NFBD1/MDC1 regulates Cav1 and Cav2 independently of DNA damage and p53

Abstract

NFBD1/MDC1 is involved in DNA damage checkpoint signaling and DNA repair. NFBD1 binds to the chromatin component γH2AX at sites of DNA damage, causing amplification of ataxia telangiectasia-mutated gene (ATM) pathway signaling and recruitment of DNA repair factors. Residues 508-995 of NFBD1 possess transactivation activity, suggesting a possible role of NFBD1 in transcription. Furthermore, NFBD1 influences p53-mediated transcription in response to adriamycin. We sought to determine the role of NFBD1 in ionizing radiation (IR)-responsive transcription and if NFBD1 influences transcription independently of p53. Using microarray analysis, we identified genes altered upon NFBD1 knockdown. Surprisingly, most NFBD1 regulated genes are regulated in both the absence and presence of IR, thus pointing toward a novel function for NFBD1 outside of the DNA damage response. Furthermore, NFBD1 knockdown regulated genes mostly independent of p53 knockdown. These genes are involved in pathways including focal adhesion signaling, carbohydrate metabolism, and insulin signaling. We found that CAV1 and CAV2 mRNA and protein levels are reduced by both NFBD1 knockdown and knockout independently of IR and p53. NFBD1-depleted cells exhibit some similar phenotypes to Cav1-depleted cells. Furthermore, like Cav1-depletion, NFBD1 shRNA increases Erk phosphorylation. Thus, Cav1 could act as a mediator of the DNA-damage independent effects of NFBD1 in mitogenic signaling.

Figure 1

Knockdown efficiencies of NFBD1b and p53 in U2OS cells. U2OS cells were infected with retroviruses expressing control, NFBD1b, or p53-targeted shRNAs. Two days later, cells were irradiated and RNA was isolated four hours after irradiation. One aliquot was used for the microarray experiment. cDNA was synthesized from the other aliquot of mRNA. Real-time PCR was carried out using primers toward A, NFBD1; B, p53; or D, p21. Error bars represent the standard error of the mean from six samples: three technical replicates from two biological replicates. C, western blots were carried out on cell lysates using the indicated antibodies.

Figure 2

Effect of NFBD1 depletion on CAV1 and CAV2 mRNA levels. A and B, U2OS cells were infected with retroviruses expressing control, NFBD1b, p53, or NFBD1a-targeted shRNAs. A, two days later, cells were irradiated and RNA was isolated four hours after irradiation. B, one day later, cells were treated with 0.6 μg/ml puromycin to select for infected cells. Four days later, RNA was isolated. A and B, cDNA was synthesized and real-time PCR was carried out using primers toward [A(i) and B(ii)] CAV1, [A(ii) and B(iii)] CAV2, or (Bi) NFBD. C, mRNA was isolated from NFBD1^+/+ or NFBD1^−/− MEFs. cDNA was synthesized and real-time PCR was carried out using primers toward (i) mNFBD1, (ii) mCAV1, or (iii) mCAV2. Error bars represent the standard error of the mean from A, six samples: three technical replicates from two biological replicates, B and C, nine samples: three technical replicates from three independent plates of cells for each condition.

Figure 3

Effect of NFBD1 depletion on Cav1 and Cav2 protein levels. A and B, U2OS cells or C, HFF cells were infected with the indicated shRNAs. A–C, two days after infection, cells were lysed. A, the NFBD1, p53, and beta–tubulin loading control blots are also shown in Fig. 1C (four left-most lanes). D, NFBD1^+/+ and NFBD1^−/− MEFs were lysed. A–D, western blots were carried out on lysates using the indicated antibodies.

Figure 4

Effect of NFBD1 depletion on focal adhesions and actin cytoskeleton. A–C, U2OS cells were infected with control, Cav1, or NFBD1a shRNA-expressing retroviruses. A and B, two days after infection, cells were plated on glass chamber slides coated with fibronectin. D, NFBD1^+/+ and NFBD1^−/− MEFs were plated on fibronectin-coated glass chamber slides. A, B, D, immunofluorescence was carried out using vinculin, NFBD1, or Cav1 antibodies. Actin was stained with Texas Red–phalloidin. C, western blots were carried out on lysates using the indicated antibodies.

Figure 5

Effect of NFBD1 depletion on scratch filling. A, U2OS cells were infected with control, Cav1, or NFBD1a shRNA-expressing retroviruses. Cells were allowed to grow three days after infection to reach confluency. B, NFBD1^+/+ and NFBD1^−/− MEFs were plated to reach confluency. A and B, a scratch was made with a pipette tip. Cells were immediately washed twice with DMEM with 0.1% FBS and then incubated in Dulbecco’s modified eagle medium (DMEM) with 0.1% FBS for the indicated time points. The horizontal line represents a scratch on the outside of the plate to ensure that the same part of the scratch was examined at both time points. The percentage difference between the width of the scratch at the 0-hour time point and the 24-hour or 48-hour time point is plotted. Error bars represent the standard deviation among six samples, three different scratches from each of A, three or B, two biological replicates.

Figure 6

Effect of NFBD1 depletion on Erk phosphorylation. U2OS cells were infected with the indicated shRNA-expressing retroviruses. Three days after infection, cells were replated to a lower density (30%–50% confluency) then lysed one day later. Western blots were carried out on lysates using the indicated antibodies.

Figure 7

Effect of restoring Cav1 levels on NFBD1 depletion. U2OS cells were stably transfected with control or an HA-Cav1 plasmid. These cells were infected with either control or NFBD1a shRNA-expressing retroviruses. A and D, western blots were carried out on cell lysates using the indicated antibodies. B, cells were plated onto fibronectin coated chamber slides and immunofluorescent analysis was carried out using the indicated antibodies. Actin was stained with Texas Red-phalloidin. Scale bars are 100 μm. C, scratch assays were carried out as in Fig. 5 on the cells. (Top) Results are quantified, and error bars represent the standard deviation of three biological replicates. Representative images 48 hours after the scratches were made (bottom).