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First published online 16 March 2004
doi: 10.1242/jcs.01026

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Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism

¹ Department of Pediatric Oncology/Hematology, Charité Campus Virchow-Klinikum, Medical University of Berlin, 13353 Berlin, Germany
² Department of Neonatology, Charité Campus Virchow-Klinikum, Medical University of Berlin, 13353 Berlin, Germany
³ Department of Clinical Molecular Biology, Kyoto University, Kyoto 606-8507, Japan

View larger version (49K): [in a new window]   Fig. 1. Expression of genes encoding HIF-1 (HIF1A) and HIF-2 (EPAS1) in Z-33, REH and Hep3B cells. The human B-cell acute lymphoblastic leukemia cell lines Z-33 and REH as well as the hepatoblastoma cell line Hep3B (Hep) were cultured in normoxia. Ribonuclease protection analysis using 30 µg of total RNA revealed no expression of HIF1A RNA in Z-33 in contrast to REH and Hep3B. The latter depicted strong signal intensity for EPAS1 whereas very little EPAS1 signal was obtained in Z-33 or REH cells. Probing against U6 small nuclear RNA (RNU6) was used for loading control. Complete ribonuclease digestion of 32P-riboprobes in the absence of added RNA was demonstrated on the right lane (Con).

View larger version (38K): [in a new window]   Fig. 2. Agarose gel electrophoresis of PCR products indicating homozygous deletion of the HIF-1 gene in Z-33. (A) Genomic PCR of DNA of Z-33 and REH was performed for the HIF-1 gene (HIF1A exon/intron-3, lane 3; HIF1A exon-12, lane 4) and HIF-1 flanking gene regions: proximal (prox) to the HIF-1 locus marker D14S592, lane 1 and D14S1258, lane 2; distal (dist) D14S1334, lane 5 and D14S183, lane 6; ß-globin, lane 7; and 100-bp DNA size marker, lane M. Fig. 2B shows the positions of the different markers relative to the HIF-1 gene on the chromosome band 14 q23.1 and q23.2, numbered according to the lanes.

View larger version (28K): [in a new window]   Fig. 3. Hypoxia induces RBM3 and CIRP in HIF-1-deficient Z-33 cells as well as in murine ARNT-deficient Hepa-1 c4 cells. Z-33, REH, Hepa-1 wt and c4 cells were cultured in parallel in normoxia or 1% oxygen for 24 hours. (A,B) Whole-cell lysates were isolated and subjected to western blot analysis for RBM3, CIRP and HIF-1 in Z-33 and REH cells. To control sample loading and transfer, the blots were stripped and reprobed for ß-actin. (C) RNA was isolated and subjected to real-time RT-PCR analysis. RBM3 (R), CIRP (C), and VEGF (V) RNA levels were normalized to RPL13a RNA levels and are expressed as fold change in the different experiment samples compared with the corresponding normoxic samples (mean±s.e.m., n=3).

View larger version (38K): [in a new window]   Fig. 4. Mild and severe hypoxia as well as hypothermia induce RBM3 and CIRP expression. HeLa and Hep3B cells were exposed to either normoxia (N), 1% oxygen (1%), 8% oxygen (8%), desferrioxamine (D) or 32°C-hypothermia (C) for 24 hours. RNA was isolated and subjected to real-time RT-PCR analysis. RBM3, CIRP and VEGF RNA levels were normalized to B2M RNA levels and are expressed as fold change in the different experiment samples compared with the corresponding normoxic samples (mean±s.e.m., n=3).

View larger version (56K): [in a new window]   Fig. 5. Mild hypoxia as well as hypothermia increase RBM3 and CIRP protein but not HIF-1, whereas desferrioxamine (DFO) does not affect RBM3 and CIRP protein. HeLa cells were cultured in parallel in normoxia (N), 8% oxygen (8%), DFO or 32°C-hypothermia (cold) for 24 hours. Whole-cell lysates were isolated and subjected to western blot analysis for RBM3, CIRP and HIF-1. To control sample loading and transfer, the blots were stripped and reprobed for ß-actin.

View larger version (22K): [in a new window]   Fig. 6. Hypoxia induces a persistent increase in RBM3 and CIRP protein levels via de novo mRNA synthesis. (A) HeLa cells were cultured in parallel in normoxia or 1% oxygen as indicated. Whole-cell lysates were isolated and subjected to western blot analysis for RBM3 and CIRP. To control sample loading and transfer, the blots were stripped and reprobed for ß-actin. (B) HeLa cells were cultured in parallel in normoxia or 1% oxygen exposed to actinomycin-D for 24 hours. RNA was isolated and subjected to real-time RT-PCR analysis. RBM3 and CIRP mRNA levels were normalized to ß2-microglobulin (B2M) mRNA levels and are expressed as fold change in the different experiment samples compared with the corresponding normoxic samples (mean±s.e.m., n=3). (C) HeLa cells were cultured in parallel in normoxia or 1% oxygen as indicated for 24 hours. Nuclei were isolated, and in vitro transcription was allowed to resume for 40 minutes. RNA was isolated before and after in vitro transcription and subjected to real-time RT-PCR analysis. The extent of RBM3 and CIRP mRNA transcription was determined by subtracting the amount of RBM3 and CIRP mRNA standardized to B2M prior to transcription from the amounts post transcription (mean±s.e.m., n=3). Results are given as a ratio of copies of target gene (RBM3 or CIRP) to copies of the reference gene (B2M).

View larger version (37K): [in a new window]   Fig. 7. The mitochondrial inhibitors NaN3 and CCCP inhibit RBM3 and CIRP induction by hypoxia but superinduce VEGF expression by hypoxia. HeLa cells were cultured in parallel in normoxia or 1% oxygen as indicated for 24 hours. Different concentrations of either NaN3 or CCCP were added as indicated. (A) RNA was isolated and subjected to real-time RT-PCR analysis. RBM3, CIRP and VEGF RNA levels were normalized to B2M RNA levels and are expressed as fold change in the different experiment samples compared with the corresponding normoxic samples (mean±s.e.m., n=3). To control for cell viability, cells were harvested and incubated with annexin V and propidiun iodide. Cell viability is given as the percentage of cells negative for both annexin V and propidium iodide staining (mean±s.e.m., n=3). (B) Annexin V reactivity and propidium iodide uptake of control HeLa cells (left dot blot), HeLa cells exposed to 5 mM NaN3 (center dot blot) or HeLa cells exposed to 20 µM CCCP (right dot blot) for 24 hours.

View larger version (19K): [in a new window]   Fig. 8. Induction of RBM3 and CIRP in response to hypoxia does not require mitochondria. mtDNA was depleted from HeLa cells with EB in two different concentrations for 6 days as indicated. Subsequently cells were cultured in parallel in normoxia or 1% oxygen as indicated for 24 hours. Parental (WT) and rho0 143B cells, which are devoid of mtDNA, were used as control. Total DNA and RNA was isolated. The upper diagram shows the relative percentage (amount relative to the untreated control cell lines) of mtDNA, assessed by quantitative real-time PCR (see Materials and Methods). RNA was subjected to real-time RT-PCR analysis; RBM3, CIRP and VEGF RNA levels were quantitated, normalized to B2M RNA levels and are expressed as fold change in the different experiment samples compared with the corresponding normoxic samples (mean±s.e.m., n=3).

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