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1 Department of Pathology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan
2 Department of Oral Maxillofacial Diagnostic Science, Division of Pathology and High-Tech Research Center, Kanagawa Dental College, Kanagawa, 238-8580, Japan
3 Department of Dental and Medical Biochemistry, Hiroshima University Graduate School of Biomedical Sciences, Hiroshima 734-8553, Japan
4 Department of Vascular Biology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan
5 Department of the Biology and Medicine of the Stem Cell, Hiroshima University Graduate School of Medical Science, Hiroshima 734-8553, Japan
6 Department of Medico-Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan
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Abstract |
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(HIF-1) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1 but not with ARNT1. The binding of HIF-1 to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over-expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved. |
Introduction |
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Hypoxia is a physiological stress, which induces the expression of VEGF. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor, which plays a central role in the gene expression induced by hypoxia, and also in the development of cancer (Forsythe et al. 1996). HIF-1 is a heterodimer of HIF-1 and aryl hydrocarbon receptor nuclear translocator 1 (ARNT1; also known as HIF-1β). The expression of HIF-1 is regulated by oxygen concentrations while ARNT1 is constitutively expressed. There is a hypoxia response element (HRE) in the promoter region of the VEGF gene (Forsythe et al. 1996), and the binding of HIF-1/ARNT1 to the HRE initiates the transcription of VEGF mRNA. Mutation of the HRE abrogates the gene transcription induced by hypoxia (Kimura et al. 2000).
It is reported that expression of the VEGF gene was regulated by circadian rhythms (Koyanagi et al. 2003). Mammalian circadian rhythms are regulated by molecular clockwork systems based on a negative feedback loop in normal and tumor cells. Clock and Brain-muscle-arnt-like-protein 1/2 (Bmal 1/2) are well known clock genes that positively regulate the expression of target genes. Period 1/2/3 (Per 1/2/3), Cryptochromes 1/2/3 (Cry 1/2/3) and Differentiated embryo-chondrocyte 1 (Dec1; also named as Bhlhb2/Sharp2/Stra13) and Dec2 (Bhlhb3/Sharp1) are also designated as clock genes, but negatively regulate the expression of their targets. Circadian rhythms are tightly regulated by these clock genes. The heterodimer comprising CLOCK and BMAL 1/2 (CLOCK/BMAL 1/2) enhances transcription of Dec, Per and Cry via CACGTG E-boxes, and the products of these genes, DEC, PER and CRY, suppress the transactivation by CLOCK/BMAL 1/2 (Sangoram et al. 1998; Hamaguchi et al. 2004). It was also reported that PER, but not CRY, regulated the expression of the VEGF gene induced by HIF-1/ARNT1 (Koyanagi et al. 2003). However, there have been no reports about the regulation of VEGF by other clock genes.
The basic-helix-loop-helix (bHLH) transcription factors are involved in the expression of various genes. We reported that DEC1 and DEC2 are bHLH transcription factors, and play an important role in the circadian rhythm of the suprachiasmatic nucleus (SCN) (Honma et al. 2002) or peripheral tissue (Hamaguchi et al. 2004; Noshiro et al. 2004; Kondo et al. 2006) as a negative regulator. These two DECs were shown to regulate the differentiation of chondrocytes, skeletal muscles and nervous systems (Boudjelal et al. 1997; Shen et al. 1997; Fujimoto et al. 2007). Recently, we reported that the expression of DEC1 and DEC2 is induced by hypoxia (Miyazaki et al. 2002). This suggests that DEC1 and DEC2 are involved in the expression of other hypoxia-inducible genes. In the present study, we investigated the role of DEC1 and DEC2 in hypoxia-induced VEGF expression. Our results showed that DEC2 regulated the expression of the VEGF gene induced by HIF-1/ARNT1 transactivation by interacting with HIF-1 and interfered with the binding of HIF-1 to the VEGF promoter under hypoxic conditions, and that DEC2 showed the opposite phase to VEGF in circadian rhythm.
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Results |
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, ARNT1, DEC1, DEC2, and PER2 in NIH3T3 and Sarcoma 180 cells
We previously showed that the mRNA expression of DEC1 and DEC2 was induced by hypoxia in 293 T and HeLa cells (Miyazaki et al. 2002). In the present study, we first examined the effect of hypoxia on the expression of mRNAs for VEGF, HIF-1, ARNT1, DEC1, DEC2, and PER2 in NIH3T3 (mouse fibroblast) and Sarcoma 180 (mouse Sarcoma) cells. Cells were incubated under normoxic or hypoxic conditions for 24 h, and mRNA was prepared. The mRNA was subjected to semi-quantitative RT-PCR analyses. The expression of mRNA for VEGF, DEC1 and DEC2 was faint under normoxic conditions in NIH3T3 cells, but was significantly up-regulated by hypoxia (Fig. 1, left panel). The level of PER2 mRNA was low during normoxia and not changed by hypoxia in NIH3T3 cells, whereas the mRNA of HIF-1 and ARNT1 was abundantly expressed under normoxic conditions and was not changed by hypoxia. In Sarcoma 180 cells, substantial amounts of mRNAs for VEGF, HIF-1, ARNT1, DEC1 and PER2 were detected under normoxic conditions (Fig. 1, right panel). Hypoxia did not induce a significant change in the expression of mRNA for VEGF, HIF-1, ARNT1 and DEC1, but inhibited the expression of PER2 mRNA. On the other hand, the expression of DEC2 mRNA was induced by hypoxia also in this cell type. These results are consistent with previous finding in other tumor cell lines (Ishibashi et al. 2001; Ivanova et al. 2001; Qin et al. 2001; Li et al. 2003).
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We next examined if DEC1 or DEC2 affects the promoter activity of Vegf. Cells were transiently transfected with a full-length Vegf promoter containing a HRE (Fig. 2A1) together with the expression vector for DEC1 or DEC2. The promoter activity of Vegf was increased by hypoxia about 100-fold and the increase was not affected by DEC1 in NIH3T3 cells (Fig. 2B, upper panel). In Sarcoma 180 cells, the promoter activity of Vegf was strong even under normoxic conditions, and was not altered by DEC1 (Fig. 2B, lower panel). The promoter activity of Vegf under hypoxia was not suppressed by DEC1. On the other hand, DEC2 suppressed, in a dose-dependent manner, the promoter activity of Vegf induced by hypoxia in NIH3T3 cells (Fig. 2C, upper panel) or the activity in Sarcoma 180 cells under both normoxia and hypoxia (Fig. 2C, lower panel). We confirmed, by Western blot analysis, that over-expression of DEC1 or DEC2 does not alter the endogenous levels of HIF-1 and ARNT1 proteins in cells.
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/ARNT1 transactivation
The HIF-1/ARNT1 heterodimer plays a critical role in the transcription of Vegf induced by hypoxia (Forsythe et al. 1996). We next examined if DEC2 affects the promoter activity of Vegf induced by HIF-1/ARNT1 transactivation. When the constructs for HIF-1 and ARNT1 were co-transfected into NIH3T3 or Sarcoma 180 cells, the promoter activity of Vegf increased about 100-fold even under normoxic conditions (Fig. 3A upper and lower panel), and the increase was suppressed by DEC2 in a dose-dependent manner.
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and ARNT1 were co-transfected into NIH3T3 cells, the promoter activity of VEGF-HRE increased about fivefold under normoxic conditions (Fig. 3B, upper panel). Co-transfection of the PER2 construct slightly suppressed the promoter activity of VEGF-HRE as previously described (Koyanagi et al. 2003). When the DEC2 construct was co-transfected, the promoter activity of VEGF-HRE was suppressed in a dose-dependent manner (Fig. 3B, upper panel). The inhibitory effect of DEC2 on the promoter activity was more significant than that of PER2. The effect of the DEC1 construct was weak. Co-expression of HIF-1 and ARNT1 did not induce the promoter activity of VEGF-HREm. The activity of VEGF-HREm was not changed by the co-transfection of DEC2, DEC1 or PER2.
Dec1 is a target of HIF-1 like Vegf (Miyazaki et al. 2002). We next examined whether DEC2 suppresses the activity of the DEC1-HRE promoter (Fig. 2A4 and A5) induced by HIF-1/ARNT1 transactivation. The promoter activity of DEC1-HRE induced by the transactivation was also suppressed by the co-transfection of DEC2 (Fig. 3B, lower panel). On the other hand, the promoter activity of DEC1-HREm was not induced by HIF-1/ARNT1 transactivation, similar to VEGF-HREm, and the activity of DEC1-HREm was not changed by co-transfection of DEC2.
DEC2 protein interacts with HIF-1 protein in cells
We performed an immunoprecipitation assay in order to examine the interaction between DEC2 and HIF-1 or ARNT1 (Fig. 4). COS-7 cells were transfected with expression vectors for ARNT1, FLAG, FLAG-HIF-1, FLAG-DEC1 or FLAG-DEC2. After 40 h of transfection, the cells were lysed and the expression of ARNT1, DEC2 or DEC1 protein was confirmed by immunoblotting using aliquots of total cell lysate. FLAG-tagged proteins in the lysates were immunoprecipitated with anti-FLAG antibody, and immunoblotted with antibodies against ARNT1 (Fig. 4A), DEC2 or DEC1 (Fig. 4B). HIF-1 was co-immunoprecipitated with ARNT1 as previously described (Jiang et al. 1996). DEC2 was co-precipitated with HIF-1 but not with ARNT1. On the other hand, DEC1 was not bound to HIF-1 or ARNT1. These results demonstrated that DEC2 protein could physically bind to HIF-1 in cells.
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We further examined if DEC2 suppresses the endogenous VEGF expression induced by hypoxia. NIH3T3 cells were transfected with the expression vector for DEC2. After 16 h of transfection, the cells were incubated for an additional 24 h under hypoxic conditions. The expression level of VEGF mRNA was examined by RT-PCR (Fig. 5A), and the amount of VEGF protein in the conditioned medium was measured by an ELISA (Fig. 5B). DEC2 suppressed, in a dose-dependent manner, the endogenous expression of VEGF mRNA and protein induced by hypoxia, but had no effect on the expression under normoxic conditions. In Sarcoma 180 cells, DEC2 suppressed the endogenous expression of VEGF during normoxia in a dose-dependent manner (Fig. 5C and D).
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and DEC2 proteins in HSC-3 cells
NIH3T3 and Sarcoma 180 cells were useful for plasmid transfection studies, but the endogenous expression of DEC2 protein was weak in these cells. Thus, in order to observe the expression of endogenous DEC2 protein, we used another cell line, human squamous cell carcinoma (HSC-3) cells. We examined the effect of hypoxia on the expression of endogenous HIF-1, DEC2, DEC1, PER2 or actin protein in HSC-3 cells (Fig. 6A). Under normoxic conditions, the expression of HIF-1, DEC2 and DEC1 protein was faint, but PER2 protein was abundantly expressed. The cells were exposed to hypoxia for 4, 12 or 24 h. The amount of HIF-1 protein was increased markedly when exposed to hypoxia for 4 h, and decreased thereafter. In contrast, the amount of DEC2 protein increased gradually and was highest after the expose of hypoxia for 24 h. The expression of DEC1 protein was also increased when exposed to hypoxia, and peaked at 12 h. The level of PER2 protein was little changed by hypoxia for up to 12 h, but was decreased markedly by hypoxia for 24 h. These results demonstrated that the expression of HIF-1, DEC1, DEC2 and PER2 is differentially regulated by hypoxia. We further examined the effect of the knockdown of endogenous DEC2 by siRNA in HSC-3 cells. Inhibition of DEC2 expression by siRNA resulted in the increased expression of VEGF mRNA (Fig. 6B). We next performed a chromatin immunoprecipitation (ChIP) assay to examine if DEC2 interferes with the binding of HIF-1 to the VEGF-promoter in Sarcoma 180 and HSC-3 cells. As shown in Fig. 6C, DEC2 over-expression decreased the binding of HIF-1 to the VEGF promoter in Sarcoma 180 cells exposed to hypoxia. In addition, siRNA knockdown of DEC2 markedly increased the binding of HIF-1 to the VEGF promoter in HSC-3 cells under hypoxic conditions. These results demonstrated that DEC2 expression regulates the binding of HIF-1 to the VEGF promoter during hypoxia.
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We next examined the circadian rhythm of VEGF and DEC2 protein expression in vivo. Samples of mouse rib cartilage were prepared at ZT 2 (10 : 00) and at ZT 14 (22 : 00), light-dark (LD) conditions, and we performed an immunohistochemical analysis of VEGF, DEC2, HIF-1 and ARNT1 in cartilage. At ZT2, VEGF protein was observed in the terminal zone (T), but not in the middle zone (M) (Fig. 7A). On the other hand, DEC2 was stained intensely in the middle zone, but weakly in the terminal zone. At ZT14, VEGF was strongly expressed in the middle zone, but DEC2 was dominant in the terminal zone (Fig. 7B). These results indicate that the expression of VEGF shows a reciprocal pattern to that of DEC2 in circadian rhythm. HIF-1 and ARNT1 were positively stained in both the terminal and middle zones (Fig. 7C and D).
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As shown in Fig. 8, mice were housed under a LD cycle and a 50 µL volume containing 1.5 x 106 viable Sarcoma 180 cells was injected into the right hind footpad of each mouse. The tumor volume was estimated as previously described (Koyanagi et al. 2003). The mRNA levels for DEC2 and VEGF in implanted Sarcoma 180 cells exhibited an opposite circadian phase at ZT2 and ZT14 (P < 0.01), but HIF-1 did not show a circadian rhythm.
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Discussion |
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is the key regulator in cellular adaptation to hypoxia. Hypoxia stabilizes HIF-1 allowing it to form a heterodimer with ARNT1 (Forsythe et al. 1996): The heterodimer HIF-1/ARNT1 binds to the HRE, and causes an increase in the expression of target genes such as the VEGF and erythropoietin genes (Grimm et al. 2002). In the present study, we demonstrated that DEC2 interacts with HIF-1, and interferes with the binding of HIF-1 to the HRE, resulting in down-regulation of the expression of HIF-1-inducible genes such as the VEGF gene during hypoxia. Recent studies have shown that both DEC1 and DEC2 were induced to express by hypoxic conditions (Miyazaki et al. 2002; Yun et al. 2002; Chakrabarti et al. 2004; Ivanov et al. 2007). Here we showed that the expression of DEC1 and DEC2 was also induced by hypoxia in mouse fibroblasts, Sarcoma 180 cells, and human squamous cell carcinoma cells. Although HIF-1 was activated by hypoxia in these cells, the induction of DEC2 expression by hypoxia was slower than that of HIF-1 and DEC1 expression.
This is a new molecular mechanism in which the DEC2-mediated feedback loop participates in the regulation of gene expression induced by hypoxia. We also found that DEC1 did not bind to HIF-1, and had little effect on the promoter activity of the VEGF gene. This finding indicates that DEC1 and DEC2, which differ in structure of the C-terminal, have distinct roles in adaptation to hypoxia: DEC2 has the alanine and glycine-rich region in its C-terminal half, but DEC1 does not.
In contrast to DEC1 and DEC2 expression, PER2 expression was down-regulated by hypoxia in mouse Sarcoma 180 and human squamous cell carcinoma cells. It was reported that PER2 also inactivates HIF-1 in a similar manner to DEC2 (Koyanagi et al. 2003): PER2 inhibited HIF-1/ARNT1-induced VEGF promoter activity in Sarcoma 180 cells by interacting with HIF-1 protein. The different expression profiles of DEC2 and PER2 under hypoxic conditions suggest distinct functions in the transcriptional regulation of target genes of HIF-1.
Both DEC1 and DEC2 show a circadian expression in many tissues (Honma et al. 2002; Noshiro et al. 2004; Furukawa et al. 2005). We also showed, in the present study, that DEC2 and VEGF protein expression had a circadian rhythm in cartilage. The expression of VEGF showed a reciprocal pattern to that of DEC2 at ZT2 and ZT14. Furthermore, both DEC2 and VEGF had circadian oscillations in implanted Sarcoma 180 cells, and the expression of VEGF mRNA showed the opposite circadian phase to that of DEC2 mRNA. These findings indicate that DEC2 regulates VEGF expression in both normal and tumorous tissues.
Several recent reports showed that clock genes play important roles in cancer biology: (i) The mRNA expression of DEC1 was suppressed by the over-expression of VHL in renal cell carcinoma (Ivanova et al. 2001), (ii) Knockdown of BMAL2 enhanced cell proliferation in hepatocellular carcinoma (Yeh et al. 2003), and (iii) PER1 plays an important role in cell growth and DNA damage control in human cancer cells (Gery et al. 2006). DEC2 may contribute to the regulatory mechanism in tumor cell biology.
In summary, we revealed that DEC2 negatively regulates VEGF expression by interacting with HIF-1, and interferes with the binding of HIF-1 to the HRE in the VEGF promoter under hypoxic conditions. Thus, DEC2 may regulate the other genes targeted by HIF-1. In addition, DEC2 regulates the transcription of its targets by interacting with E-box in a circadian fashion (Fujimoto et al. 2007). DEC2 may be a key transcription factor, which plays an important role in the cross-talk between the hypoxia-response system and circadian regulation.
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Experimental procedures |
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NIH3T3, Sarcoma 180 and COS-7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HSC-3 cells were obtained from the Japanese Cancer Research Resources Bank. Cells were cultured in Dulbecco's Modified Eagle's Medium-high glucose (Sigma Chemical Co., St. Louis, MO) supplemented with 10% fetal bovine serum. The cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2 (20% O2). Hypoxic exposure was performed in a hypoxic chamber (1% O2 for NIH3T3 or HSC-3 cells, and 1% O2 for 4 h or 3% O2 for 24 h for Sarcoma 180 cells; Sarcoma 180 cells died in the presence of 1% O2 for 24 h).
Construction of reporter and expression plasmids
The luciferase reporter plasmid, Vegf-luc, containing a 1289-bp fragment of the mouse Vegf gene in pGL3-basic (Fig. 2A1), was previously described (Koyanagi et al. 2003). A 34-bp fragment containing the hypoxia-response element (HRE) of Vegf with the flanking sequence was made by annealing the oligonucleotides 5'-CTAGTGTGCATACGTGGGTTTCCACAGGTCGTCT-3' and 5'-TCGAAGACGACCTGTGGAAACCCACGTATGCACA-3'. Another 34-bp fragment which has a mutation in the HREm of Vegf was made in a similar manner using 5'-CTAGTGTGCATGATATGGTTTCCACAGGTCGTCT-3' and 5'-TCGAAGACGACCTGTGGAAACCATATCATGCACA-3'. These fragments were ligated into the NheI and XhoI sites of pTK-Luc upstream of the TK promoter (Fig. 2A; pVEGF-HRE-TK-Luc and pVEGF-HREm-TK-Luc). The promoter constructs of HRE and HREm of DEC1 (Fig. 2A; pDEC1-HRE-TK-Luc and pDEC1-HREm-TK-Luc), and the expression plasmids for FLAG-HIF-1, FLAG-DEC2, FLAG-DEC1, DEC2 pcDNA, DEC1 pcDNA and PER2 pcDNA were previously described (Honma et al. 2002; Miyazaki et al. 2002; Sato et al. 2004; Fujimoto et al. 2007). Expression constructs encoding full-length mouse ARNT1 were obtained by subcloning PCR-generated fragments into the vector pcDNA3.1.
Transient transfection and luciferase assay
NIH3T3 or Sarcoma 180 cells were seeded at 1 x 104 cells per 16-mm well 24 h before transfection. The transfection of plasmid DNA was performed using Lipofectamine 2000 Reagent (Invitrogen Corp., Carlsbad, CA) as previously described (Kondo et al. 2006). After the transfection, the cells were incubated under normoxic conditions for 16 h. Then, these cells were incubated for an additional 24 h under hypoxic or normoxic conditions. The cells were harvested and the luciferase activity in the cell lysate was determined using a Dual Luciferase Reporter Assay System (Promega, Madison, WI). Luciferase activity was normalized using pTK-luc activity as a control. Values are mean ± SD for three wells.
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated using an RNEasy RNA Isolation Kit (Qiagen, Hilden, Germany). First-strand cDNA was synthesized from 1 µg of total RNA, using ReverTra Ace (Toyobo, Osaka, Japan). PCR was performed using an aliquot of first-strand cDNA as a template under standard conditions with ExTaq DNA polymerase (Takara, Siga, Japan). The cDNA for mouse VEGF, ARNT1, DEC1, DEC2, PER2 and GAPDH were amplified up to 30 cycles. The cDNA for mouse HIF-1 was amplified 32 cycles. The primers used were as follows: VEGF-F: 5'-ATGAACTTTCTGCTCTCTTGG-3', VEGF-R: 5'-TCACCGCCTTGGCTTGTCACATC-3', HIF-1-F: 5'-AGCCCTAGATGGCTTTGTGA-3', HIF-1-R: 5'-TATCGAGGCTGTGTCGACTG-3', ARNT1-F: 5'-GCTATAATCATTCCCAGGTTTCT-3', ARNT1-R: 5'-CATTGTTGTAGGTGTTGCTTTGG-3', DEC1-F: 5'-GTGAGTCACTCTCCAGTTT-3', DEC1-R: 5'-ATCCGTGTCTAGCTGTGCAAT-3', DEC2-F: 5'-ATGCTCGACAGGCTTAGGACA-3', DEC2-R: 5'-TGTGTGAGCTGAGACATGAAAC-3', PER2-F: 5'-CAGACTCATGATGACAGAGG-3', PER2-R: 5'-AGGGCTGGCTCTCACTGGACA-3', GAPDH-F: 5'-ACATTCGAGGCTCCAGTGAATTCGG-3' and GAPDH-R: 5'-ACGACATACTCAGCACCGGCCTCAC-3'. As a result of alternative splicing, there are three transcripts encoding VEGF containing 120, 164 and 188 amino acid residues (VEGF120, VEGF164 and VEGF188). The predicted size of the amplified product was 440 bp for VEGF120 and 572 bp for VEGF164. The band corresponding to VEGF188 was not amplified. The products for HIF-1, ARNT1, DEC1, DEC2, PER2 and GAPDH were 466, 768, 534, 612, 253 and 612 bp, respectively. The PCR products were separated on 1.5% (w/v) agarose gels.
SiRNA and real-time RT-PCR
Short interference RNA (siRNA) against DEC2 was synthesized by Qiagen. The sequences for the sense and anti-sense DEC2 siRNAs were 5'-r (CGUUGCAACCUAUUCUGAA) d (TT)-3' and 5'-r (UUCAGAAUAGGUUGCAACG) d (TG)-3'. The negative control siRNAs were 5'-r (UUCUCCGAACGUGUCACGU) d (TT)-3' and 5'-r (ACGUGACACGUUCGGAGAA) d (TT). The siRNA was transfected into cells using Lipofectamine 2000 reagent. After 50 h under normoxic conditions, the cells were incubated for an additional 12 h under hypoxic or normoxic conditions. RNA was prepared and subjected to a real-time quantitative-PCR analysis. The real-time PCR was performed using the SYBR Green Master Mix (Applied Bio-Science) and specific primers. The primer sets are as follows: DEC2-qF: 5'-CGCCCATTCAGTCCGACTT-3', DEC2-qR: 5'-CGGGAGAGGTATTGCAAGACTT-3', VEGF-qF: 5'-TACCTCCACCATGCCAAGTG-3', VEGF-qR: 5'-ATGATTCTGCCCTCCTCCTTCT-3', GAPDH-qF: 5'-GCACCGTCAAGGCTGAGAAC-3', GAPDH-qR: 5'-ATGGTGGTGAAGACGCCAGT-3'.
Enzyme-linked immunosorbent assay (ELISA)
The concentration of mouse VEGF in the conditioned medium was measured using an ELISA kit (R&D systems, Minneapolis, MN). This assay recognizes the soluble forms of VEGF (mouse VEGF120 and VEGF164). The values were represented as the mean ± SD for the three wells.
Western blot analysis
The cells were seeded at 1 x 105 cells per 35-mm well and lysed using M-PER lysis buffer (Pierce, Rockford, IL). Protein concentrations were determined by BCA assay. The lysates (30 µg protein) were subjected to SDS/PAGE, and proteins were transferred to PVDF membranes. The membranes were incubated with antibodies specific for DEC2 (1 : 2000) (Kondo et al. 2006), DEC1 (1 : 1000) (Sato et al. 2004), HIF-1 (1 : 1000, Abcam, Cambridge, England), ARNT1 (1 : 3000, Abcam), PER2 (1 : 500, Trans Genic Inc, Kobe, Japan) or actin (1 : 30 000) followed by a horseradish peroxidase-conjugated secondary antibody (1 : 5000). An ECL Advance Western Blotting Detection kit (Amersham, Uppsala, Sweden) was used for detection.
Immunoprecipitation
COS-7 cells were seeded at 1 x 106 cells per 100-mm well 24 h before transfection, and transfected with expression vectors using Lipofectamine 2000 reagent. After 40 h, cells were lysed in lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 1% Triton X-100. The lysates were incubated with anti-FLAG M2 agarose affinity gel (Sigma) overnight at 4 °C. Immunoprecipitates were washed 3 times with buffer containing 0.5 M Tris–HCl (pH7.4) and 1.5 M NaCl, and then were eluted in SDS sample buffer. The immunoprecipitated proteins were analyzed by Western blotting.
ChIP
ChIP was done using a kit from Upstate (Lake Placid, NY). Briefly, Sarcoma 180 and HSC-3 cells growing in 100-mm dishes under normoxic or 1% hypoxic conditions (4 h for Sarcoma 180 cells or 12 h for HSC-3 cells) were cross-linked using 1% formaldehyde at 37 °C for 7 min. After two washes with cold-PBS, cells were resuspended in 300 µL of lysis buffer (50 mM Tris–HCl (pH 8.1), 10 mM EDTA, 1% SDS and protease inhibitor cocktail mixture) and incubated on ice for 15 min. Cell lysate was sonicated to give DNA ranging in size from 200 to 900 bp. Samples were centrifuged for 10 min at 4 °C. The supernatants were diluted tenfold with dilution buffer (167 mM NaCl, 16.7 mM Tris–HCl (pH 8.1), 1.2 mM EDTA, 1.1% Triton X-100 and 0.01% SDS), pre-cleared with 60 µL of Protein G agarose for 1 h at 4 °C, and then incubated with either anti-HIF-1 antibody or normal rabbit IgG antibody over-night at 4 °C. The beads were washed with low-salt, high-salt, LiCl and TE buffers. Immunocomplexes were extracted from the beads with 1% SDS and 0.1 M NaHCO3. Cross-linking was reversed by heating the eluates at 65 °C for 10 h. DNA was purified by ethanol precipitation, and the fragment of the mouse or human Vegf promoter containing the HRE, was amplified by PCR. The primers were designed to amplify the DNA fragment containing the HRE as shown in Fig. 6C. The sequences of the primers were mouse Vegf-promoter-F: 5'-CAGTTCCCTGGCAACATCTCTG-3', mouse Vegf-promoter-R: 5'-CCAAATTTGTGGCACTGA-3', human Vegf-promoter-F: 5'-CCTTTGGGTTTTGCCAGA-3' and human Vegf-promoter-R': 5'-CCAAGTTTGTGGAGCTGA-3'.
The sizes of products were 208 and 119 bp, respectively.
Animals and immunohistochemistry
Six-week-old male C57/BL6 mice (Crea Japan, Tokyo, Japan) were housed under 12 : 12-h LD conditions for 19 days, and killed at the indicated zeitgeber (ZT-an environmental agent or event that provides the setting or resetting of the biological clock of an organism: ZT-0 corresponds to 8:00 am lights on) time on day 20, and mouse rib cartilage samples were placed within phosphate-buffered saline (PBS) and stored in a deep freezer. Mice were housed under LD conditions. Sarcoma 180 cells were implanted into the mice and RNA was prepared as previously described (Koyanagi et al. 2003). The cDNA of HIF-1, DEC2, VEGF and GAPDH was amplified by RT-PCR. To evaluate the quantitative reliability of RT-PCR, we performed a kinetic analysis of amplified products to ensure that signals were derived only from the exponential phase of amplification. The density of each band was analyzed with the use of NIH image software.
Serial paraffin sections of cartilage in rib were immunostained using DAKO ENVISION Kit/HRP (DAB) (Dako Cytomation, Kyoto, Japan). Endogenous peroxidase was blocked with 0.03% hydrogen peroxidase containing sodium azide for 10 min. This was followed by incubation for 1 h at room temperature with rabbit polyclonal anti-DEC2 antibody (1 : 50), VEGF (1 : 100; Santa Cruz, CA), HIF-1 (1 : 200) and ARNT1 (1 : 200) diluted in antibody diluent. The sections were then sequentially incubated with the Labeled Polymer, HRP for 30 min, and with a ready-to-use DAB + substrate-chromogen solution (1–3 min). Finally, the sections were counterstained with Mayer's hematoxylin. Specificity was confirmed by applying PBS instead of the primary antibodies, or by replacing the primary antibodies with non-immune serum.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: fsato{at}cc.hirosaki-u.ac.jp
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References |
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Accepted: 6 November 2007
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