The Impact of HIF1α on the <i>Per2</i> Circadian Rhythm in Renal Cancer Cell Lines

<div><p>In mammals, the circadian rhythm central generator consists of interactions among clock genes, including <i>Per1/2/3</i>, <i>Cry1/2</i>, <i>Bmal1</i>, and <i>Clock</i>. Circadian rhythm disruption may lead to increased risk of cancer in humans, and deregulation of clock genes has been implicated in many types of cancers. Among these genes, <i>Per2</i> is reported to have tumor suppressor properties, but little is known about the correlation between <i>Per2</i> and HIF, which is the main target of renal cell carcinoma (RCC) therapy. In this study, the rhythmic expression of the <i>Per2</i> gene was not detectable in renal cancer cell lines, with the exception of Caki-2 cells. In Caki-2 cells, HIF1α increased the amplitude of <i>Per2</i> oscillation by directly binding to the HIF-binding site located on the <i>Per2</i> promoter. These results indicate that HIF1α may enhance the amplitude of the <i>Per2</i> circadian rhythm.</p></div

The impact of HIF1α/ARNT on <i>Per2</i> transcriptional activity.

<p>(<b>A</b>) NIH3T3 cells were co-transfected with the <i>Per2</i> promoter reporter (400 ng) and the indicated expression plasmids (300 ng) for HIF1α/ARNT or empty vector pcDNA3 (600 ng) as a control. Bioluminescence was then measured using a real-time monitoring assay. Control, transfected with empty vector pcDNA3 (uncloned-vector control); + HIF1α/ARNT, transfected with the expression plasmids. Luciferase activities of four replicate samples are shown. (<b>B</b>) Detrended bioluminescence is shown. Period, amplitude, and acrophase of the oscillations were measured on days 2 to 5 using the Cosinor software (Circadian Rhythm Laboratory). Amplitude significantly increased (mean ± SEM, <i>n</i> = 4) compared to the control (<i>p</i><0.01, Student's <i>t-</i>test). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109693#pone-0109693-t002" target="_blank">Table 2</a>. (<b>C</b>) U2OS cells were co-transfected with the <i>Per2</i> promoter reporter (400 ng) and the indicated expression plasmids (300 ng) for HIF1α/ARNT or empty vector pcDNA3 (600 ng) as a control. Bioluminescence was then measured using a real-time monitoring assay. Control, transfected with empty vector pcDNA3 (uncloned-vector control); + HIF1α/ARNT, transfected with the expression plasmids. Luciferase activities of four replicate samples are shown. (<b>D</b>) Detrended bioluminescence is shown. Period, amplitude, and acrophase of the oscillations were measured on days 2 to 5 using the Cosinor software (Circadian Rhythm Laboratory). Amplitude significantly increased (mean ± SEM, <i>n</i> = 4) compared to the control (<i>p</i><0.01, Student's <i>t-</i>test). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109693#pone-0109693-t002" target="_blank">Table 2</a>. (<b>E</b>) Caki-2 cells were co-transfected with the <i>Per2</i> promoter reporter (2 µg) and the indicated expression plasmids (1.5 µg) for HIF1α/ARNT or empty vector pcDNA3 (3 µg) as a control. The bioluminescence was then measured using a real-time monitoring assay. Control, transfected with empty vector pcDNA3 (uncloned-vector control); + HIF1α/ARNT, transfected with the expression plasmids. The luciferase activities of four replicate samples are shown. (<b>F</b>) Detrended bioluminescence is shown. Period, amplitude, and acrophase of the oscillations were measured from days 2 to 5 using the Cosinor software (Circadian Rhythm Laboratory). Amplitude significantly increased (mean ± SEM, <i>n</i> = 4) compared to the control (<i>p</i><0.01, Student's <i>t</i>-test). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109693#pone-0109693-t002" target="_blank">Table 2</a>.</p

Rhythmic expression of <i>Per2</i> in Caki-2 cells.

<p>(<b>A</b>) All renal cancer cell lines were transfected with the Per2 promoter reporter (2 µg) and the bioluminescence was then measured using a real-time monitoring assay. Real-time monitoring of luciferase activity of the <i>Per2</i> promoter showed that activity oscillated over an approximately 24-h cycle. The luciferase activities of four replicate samples are shown. These cultures showed significant circadian rhythms (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109693#pone-0109693-t001" target="_blank">Table 1</a>). (<b>B</b>) mRNA levels of <i>Per2</i> were determined by real-time PCR for six plates at each time point. Total RNA was extracted every 4 h, beginning 24 h after treatment with dexamethasone for one 24-h cycle, and <i>Per2</i> transcripts were quantified. Error bars indicate the standard errors of the mean values (<i>n</i> = 6). The data from a single 24 hours after dexamethasone treatment were analyzed using the Cosinor software for rhythmicity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109693#pone-0109693-t001" target="_blank">Table 1</a>). (<b>C</b>) The structure of the <i>Per2</i> promoter and an analysis of the potential transcription factor-binding motifs in this region. The 2,994-bp region contains one E-box-like sequence (CACGTT) and one HRE-like sequence (ATGTG), similar to the consensus HRE sequence (ACGTG) located upstream of the transcription start site (TSS). (<b>D</b>) Sequence comparisons: upper line, mouse sequence; lower line, human sequence. The nucleotide sequence of potential transcription factor-binding motifs for E-box-like sequence and HRE-like sequence are 100% conserved between mouse and human.</p

The effect of HIF1α inhibition on <i>Per2</i> circadian rhythm in Caki-2 cells.

<p>(<b>A</b>) Caki-2 cells were cultured to 60–70% confluence. The cells were treated with DMSO as a control, or different chrysin concentrations (1, 10, 100 µM) for 2 h. (<b>B</b>) HIF1α protein levels were measured in optical density values normalized to their respective GAPDH loading control, then averaged ± SEM, and graphed (relative expression) to semiquantitatively compare protein levels (<i>n</i> = 4). HIF1α expression was significantly suppressed by 100 nM chrysin compared to the control incubated with DMSO (<i>p</i><0.05, one-way ANOVA followed by Tukey's post hoc test). (<b>C</b>) Caki-2 cells were transfected with the <i>Per2</i> promoter reporter (2 µg). Twenty-four hours after the transfection, cells were incubated with 100 µM chrysin or DMSO for 2 h. Bioluminescence was then measured using a real-time monitoring assay. The luciferase activities of four replicate samples are shown. (<b>D</b>) Detrended bioluminescence is shown. Period, amplitude, and acrophase of the oscillations were measured on days 2 to 5 using the Cosinor software (Circadian Rhythm Laboratory). Amplitude significantly decreased (mean ± SEM, <i>n</i> = 4) compared to the control (<i>p</i><0.05). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109693#pone-0109693-t003" target="_blank">Table 3</a>.</p

<b>Table 3.</b> Circadian parameters of <i>Per2</i> promoter activities based on four days of data in Caki-2 cells.

<p>Period, amplitude, and acrophase of the oscillations were measured on days 2 to 5 using the Cosinor software (Circadian Rhythm Laboratory). Amplitude significantly decreased (mean ± SEM, <i>n</i> = 4) compared to the control (* <i>p</i><0.05).</p><p><b>Table 3.</b> Circadian parameters of <i>Per2</i> promoter activities based on four days of data in Caki-2 cells.</p

<b>Table 2.</b> Circadian parameters of <i>Per2</i> promoter activities based on four days of data.

<p>Period, amplitude, and acrophase of the oscillations were measured on days 2 to 5 using the Cosinor software (Circadian Rhythm Laboratory). Amplitude significantly increased (mean ± SEM, <i>n</i> = 4) compared to the control (** <i>p</i><0.01, Student's <i>t-</i>test).</p><p><b>Table 2.</b> Circadian parameters of <i>Per2</i> promoter activities based on four days of data.</p

Western blot analysis of the indicated proteins.

<p>All cell lines were lysed and harvested 24 h after synchronization by 2-h dexamethasone treatment. We performed four replicate Western blots; a representative blot is shown. (<b>A</b>) Western blots of renal cancer whole-cell extracts (20 µg) with BMAL1, CLOCK, PER2, CRY1 and GAPDH antibodies are shown. Full-length blots of PER2 and BMAL1, in addition to a positive control, are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109693#pone.0109693.s002" target="_blank">Figure S2</a>, 3. (<b>B</b>) Western blots of renal cancer whole-cell extracts (20 µg) with HIF1α and GAPDH antibodies are shown.</p

The effects of HIF1α/ARNT on the <i>Per2</i> promoter.

<p>(<b>A</b>) Schematic representation of the mouse <i>Per2</i> promoter. The upper area represents the wild-type mouse <i>Per2</i> promoter and the lower area represents the HRE-mutant <i>Per2</i> promoter. (<b>B</b>) HIF1α/ARNT potently induced <i>Per2</i> promoter activity. The <i>Per2</i> promoter and the HRE-mutant <i>Per2</i> promoter reporter (60 ng) were co-transfected with the indicated expression plasmids (+; 50 ng). <i>Per2</i> promoter activities were significantly increased (mean ± SEM, <i>n</i> = 4, <i>p</i><0.01, Student's <i>t</i> test) compared to the control (without the expression plasmid), but the HRE-mutant <i>Per2</i> promoter was not affected. (<b>C</b>) Twenty-four hours after treatment with CoCl₂ (10, 30, 100 µM for 6 h), luciferase activity was measured. <i>Per2</i> promoter activities were significantly increased (mean ± SEM, <i>n</i> = 4, <i>p</i><0.05, one-way ANOVA followed by Tukey's post hoc tests) concentration-dependently compared to the control, but the HRE-mutant <i>Per2</i> promoter was not affected. (<b>D</b>) HIF1α specifically interacts with the HRE-like sequence within the <i>Per2</i> promoter. Caki-2 cells were cross-linked, lysed, and immunoprecipitated with anti-HIF1α antibody or normal rabbit IgG (negative control). The precipitated DNA was subjected to PCR with primers specific for the target region (−476/−284). One aliquot of input DNA was used as a positive control. PCR product was observed in the anti-HIF1α ChIP (lane 3) and 10% Input DNA (lane 4). Substantially less was detected in the no antibody ChIP (lane 1) and normal rabbit IgG ChIP (lane 2) lanes.</p

<b>Table 1.</b> Circadian parameters of <i>Per2</i> promoter activities and mRNA in Caki-2 cells.

<p>Promoter activity and mRNA levels of <i>Per2</i> showed significant circadian rhythms (<i>p</i><0.000001, <i>p</i><0.05, by Cosinor).</p><p><b>Table 1.</b> Circadian parameters of <i>Per2</i> promoter activities and mRNA in Caki-2 cells.</p

The effects of HIF1α/ARNT on the number of NIH3T3 cells.

<p>NIH3T3 cells were co-transfected with the Per2 promoter reporter (400 ng) and the indicated expression plasmids (300 ng) for HIF1α/ARNT or empty vector pcDNA3 (600 ng) as a control. Plates were read on the ArrayScan XTI (Thermo Scientific) for cell count indicated time after dexamethasone treatment. Numbers of viable cells were not affected by HIF1α/ARNT at all time points (mean ± SEM, <i>n</i> = 6, Student's <i>t</i>-test).</p