conditions. To further investigate changes in the mRNA and protein levels of Hedgehog/Gli1 target genes in the presence of VHL, RT-PCR and Western blot analyses were performed. VHL also inhibited the expression of PTCH1 and BCL2 at the mRNA and protein levels under control and Hedgehog signaling activated conditions (Fig. 2B and C). These results suggest that VHL suppresses the Hedgehog signaling pathway via repression of Gli1-mediated target genes. Further, protein and mRNA expression of Hedgehog/Gli target genes were increased by knock-down of the VHL gene (Fig. 2D). These results provide strong evidence that VHL decreases the expression of Hedgehog/Gli target genes through inactivation of the Hedgehog/Gli signaling pathway. 3.3. VHL interacts with Gli1 protein To determine the molecular mechanism through which VHL represses Gli1 transcriptional activity, a co-immunoprecipitation
Fig. 3. VHL interacts with Gli1. (A) Interaction between GFP-Gli1 and HA-VHL by co-immunoprecipitation assay. HEK293 cells were transiently transfected with HA-VHL30, HA-VHL19, or HA vector, along with GFP-Gli1 plasmid (left), or with GFP-Gli1 or GFP vector, along with HA-VHL30 or HA-VHL19 (right). After 24 h of transfection, cell lysates were subjected to immunoprecipitation (IP) with anti-HA (left) antibody or anti-GFP (right) antibody, followed by Western blotting using anti-GFP antibody (left) or anti-HA antibody (right). (B) Interaction between GFP-Gli1 and HA-VHL by in vivo GST pull-down assay. HEK293 cells were transfected with vector expression GST alone or GST-VHL protein, along with GFP-Gli1 expression vector. After 24 h of transfection, cell lysates were obtained and immobilized onto glutathione-Sepharose beads, followed by Western blotting using the indicated antibodies.
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assay was performed. GFP-Gli1 proteins were co-immunoprecipitated with HA-VHL30 or HA-VHL19, but not with the control protein HA, as shown by anti-GFP Western blotting (Fig. 3A, left panel). Inversely, HEK293 cells were co-transfected with GFPGli1 or GFP along with HA-VHLs, followed by gel-fractionation and Western blotting analysis. VHL30 and VHL19 were co-immunoprecipitated with GFP-Gli1 protein only (Fig. 3A, right panel). In addition, to confirm a physical interaction between VHL and Gli1 protein, in vivo GST pull-down assay was performed. The GST-VHL-bound GFP-Gli1 protein was detected via immunoblotting using anti-GFP antibody. As shown in Fig. 3B, GFP-Gli1 bound to GST-VHL, but not to GST protein. The results of the co-immunoprecipitation and in vivo GST pull-down assays imply that VHL specifically interacts with Gli1 protein. 3.4. VHL co-localizes with Gli1 in the cytoplasm and disturbs Gli1 nuclear localization Since Gli1 is a nuclear-cytoplasmic shuttling protein [34], Gli1 proteins can be found in the cytoplasm as well as the nucleus depending on the context [4,22]. To investigate whether or not VHL co-localizes with Gli1 protein, SKBR3 cells were co-transfected with GFP-Gli1 and RFP-VHL expression vectors, after which subcellular localization was examined by fluorescence microscopy in living cells. The subcellular localization pattern of Gli1 is defined as N,
nucleus; C, cytoplasm; and N/C, nucleus and cytoplasm. As shown in Fig. 4A, GFP-Gli1 co-localized with RFP-VHL in the cytoplasm, but not with RFP protein. These results indicate that VHL physically interacts with Gli1 protein, suggesting that VHL may play an important role as a negative regulator of Gli1 nuclear localization. Previous studies have reported that although VHL is predominantly cytoplasmic, it can shuttle between the cytoplasm and nucleus [35]. The biological function of VHL nuclear-cytoplasmic shuttling could be important for its anti-tumor effects, since the same exon (exon 2) that controls shuttling is also a b-domain-binding surface for VHL-E3 ligase [36]. To more directly confirm the role of VHL in the Gli1–VHL interaction, a nuclear export inhibitor was used. It is known that Gli1 protein activity can be suppressed by altering its subcellular localization from the nucleus to the cytoplasm, and nuclear export of Gli1 protein is chromosome region maintenance homologue 1 (CRM1)-dependent [34]. Leptomycin B (LMB), a CRM1-dependent export inhibitor, induced an increase in the nuclear localization of Gli1. On the other hand, co-transfection of RFP-VHL but not RFP disturbed this LMB-dependent nuclear accumulation of GFP-Gli1 protein (Fig. 4B). To confirm these results, Gli1-NESmut, in which two amino acids (Leu501 and Leu503) in the nuclear export sequence (NES) are replaced with alanine residues, was used. As shown in Fig. 4C, VHL inhibited the nuclear localization of Gli1-NESmut protein. The nuclear localization pattern of Gli1 was reduced by about 40% upon VHL co-transfection
Fig. 4. VHL co-localizes with Gli1 in the cytoplasm and disturbs Gli1 nuclear localization. (A) Co-localization of Gli1 and VHL in the cytoplasm. SKBR3 cells were cotransfected with RFP or RFP-VHL vectors, along with GFP-Gli1 expression vector and were counterstained with Hoechst to label nuclei. Cell imaging was assessed by fluorescence microscopy of living cells. Histogram of the quantifying changes in Gli1 subcellular localization after RFP-VHL co-expression is shown in the right panel. (B) Effect of LMB on VHL-mediated Gli1 subcellular localization. After 24 h of transfection, cells were treated with 5 nM LMB for 8 h. The cells were counterstained with Hoechst to label nuclei, and cell imaging was assessed by fluorescence microscopy of living cells. (C) Effect of Gli1-NES mutant on VHL-mediated Gli1 subcellular localization. GFPGli1-NESmut expression vectors were co-transfected along with RFP or RFP-VHL expression vector. After 24 h of transfection, the cells were counterstained with Hoechst to label nuclei, and cell imaging was assessed by fluorescence microscopy. (D) Histogram of the quantifying changes in GFP-Gli1 subcellular localization in LMB-treated cells (left) and Gli1-NESmut (right) after RFP-VHL co-expression.
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(Fig. 4D). These results demonstrate that VHL may function as a negative regulator of the Hedgehog/Gli pathway. 4. Discussion This study focuses on the function of VHL in Hedgehog signaling pathway. Herein, VHL30 and VHL19 showed a similar ability to down-regulate the expression of Hedgehog target genes. Hergovich et al. showed that VHL30 localizes to both nuclear and cytoplasmic compartments, whereas VHL19 predominantly localizes to the nucleus [35]. Despite a difference in localization, the VHLs have a similar ability to down-regulate the expression of Hedgehog target genes, implying that they may have other mechanisms that inhibit the Hedgehog signaling. For example, VHL30 or VHL19 mainly interact with cytoplasmic or nuclear Gli1, and thereby may inhibit Gli1 transcriptional activity. It is well known that VHL interacts with and targets HIF-1a for ubiquitin-mediated proteolysis under normal oxygen conditions [36]. This VHL-mediated proteolytic degradation of HIF suppresses a transcription program that is associated with the cellular adaptive response to hypoxia [37]. However, under hypoxic conditions, the ubiquitin ligase activity of VHL and the interaction between VHL and HIF-1a are inhibited [28]. Stabilized HIF-1a promotes the expression of a large panel of target genes involved in growth, motility, metabolism, and angiogenesis, including vascular endothelium growth factor (VEGF), parathyroid hormone-related protein (PTHrP), tumor growth factors (TGFs), and glucose transporters [30]. Recently, it has become clear that the Hedgehog pathway is activated in ischaemic adult tissues, including astrocytes, cardiomyoblast cells, neural progenitor cells, and human pulmonary arterial smooth muscle cells (HPASMCs), in response to hypoxia [38]. A recent investigation by Bijlsma et al. showed that HIF-1a accumulation lead to the activated Hedgehog response [31]. Further, an increase of HIF-1a due to hypoxic conditions can lead to the de novo expression of Shh and PTCH1, followed by Hedgehog pathway activation [31]. The present data show that VHL directly binds to Gli1 protein and suggest two molecular models for Gli1 inhibition by VHL. One is a direct model in which VHL directly binds to Gli1 protein and suppresses Gli1 nuclear localization. The other is an indirect model wherein HIF-1a acts as a mediator of Gli1 inhibition by VHL. Under normal conditions, the VHL-E3 ubiquitin protein ligase complex degrades HIF-1a [28]. Under hypoxic conditions, the ubiquitin ligase activity of VHL is inhibited, resulting in sequential activation of HIF-1a and up-regulation of Gli1 transcription, leading to tumor progression. However, we think that inhibition of Gli1 transcriptional activity by VHL is unlikely to occur through ubiquitin ligase activity of VHL. Therefore, we suppose that if VHL is over-expressed in hypoxic conditions, it will attenuate HIF-1a-induced Hedgehog response. Further, we suppose that mutated VHL with impaired ubiquitin ligase activity can attenuate the Hedgehog signaling, leading to down-regulation of Gli1, PTCH1, and Bcl2 expression. These suggestion needs to be confirmed in further experiments. So far more than 400 mutations in VHL have been identified, comprising for more than 150 independent intragenic mutation events. The study about correlation between VHL mutations and Hedgehog signaling activation has not yet been performed; however, we suppose that there is Hedgehog signaling activation-related cancer among the VHL mutations. Because pancreatic cancer is related to both VHL and Hedgehog signaling, the study about relation between VHL mutation and Hedgehog signaling need to be performed in patients with pancreatic cancer as the priority. In summary, the present study determined if and how VHL suppresses the Hedgehog/Gli signaling pathway. These data indicate
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that VHL inhibits the expression of Gli1 target genes as well as nuclear localization of Gli1 via protein–protein interactions. These results might provide evidence for VHL as a tumor suppressor in Hedgehog pathway-related cancers, especially pancreatic cancer. Acknowledgment This study was supported by a Grant of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A101031). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.febslet.2013. 01.050. References [1] Carol, W., Ian, S. and Allen, B. (1999) The hedgehog signalling pathway in tumorigenesis and development. Oncogene 18, 7844–7851. [2] Ruiz i Altaba, A. (1999) Gli proteins encode context-dependent positive and negative functions: implications for development and disease. Development 126, 3205–3216. [3] Kasper, M., Schnidar, H., Neill, G.W., Hanneder, M., Klingler, S., Blaas, L., Schmid, C., Hauser-Kronberger, C., Regl, G., Philpott, M.P. and Aberger, F. (2006) Selective modulation of Hedgehog/GLI target gene expression by epidermal growth factor signaling in human keratinocytes. Mol. Cell. Biol. 26, 6283–6298. [4] Ruiz i Altaba, A., Sanchez, P. and Dahmane, N. (2002) Gli and hedgehog in cancer: tumours, embryos and stem cells. Nat. Rev. Cancer 2, 361–372. [5] Ramalho-Santos, M., Melton, D.A. and McMahon, A.P. (2000) Hedgehog signals regulate multiple aspects of gastrointestinal development. Development 127, 2763–2772. [6] Taylor, M.D., Liu, L., Raffel, C., Hui, C.C., Mainprize, T.G., Zhang, X., Agatep, R., Chiappa, S., Gao, L., Lowrance, A., Hao, A., Goldstein, A.M., Stavrou, T., Scherer, S.W., Dura, W.T., Wainwright, B., Squire, J.A., Rutka, J.T. and Hogg, D. (2002) Mutations in SUFU predispose to medulloblastoma. Nat. Genet. 31, 306–310. [7] Tostar, U., Malm, C.J., Meis-Kindblom, J.M., Kindblom, L.G., Toftgard, R. and Unden, A.B. (2006) Deregulation of the hedgehog signalling pathway: a possible role for the PTCH and SUFU genes in human rhabdomyoma and rhabdomyosarcoma development. J. Pathol. 208, 17–25. [8] Hahn, H., Wicking, C., Zaphiropoulous, P.G., Gailani, M.R., Shanley, S., Chidambaram, A., Vorechovsky, I., Holmberg, E., Unden, A.B., Gillies, S., Negus, K., Smyth, I., Pressman, C., Leffell, D.J., Gerrard, B., Goldstein, A.M., Dean, M., Toftgard, R., Chenevix-Trench, G., Wainwright, B. and Bale, A.E. (1996) Mutations of the human homolog of Drosophila patched in the nevoid basal cell carcinoma syndrome. Cell 85, 841–851. [9] Kubo, M., Nakamura, M., Tasaki, A., Yamanaka, N., Nakashima, H., Nomura, M., Kuroki, S. and Katano, M. (2004) Hedgehog signaling pathway is a new therapeutic target for patients with breast cancer. Cancer Res. 64, 6071–6074. [10] Watkins, D.N., Berman, D.M., Burkholder, S.G., Wang, B., Beachy, P.A. and Baylin, S.B. (2003) Hedgehog signalling within airway epithelial progenitors and in small-cell lung cancer. Nature 422, 313–317. [11] Huang, S., He, J., Zhang, X., Bian, Y., Yang, L., Xie, G., Zhang, K., Tang, W., Stelter, A.A., Wang, Q., Zhang, H. and Xie, J. (2006) Activation of the hedgehog pathway in human hepatocellular carcinomas. Carcinogenesis 27, 1334–1340. [12] Qualtrough, D., Buda, A., Gaffield, W., Williams, A.C. and Paraskeva, C. (2004) Hedgehog signalling in colorectal tumour cells: induction of apoptosis with cyclopamine treatment. Int. J. Cancer 110, 831–837. [13] Thayer, S.P., di Magliano, M.P., Heiser, P.W., Nielsen, C.M., Roberts, D.J., Lauwers, G.Y., Qi, Y.P., Gysin, S., Fernández-del Castillo, C., Yajnik, V., Antoniu, B., McMahon, M., Warshaw, A.L. and Hebrok, M. (2003) Hedgehog is an early and late mediator of pancreatic cancer tumorigenesis. Nature 425, 851–856. [14] Sanchez, P., Hernández, A.M., Stecca, B., Kahler, A.J., DeGueme, A.M., Barrett, A., Beyna, M., Datta, M.W., Datta, S. and Ruiz i Altaba, A. (2004) Inhibition of prostate cancer proliferation by interference with SONIC HEDGEHOG–GLI1 signaling. Proc. Natl. Acad. Sci. USA 101, 12561–12566. [15] Berman, D.M., Karhadkar, S.S., Maitra, A., Montes De Oca, R., Gerstenblith, M.R., Briggs, K., Parker, A.R., Shimada, Y., Eshleman, J.R., Watkins, D.N. and Beachy, P.A. (2003) Widespread requirement for Hedgehog ligand stimulation in growth of digestive tract tumours. Nature 425, 846–851. [16] Kim, Y., Yoon, J.W., Xiao, X., Dean, N.M., Monia, B.P. and Marcusson, E.G. (2007) Selective down-regulation of glioma-associated oncogene 2 inhibits the proliferation of hepatocellular carcinoma cells. Cancer Res. 67, 3583–3593. [17] Bigelow, R.L., Chari, N.S., Unden, A.B., Spurgers, K.B., Lee, S., Roop, D.R., Toftgard, R. and McDonnell, T.J. (2004) Transcriptional regulation of bcl-2 mediated by the sonic hedgehog signaling pathway through gli-1. J. Biol. Chem. 279, 1197–1205.
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[18] Wang, K., Pan, L., Che, X., Cui, D. and Li, C. (2010) Gli1 inhibition induces cellcycle arrest and enhanced apoptosis in brain glioma cell lines. J. Neurooncol. 98, 319–327. [19] Tsuda, N., Ishiyama, S., Li, Y., Ioannides, C.G., Abbruzzese, J.L. and Chang, D.Z. (2006) Synthetic microRNA designed to target glioma-associated antigen 1 transcription factor inhibits division and induces late apoptosis in pancreatic tumor cells. Clin. Cancer Res. 12, 6557–6564. [20] Stone, D.M., Murone, M., Luoh, S., Ye, W., Armanini, M.P., Gurney, A., Phillips, H., Brush, J., Goddard, A., de Sauvage, F.J. and Rosenthal, A. (1999) Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli. J. Cell Sci. 112, 4437–4448. [21] Sheng, T., Chi, S., Zhang, X. and Xie, J. (2006) Regulation of Gli1 localization by the cAMP/protein kinase a signaling axis through a site near the nuclear localization signal. J. Biol. Chem. 281, 9–12. [22] Rahnama, F., Shimokawa, T., Lauth, M., Finta, C., Kogerman, P., Teglund, S., Toftgard, R. and Zaphiropoulos, P.G. (2006) Inhibition of GLI1 gene activation by Patched1. Biochem. J. 394, 19–26. [23] Kim, K., Kim, K.H., Cho, H.K., Kim, H.Y., Kim, H.H. and Cheong, J. (2010) SHP (small heterodimer partner) suppresses the transcriptional activity and nuclear localization of Hedgehog signalling protein Gli1. Biochem. J. 427, 413–422. [24] Latif, F., Tory, K., Gnarra, J., Yao, M., Duh, F.M., Orcutt, M.L., Stackhouse, T., Kuzmin, L., Modi, W. and Geil, L. (1993) Identification of the von Hippel– Lindau disease tumor suppressor gene. Science 260, 1317–1320. [25] Kim, W.Y. and Kaelin, W.G. (2004) Role of VHL gene mutation in human cancer. J. Clin. Oncol. 22, 4991–5004. [26] Blankenship, C., Naglich, J.G., Whaley, J.M., Seizinger, B. and Kley, N. (1999) Alternate choice of initiation codon produces a biologically active product of the von Hippel Lindau gene with tumor suppressor activity. Oncogene 18, 1529–1535. [27] Cohen, H.T. (1999) Advances in the molecular basis of renal neoplasia. Curr. Opin. Nephrol. Hypertens. 8, 325–331. [28] Czyzyk-Krzeska, M.F. and Miller, J. (2004) von Hippel–Lindau tumor suppressor: not only HIF’s executioner. Trends Mol. Med. 10, 146–149.
[29] Ohh, M., Yauch, R.L., Lonergan, K.M., Whaley, J.M., Stemmer-Rachamimov, A.O., Louis, D.N., Gavin, B.J., Kley, N., Kaelin Jr., W.G. and Iliopoulos, O. (1998) The von Hippel–Lindau tumor suppressor protein is required for proper assembly of an extracellular fibronectin matrix. Mol. Cell 1, 959–968. [30] Cockman, M.E., Masson, N., Mole, D.R., Jaakkola, P., Chang, G.W., Clifford, S.C., Maher, E.R., Pugh, C.W., Ratcliffe, P.J. and Maxwell, P.H. (2000) Hypoxia inducible factor-alpha binding and ubiquitylation by the von Hippel–Lindau tumor suppressor protein. J. Biol. Chem. 275, 25733–25741. [31] Bijlsma, M.F., Groot, A.P., Oduro, J.P., Franken, R.J., Schoenmakers, S.H., Peppelenbosch, M.P. and Spek, C.A. (2009) Hypoxia induces a hedgehog response mediated by HIF-1alpha. J. Cell. Mol. Med. 13, 2053–2060. [32] Chen, J.K., Taipale, J., Young, K.E., Maiti, T. and Beachy, P.A. (2002) Small molecule modulation of smoothened activity. Proc. Natl. Acad. Sci. USA 99, 14071–14076. [33] Katoh, Y. and Katoh, M. (2009) Hedgehog target genes: mechanisms of carcinogenesis induced by aberrant hedgehog signaling activation. Curr. Mol. Med. 9, 873–886. [34] Kogerman, P., Grimm, T., Kogerman, L., Krause, D., Undén, A.B., Sandstedt, B., Toftgård, R. and Zaphiropoulos, P.G. (1999) Mammalian suppressor-of-fused modulatesn nuclear-cytoplasmic shuttling of Gli-1. Nat. Cell Biol. 1, 312–319. [35] Hergovich, A., Lisztwan, J., Barry, R., Ballschmieter, P. and Krek, W. (2003) Regulation of microtubule stability by the von Hippel–Lindau tumour suppressor protein pVHL. Nat. Cell Biol. 5, 64–70. [36] Maxwell, P.H., Wiesener, M.S., Chang, G.W., Clifford, S.C., Vaux, E.C., Cockman, M.E., Wykoff, C.C., Pugh, C.W., Maher, E.R. and Ratcliffe, P.J. (1999) The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygendependent proteolysis. Nature 399, 271–275. [37] Barry, R.E. and Krek, W. (2004) The von Hippel–Lindau tumour suppressor: a multi-faceted inhibitor of tumourigenesis. Trends Mol. Med. 10, 466–472. [38] Wang, G., Zhang, Z., Xu, Z., Yin, H., Bai, L., Ma, Z., Decoster, M.A., Qian, G. and Wu, G. (2010) Activation of the sonic hedgehog signaling controls human pulmonary arterial smooth muscle cell proliferation in response to hypoxia. Biochim. Biophys. Acta 1803, 1359–1367.
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Tumor suppressor protein VHL inhibits Hedgehog–Gli activation through suppression of Gli1 nuclear localization Hyun Kook Cho a, So Young Kim a, Kook Hwan Kim a, Hyeong Hoe Kim b, JaeHun Cheong a,⇑ a b
Department of Molecular Biology, College of Natural Sciences, Pusan National University, Busan 609-735, Republic of Korea Department of Experimental Medicine, Pusan National University Hospital, Busan 602-739, Republic of Korea
a r t i c l e
i n f o
Article history: Received 18 November 2012 Revised 19 January 2013 Accepted 23 January 2013 Available online 8 February 2013 Edited by Lukas Huber Keywords: VHL Gli Hedgehog signaling Tumor suppression
a b s t r a c t The transcription factor Gli1 acts in the last known step of the Hedgehog signaling, and deregulation of Gli1 is implicated in human cancers. VHL protein is widely expressed in both fetal and adult tissues and acts as a tumor suppressor. Here, we demonstrate the molecular mechanism through which VHL inhibits the Hedgehog–Gli pathway. VHL decreased Gli1-mediated promoter transactivation as well as the expression of Hedgehog/Gli pathway target genes. Nuclear translocation of cytosolic Gli1 protein was inhibited by VHL via protein–protein interaction. These results indicate that overexpression of VHL may antagonize Hedgehog–Gli activation at the post-translational level in Hedgehog pathway-induced cancers. Structured summary of protein interactions: VHL-30 physically interacts with GLI1 by anti tag coimmunoprecipitation (View Interaction: 1, 2) GLI1 and VHL colocalize by fluorescence microscopy (View interaction) VHL-19 physically interacts with GLI1 by anti tag coimmunoprecipitation (View Interaction: 1, 2) VHL physically interacts with GLI1 by pull down (View interaction) Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
1. Introduction The Hedgehog signaling pathway is one of the most fundamental signal transduction pathways in embryonic development and is highly conserved from flies up through humans [1]. The pathway is initiated by the binding of Hedgehog (Hh) ligand to PTCH1 receptor on the target cell surface, resulting in activation of SMO and transmission of the Hh signal through the protein complex [2]. Finally, activated glioma-associated oncogene homologue (Gli), a zinc finger transcription factor, translocates to nucleus whereupon it increases the expression of Gli target genes such as Gli1, PTCH1, Cyclin D1, Snail, BCL2, VEGF, and so on [3]. Gli1 and Gli2 mostly function as transcriptional activators while Gli3 functions as a repressor [4]. Recent findings have shown that Hh signaling is also important in the regulation of proliferation, survival, and growth of adult tissues [5]. Aberrant activation of the Hedgehog signaling pathway has been implicated in several human cancers, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, breast cancer, small-cell lung carcinoma, hepatocellular carcinoma, colon cancer, pancreatic cancer, prostate cancer, and digestive tract cancer [6–15]. Furthermore, various studies have shown that transcriptional activation of Gli1 and Gli2 is important for cell ⇑ Corresponding author. Fax: +82 51 513 9258. E-mail address: [email protected] (J. Cheong).
proliferation, cell cycle progression, and anti-apoptosis [14,16– 19]. Other studies have identified several negative regulators of Gli proteins. According to recent models, Gli1 activity is mainly regulated by nuclear-cytoplasmic shuttling [20–23]. Von Hippel–Lindau (VHL) disease is the result of inheriting a mutation in the VHL tumor-suppressor gene [24]. VHL mutations, including missense, non-sense, splice site, deletion, and insertion mutations, are associated with several types of tumors, which suggests that VHL protein exerts tumor suppressor activity in various cancers [25]. The VHL gene located on chromosome locus 3p25 is translated into two biological active proteins, pVHL30 and pVHL19. Internal translation is initiated at the 54th codon of VHL mRNA, thus generating pVHL19 [26]. Signs of VHL disease, including retinal angiomas, endolymphatic sac tumors, central nervous system hemangioblastomas, clear-cell renal cancers, and pancreatic neuroendocrine tumors, suggest that VHL protein has multiple functions [27]. Specifically, VHL protein interacts with several proteins and acts as a tumor suppressor [28]. The best characterized target of VHL protein is the a-subunit of HIF-1a and HIF-2a, which are hypoxia-inducible factor (HIF) family members [29]. Interaction of VHL protein with the HIF-1a subunit results in ubiquitylation and degradation of HIF-1a under normoxic conditions [30]. A recent study indicated that HIF-1a activates the Hedgehog pathway under hypoxic conditions [31]. This suggests that the tumor suppressor VHL may regulate the Hedgehog signaling
0014-5793/$36.00 Ó 2013 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies. http://dx.doi.org/10.1016/j.febslet.2013.01.050
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pathway through suppression of HIF-1a. However, a direct effect of VHL on Hedgehog/Gli pathway-related cancer development has not yet been confirmed. Therefore, we investigated whether or not VHL can inhibit the Hedgehog pathway. We found that VHL suppressed the Hedgehog/Gli pathway, leading to repression of Hedgehog target gene expression. Furthermore, the protein–protein interaction between VHL and Gli1 inhibited nuclear localization and transcriptional activity of Gli1. These results suggest that VHL may act as a tumor suppressor in some Hedgehog-related cancers. 2. Materials and methods 2.1. Plasmid constructs and reagents The Gli1-luc reporter, PTCH1-luc reporter, and BCL2-luc reporter constructs (containing the Gli1, PTCH1, and BCL2 promoters fused to luciferase reporter, respectively) were provided by Dr. Fritz Aberger (University of Salzburg, Salzburg, Austria). The pcDNA3/HA-hGli1, pcDNA3/GFP-hGli1, pcDNA3/GFP-Gli1/NESmut expression plasmids were constructed as previously described
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[23]. VHL constructs (pcDNA3/HA-VHL19, pcDNA3/HA-VHL30, pcDNA3/RFP-VHL30, and pcDNA3/GST-VHL30) were kindly provided by Dr. K. H. Kim. LMB (leptomycin B) and SAG was purchased from Sigma–Aldrich. The transfection reagents jetPEI and jetPRIME were purchased from Polyplus Transfection. 2.2. Cell culture Huh7, HEK-293, and SKBR3 cells were maintained in DMEM containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin–streptomycin (PS) (GIBCO BRL) at 37 °C in a humid atmosphere of 5% CO2, respectively. NIH3T3 cells were cultured in DMEM containing 10% fatal calf serum (FCS) and 1% (v/v) PS (GIBCO BRL) at 37 °C in a humidified atmosphere containing 5% CO2. 2.3. SDS–PAGE and Western blotting Whole cell lysates (50 lg) were subjected to SDS–PAGE (8–12%) and transferred onto a PVDF membrane (Millipore) by semi-dry electroblotting. Membranes were then incubated with anti-actin
Fig. 1. VHL decreases Gli1 transcriptional activity. (A) Effect of VHL on Gli1 expression. Huh7 cells were transiently transfected with empty vector, VHL30, or VHL19 expression plasmids. After 12 h of transfection, the cells were treated with or without SAG (1 lM) dissolved in distilled water for 24 h. The cell lysates were analyzed for expression of Gli1, HA-VHL or Actin by Western blotting (top panels). Total RNAs were prepared from the cells, and then Gli1 or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (bottom panels). (B) Transcriptional repression of VHL on Gli1 expression. Huh7 cells were transfected with expression vectors encoding VHL30 or VHL19, along with Gli1 promoter luciferase reporter plasmid. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for luciferase activity. Luciferase activity was normalized for transfection efficiency based on corresponding b-galactosidase activity. Values are means ± S.D. (n = 3). ⁄⁄P < 0.01 compared with mock transfectants, ##P < 0.01 compared with SAG treated mock transfectants. (C) Effect of VHL-knockdown on Gli1 expression. Huh7 cells were transiently transfected with negative siRNA or siVHL. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for expression of Gli1, VHL or Actin by Western blotting (top panels). Total RNAs were prepared from the cells and then Gli1 or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (bottom panels). (D) Transcriptional activation of VHL-knockdown on Gli1 expression. Huh7 cells were transiently transfected with negative siRNA or siVHL, along with Gli1 promoter luciferase reporter plasmid. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for luciferase activity. Luciferase activity was normalized for transfection efficiency based on corresponding b-galactosidase activity. Values are means ± S.D. (n = 3). ⁄⁄P < 0.01, ⁄P < 0.05 compared with mock transfectants.
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antibody (A2066; Sigma), anti-HA antibody (1 867423; Roche), anti-Gli1 antibody (#2534; Cell Signaling), anti-patched antibody (sc-9016; Santa Cruz Biotechnology), anti-BCL2 antibody (sc-783; Santa Cruz Biotechnology), and anti-GFP antibody (sc-9996; Santa Cruz Biotechnology) in TBST (Tris-buffered saline containing 1% Tween-20) supplemented with 1% (w/v) non-fat dry milk. After washing three times with TBST, the blotted membranes were incubated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) for 30 min at room temperature. After washing three times with TBST, bands were detected using an ECL (enhanced chemiluminescence) system (Amersham Biosciences). 2.4. RNA isolation and RT-PCR Total RNAs from Huh7 or NIH3T3 cells were prepared using Trizol reagent (Invitrogen) according to the manufacturer’s recommendation. Total RNAs were converted into a single-stranded cDNA using MMLV (Moloney-murine-leukaemia virus) reverse transcriptase (RT) (Promega) with a random hexamer at 42 °C. An aliquot (1/20 vol) of the cDNA was then subjected to PCR amplification using gene-specific primers. 2.5. Transfection and luciferase assay Huh7 cells were seeded on a 24-well culture plate and transfected with reporter vector and b-galactosidase expression plasmid,
along with each indicated expression plasmid or VHL siRNA (50 GUC AUC UUC UGC AAU CGC AGU-30 , knockdown efficiency: 70%; Bioneer siRNA No. 1161840, knockdown efficiency: unchanged), using jetPEI or jetPRIME (Polyplus Transfection), respectively. The pcDNA3 plasmid DNA was then added to achieve the same total amount of plasmid DNA transfection. Luciferase activity was determined using an analytical luminometer according to the manufacturer’s instructions. Luciferase activity was then normalized for transfection efficiency based on corresponding b-galactosidase activity. All assays were performed at least in triplicate. 2.6. Co-immunoprecipitation and in vivo GST (gluthathione transferase) pull-down assay Cells were lysed in radio-immunoprecipitation assay buffer. Cell lysates were mixed with anti-HA antibody (1 867423; Roche) or anti-GFP antibody (sc-9996; Santa Cruz Biotechnology) at 4 °C for 12 h with gentle agitation. Immune complexes were then collected on protein G-Sepharose beads (Invitrogen) and incubated for 2 h in a cold-room. After washing three times extensively with RIPA buffer, the precipitates were boiled with an equal volume of 2 Laemmli sample buffer, separated by SDS–PAGE, and subjected to Western blot analysis. For GST pull-down assay, whole cell lysates were obtained by subsequent centrifugation at 13 000g for 20 min at 4 °C. Cell lysates were subjected to SDS–PAGE (6–12% gels) and transferred
Fig. 2. VHL suppresses the expression of Gli1 target genes. (A) Effect of VHL on of PTCH1 or BCL2 transcriptional activities. Huh7 cells were transfected with expression vectors encoding VHL30 or VHL19, along with PTCH1 or BCL2 promoter reporter plasmid. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for luciferase activity. Luciferase activity was normalized for transfection efficiency based on corresponding b-galactosidase activity. Values are means ± S.D. (n = 3). ⁄⁄P < 0.01, ⁄P < 0.05 compared with mock transfectants, ##P < 0.01, ##P < 0.05 compared with SAG treated mock transfectants. (B and C) Effect of VHL on PTCH1 or BCL2 expression. Huh7 cells were transiently transfected with empty vector, VHL30, or VHL19 expression plasmids. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. Total RNAs were prepared from the cells, and then PTCH1, BCL2, or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (B). The cell lysates were analyzed for expression of PTCH1, BCL2, HA-VHL, or Actin by Western blotting (C). (D) Effect of VHL-knockdown on the expression of Gli1 target genes. Huh7 cells were transiently transfected with negative siRNA or siVHL. After 12 h of transfection, the cells were treated with or without SAG (1 lM) for 24 h. The cell lysates were analyzed for expression of PTCH1, BCL2, VHL or Actin by Western blotting (top panels). Total RNAs were prepared from the cells and then PTCH1, BCL2, or VHL mRNA levels were detected by RT-PCR with b-actin as a loading control (bottom panels).
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onto a PVDF membrane (Millipore) by semi-dry electroblotting. Membranes were then incubated with anti-GST antibody (sc138; Santa Cruz Biotechnology) or anti-GFP antibody (sc-9996; Santa Cruz Biotechnology) in TBST (Tris-buffered saline containing 1% Tween-20) supplemented with 3% (w/v) non-fat dry milk. The bands were detected using an ECL (enhanced chemiluminescence) system (Amersham Biosciences). 2.7. Fluorescence microscopy in living cells Following transfection, cells were incubated for 24 h. Prior to imaging, cells were counterstained with Hoechst dye for 10 min at 37 °C in order to stain nuclei. Cells were visualized with a Zeiss Axiovert 200 M microscope. 2.8. Statistical analysis Statistical analyses were carried out by unpaired or paired t test as appropriate. All data are reported as the means ± S.D. P value of <0.05 was considered as significant. 3. Results 3.1. VHL decreases the expression of Gli1 gene To determine whether or not VHL regulates the expression of Hedgehog pathway-related target genes, the expression of Gli1 protein was investigated in Huh7 cells following transient transfection with VHL30 or VHL19 expression vector. The expression of Gli1 protein and mRNA was down-regulated by VHL30 or VHL19, and SAG (SMO agonist/Hedgehog signaling activator)-induced Gli expression was attenuated by VHL30 or VHL19 (Fig. 1A) [32]. To confirm whether or not the effect of VHL on Gli1 expression is dependent on the Gli1 promoter, luciferase assay was performed with Gli1 promoter linked to the luciferase gene. As
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shown in Fig. 1B, VHL30 or VHL19 significantly suppressed Gli1 and SAG-induced Gli1 promoter activity. Small interfering RNA (siRNA) is involved in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. To confirm the effect of VHL on expression of Gli1, we used siRNA against VHL. As shown in Fig. 1C and D, Gli1 expression and promoter activity were increased by knock-down of the VHL gene. Further, SAG-induced Gli expression and promoter activity were enhanced by knockdown of the VHL gene. These results indicate that VHL proteins inhibit Gli1 expression at the transcriptional level. 3.2. VHL represses the expression of Hedgehog target genes To investigate whether VHL30 and VHL19 affect the expression of Hedgehog/Gli target genes, a luciferase reporter assay was performed. PTCH1 and BCL2 have been well characterized as markers of Hedgehog signaling activation [33]. As shown in Fig. 2A, both VHL30 and VHL19 decreased PTCH1 and BCL2 promoter activities under control and Hedgehog signaling activated conditions. To further investigate changes in the mRNA and protein levels of Hedgehog/Gli1 target genes in the presence of VHL, RT-PCR and Western blot analyses were performed. VHL also inhibited the expression of PTCH1 and BCL2 at the mRNA and protein levels under control and Hedgehog signaling activated conditions (Fig. 2B and C). These results suggest that VHL suppresses the Hedgehog signaling pathway via repression of Gli1-mediated target genes. Further, protein and mRNA expression of Hedgehog/Gli target genes were increased by knock-down of the VHL gene (Fig. 2D). These results provide strong evidence that VHL decreases the expression of Hedgehog/Gli target genes through inactivation of the Hedgehog/Gli signaling pathway. 3.3. VHL interacts with Gli1 protein To determine the molecular mechanism through which VHL represses Gli1 transcriptional activity, a co-immunoprecipitation
Fig. 3. VHL interacts with Gli1. (A) Interaction between GFP-Gli1 and HA-VHL by co-immunoprecipitation assay. HEK293 cells were transiently transfected with HA-VHL30, HA-VHL19, or HA vector, along with GFP-Gli1 plasmid (left), or with GFP-Gli1 or GFP vector, along with HA-VHL30 or HA-VHL19 (right). After 24 h of transfection, cell lysates were subjected to immunoprecipitation (IP) with anti-HA (left) antibody or anti-GFP (right) antibody, followed by Western blotting using anti-GFP antibody (left) or anti-HA antibody (right). (B) Interaction between GFP-Gli1 and HA-VHL by in vivo GST pull-down assay. HEK293 cells were transfected with vector expression GST alone or GST-VHL protein, along with GFP-Gli1 expression vector. After 24 h of transfection, cell lysates were obtained and immobilized onto glutathione-Sepharose beads, followed by Western blotting using the indicated antibodies.
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assay was performed. GFP-Gli1 proteins were co-immunoprecipitated with HA-VHL30 or HA-VHL19, but not with the control protein HA, as shown by anti-GFP Western blotting (Fig. 3A, left panel). Inversely, HEK293 cells were co-transfected with GFPGli1 or GFP along with HA-VHLs, followed by gel-fractionation and Western blotting analysis. VHL30 and VHL19 were co-immunoprecipitated with GFP-Gli1 protein only (Fig. 3A, right panel). In addition, to confirm a physical interaction between VHL and Gli1 protein, in vivo GST pull-down assay was performed. The GST-VHL-bound GFP-Gli1 protein was detected via immunoblotting using anti-GFP antibody. As shown in Fig. 3B, GFP-Gli1 bound to GST-VHL, but not to GST protein. The results of the co-immunoprecipitation and in vivo GST pull-down assays imply that VHL specifically interacts with Gli1 protein. 3.4. VHL co-localizes with Gli1 in the cytoplasm and disturbs Gli1 nuclear localization Since Gli1 is a nuclear-cytoplasmic shuttling protein [34], Gli1 proteins can be found in the cytoplasm as well as the nucleus depending on the context [4,22]. To investigate whether or not VHL co-localizes with Gli1 protein, SKBR3 cells were co-transfected with GFP-Gli1 and RFP-VHL expression vectors, after which subcellular localization was examined by fluorescence microscopy in living cells. The subcellular localization pattern of Gli1 is defined as N,
nucleus; C, cytoplasm; and N/C, nucleus and cytoplasm. As shown in Fig. 4A, GFP-Gli1 co-localized with RFP-VHL in the cytoplasm, but not with RFP protein. These results indicate that VHL physically interacts with Gli1 protein, suggesting that VHL may play an important role as a negative regulator of Gli1 nuclear localization. Previous studies have reported that although VHL is predominantly cytoplasmic, it can shuttle between the cytoplasm and nucleus [35]. The biological function of VHL nuclear-cytoplasmic shuttling could be important for its anti-tumor effects, since the same exon (exon 2) that controls shuttling is also a b-domain-binding surface for VHL-E3 ligase [36]. To more directly confirm the role of VHL in the Gli1–VHL interaction, a nuclear export inhibitor was used. It is known that Gli1 protein activity can be suppressed by altering its subcellular localization from the nucleus to the cytoplasm, and nuclear export of Gli1 protein is chromosome region maintenance homologue 1 (CRM1)-dependent [34]. Leptomycin B (LMB), a CRM1-dependent export inhibitor, induced an increase in the nuclear localization of Gli1. On the other hand, co-transfection of RFP-VHL but not RFP disturbed this LMB-dependent nuclear accumulation of GFP-Gli1 protein (Fig. 4B). To confirm these results, Gli1-NESmut, in which two amino acids (Leu501 and Leu503) in the nuclear export sequence (NES) are replaced with alanine residues, was used. As shown in Fig. 4C, VHL inhibited the nuclear localization of Gli1-NESmut protein. The nuclear localization pattern of Gli1 was reduced by about 40% upon VHL co-transfection
Fig. 4. VHL co-localizes with Gli1 in the cytoplasm and disturbs Gli1 nuclear localization. (A) Co-localization of Gli1 and VHL in the cytoplasm. SKBR3 cells were cotransfected with RFP or RFP-VHL vectors, along with GFP-Gli1 expression vector and were counterstained with Hoechst to label nuclei. Cell imaging was assessed by fluorescence microscopy of living cells. Histogram of the quantifying changes in Gli1 subcellular localization after RFP-VHL co-expression is shown in the right panel. (B) Effect of LMB on VHL-mediated Gli1 subcellular localization. After 24 h of transfection, cells were treated with 5 nM LMB for 8 h. The cells were counterstained with Hoechst to label nuclei, and cell imaging was assessed by fluorescence microscopy of living cells. (C) Effect of Gli1-NES mutant on VHL-mediated Gli1 subcellular localization. GFPGli1-NESmut expression vectors were co-transfected along with RFP or RFP-VHL expression vector. After 24 h of transfection, the cells were counterstained with Hoechst to label nuclei, and cell imaging was assessed by fluorescence microscopy. (D) Histogram of the quantifying changes in GFP-Gli1 subcellular localization in LMB-treated cells (left) and Gli1-NESmut (right) after RFP-VHL co-expression.
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(Fig. 4D). These results demonstrate that VHL may function as a negative regulator of the Hedgehog/Gli pathway. 4. Discussion This study focuses on the function of VHL in Hedgehog signaling pathway. Herein, VHL30 and VHL19 showed a similar ability to down-regulate the expression of Hedgehog target genes. Hergovich et al. showed that VHL30 localizes to both nuclear and cytoplasmic compartments, whereas VHL19 predominantly localizes to the nucleus [35]. Despite a difference in localization, the VHLs have a similar ability to down-regulate the expression of Hedgehog target genes, implying that they may have other mechanisms that inhibit the Hedgehog signaling. For example, VHL30 or VHL19 mainly interact with cytoplasmic or nuclear Gli1, and thereby may inhibit Gli1 transcriptional activity. It is well known that VHL interacts with and targets HIF-1a for ubiquitin-mediated proteolysis under normal oxygen conditions [36]. This VHL-mediated proteolytic degradation of HIF suppresses a transcription program that is associated with the cellular adaptive response to hypoxia [37]. However, under hypoxic conditions, the ubiquitin ligase activity of VHL and the interaction between VHL and HIF-1a are inhibited [28]. Stabilized HIF-1a promotes the expression of a large panel of target genes involved in growth, motility, metabolism, and angiogenesis, including vascular endothelium growth factor (VEGF), parathyroid hormone-related protein (PTHrP), tumor growth factors (TGFs), and glucose transporters [30]. Recently, it has become clear that the Hedgehog pathway is activated in ischaemic adult tissues, including astrocytes, cardiomyoblast cells, neural progenitor cells, and human pulmonary arterial smooth muscle cells (HPASMCs), in response to hypoxia [38]. A recent investigation by Bijlsma et al. showed that HIF-1a accumulation lead to the activated Hedgehog response [31]. Further, an increase of HIF-1a due to hypoxic conditions can lead to the de novo expression of Shh and PTCH1, followed by Hedgehog pathway activation [31]. The present data show that VHL directly binds to Gli1 protein and suggest two molecular models for Gli1 inhibition by VHL. One is a direct model in which VHL directly binds to Gli1 protein and suppresses Gli1 nuclear localization. The other is an indirect model wherein HIF-1a acts as a mediator of Gli1 inhibition by VHL. Under normal conditions, the VHL-E3 ubiquitin protein ligase complex degrades HIF-1a [28]. Under hypoxic conditions, the ubiquitin ligase activity of VHL is inhibited, resulting in sequential activation of HIF-1a and up-regulation of Gli1 transcription, leading to tumor progression. However, we think that inhibition of Gli1 transcriptional activity by VHL is unlikely to occur through ubiquitin ligase activity of VHL. Therefore, we suppose that if VHL is over-expressed in hypoxic conditions, it will attenuate HIF-1a-induced Hedgehog response. Further, we suppose that mutated VHL with impaired ubiquitin ligase activity can attenuate the Hedgehog signaling, leading to down-regulation of Gli1, PTCH1, and Bcl2 expression. These suggestion needs to be confirmed in further experiments. So far more than 400 mutations in VHL have been identified, comprising for more than 150 independent intragenic mutation events. The study about correlation between VHL mutations and Hedgehog signaling activation has not yet been performed; however, we suppose that there is Hedgehog signaling activation-related cancer among the VHL mutations. Because pancreatic cancer is related to both VHL and Hedgehog signaling, the study about relation between VHL mutation and Hedgehog signaling need to be performed in patients with pancreatic cancer as the priority. In summary, the present study determined if and how VHL suppresses the Hedgehog/Gli signaling pathway. These data indicate
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