Article Figures & Data
Figures
- FIG. 1.
Cloning of the murine Dmp1 promoter and the sequence of the proximal region. The murine Dmp1 promoter was cloned from the Bacterial Artificial Chromosome library derived from 129/Svj mice (Mouse ES Release II; Genome Systems Inc.) with 60-bp synthetic oligonucleotides covering the 5′ end of the murine Dmp1 cDNA. The 1.8-kb PstI fragment hybridizing with the probe was cloned into the polylinker site of the pGL2-basic vector (Promega). (A) Deletion analysis of the Dmp1 promoter. NIH 3T3, BALB/3T3, and C33A cells were transfected with 4 μg of murine Dmp1 promoter-luciferase constructs and 4 μg of secreted alkaline phosphatase expression vector driven by the actin promoter. Relative luciferase levels corrected by the internal control alkaline phosphatase levels are shown. Deletion of the promoter up to the NsiI site did not influence the endogenous promoter activity, while deletion of the promoter to the ApaI site resulted in a 4- to 10-fold decrease in the relative luciferase levels. (B) Nucleotide sequences of the proximal region (−414 to +65). The major transcription initiation sites determined by 5′-RACE are shown in large capital letters. Consensus sequences for possible transcription factor binding are also shown.
- FIG. 2.
Responsiveness of the Dmp1 promoter to various oncogenic stimuli. (A) The Dmp1 promoter is weakly activated by oncogenic RasV12 but not by c-Myc or E2F-1 in 3T3 cells. NIH 3T3 cells were transfected with the luciferase reporter −374 NsiI with expression vectors for c-Myc, E2F-1, and Ha-RasV12 driven by the CMV promoter. The numbers show the fold activation of the luciferase reporter corrected by the internal control secreted endocrine alkaline phosphatase levels. (B) p19Arf dependence of the response of the Dmp1 promoter to RasV12. Reporter assays were performed in passage-5 wild-type MEFs (left panel), Arf-null MEFs (middle panel), and Arf-null MEFs with p19Arf expression vector (right panel). The Dmp1 promoter was efficiently activated by RasV12 in wild-type cells but not in Arf-null cells. The loss of responsiveness of the Dmp1 promoter to RasV12 in Arf-null cells was restored by cotransfecting the RasV12 and p19Arf expression vectors. (C) The Dmp1 promoter is activated by RasV12S35 but not by RasV12G37 or by RasV12C40. A Dmp1 reporter assay was performed in wild-type MEFs with RasV12 double mutants (RasV12S35, RasV12G37, and RasV12C40). The Dmp1 promoter was activated most efficiently by RasV12S35, suggesting that the MAPK pathway plays the most significant role in Dmp1 promoter activation in response to oncogenic Ras signaling. (D) Activation of the Dmp1 promoter by MAPK pathways is inhibited by the MEK/ERK inhibitor U0126. In order to study which MAPK pathway is key to RasV12-mediated Dmp1 promoter activation, reporter assays were performed with pCMV-RasV12 in the presence of 10 μM U0126, 10 μM SP600125, 10 μM SB203580, or a combination of these compounds. Activation of the Dmp1 promoter by RasV12 was efficiently blocked by U0126.
- FIG. 3.
Dmp1 plays a key role in c-Raf-induced senescence. (A) Ha-RasV12 and activated ΔRaf:ER induce Dmp1 mRNA. Passage-5 wild-type MEFs were infected with retroviruses expressing Ha-RasV12 (left panel) or ΔRaf:ER (right panel) and selected with puromycin. Northern blotting was performed with a Dmp1-specific probe with β-actin as an internal control. Expression of Ha-RasV12 increased Dmp1 mRNA by threefold at day 3 to 4 (left panel). Activation of ΔRaf:ER by 2 μM 4-HT increased Dmp1 mRNA by threefold at 8 h and by eightfold at 16 to 24 h (right panel). (B) Activated ΔRaf:ER induces the Dmp1 protein. The increase of Dmp1 mRNA resulted in Dmp1 protein induction by threefold at 8 h and eightfold by 24 h. The kinetics was slower than for phosphor-ERK accumulation but very similar to that of cyclin D1 induction. (C) Dmp1-null cells are resistant to Raf-induced cell cycle arrest. In order to study the biological effects of c-Raf activation in Dmp1−/− cells, both wild-type and Dmp1-null MEFs were infected with retroviruses encoding ΔRaf:ER or the control ER vector. After selection with puromycin, 105 cells were plated in 60-mm-diameter dishes and 1 μM 4-HT was added to activate ΔRaf:ER. (Open symbols, with 4-HT; closed symbols, without 4-HT). Note that Dmp1-null cells expressing ΔRaf:ER (triangles) grew at the same rate as those expressing ER vector alone (rectangles), while wild-type cells expressing activated ΔRaf:ER underwent irreversible cell cycle arrest. (D) p19Arf does not increase in response to ΔRaf:ER in Dmp1-null cells. None of p19Arf, p53, and p21Cip1 increased in response to ΔRaf:ER in Dmp1-null cells, while p16Ink4a levels remained constant in both wild-type and Dmp1-null cells.
- FIG. 4.
Mapping of the RasV12-responsive element on the murine Dmp1 promoter. (A) Mapping of the RasV12-responsive element on the Dmp1 promoter based on a luciferase assay. Reporter assays were performed in IMR-90 cells with the deletion mutants described in Fig. 1A and their derivatives with (white columns) or without (black columns) Ha-RasV12 expression vector. (B) Kinetics of Fos and Jun family protein accumulation in cells with activated c-Raf. Wild-type MEFs were infected with vector ER or ΔRaf:ER retroviruses and were stimulated with 4-HT. The lysates were analyzed by Western blotting with specific antibodies. (C) Identification of the transcription factors that bind to the AP-1-like sequence on the Dmp1 promoter by EMSA. Lysates were prepared from wild-type MEFs expressing ΔRaf:ER or ER alone and stimulated with 2 μM 4-HT for 16 h. Complex A formation was antagonized by a 100-fold excess of cold oligonucleotides derived from the AP-1-like sequence on the Dmp1 promoter, as well as classical AP-1 or CREB consensus sequences reported earlier. The complex was supershifted only with antibodies to c-Fos, Fra-1, c-Jun, phospho-c-Jun, JunB, and JunD (arrows, S). (D) ChIP assay of the Dmp1 promoter. Significant amounts of c-Fos, Fra-1, c-Jun, JunB, and JunD were detected on the Dmp1 promoter in response to activated Raf signaling. The specificity of the Fos/Jun signals was confirmed by control PCR amplification of the Dmp1 promoter sequence located 2 kb upstream from the transcription initiation site (24 h cont).
- FIG. 5.
Synergism of Dmp1 promoter activation by AP-1 proteins and RNAi assay. (A) Synergistic activation of the Dmp1 promoter by AP-1 family proteins. Synergism among Fos and Jun family proteins was tested in IMR-90 cells. For reporter assays, 0.5 to 1 μg of Fos/Jun expression vectors per dish were used for transfections in the left panel, while 0.25 μg of expression vectors were used per dish in the right panel to study synergism. Significant synergism was found between c-Jun and c-Fos or Fra-1, JunB and c-Fos or Fra-1. (B) Downregulation of Fos and Jun family proteins by siRNA. Wild-type MEFs were infected with retroviruses that produce siRNA, and puromycin-resistant cells were expanded. The lysates were studied for target protein expression with specific antibodies. (C) RasV12 does not activate the Dmp1 promoter in cells with downregulated c-Jun or JunB. Reporter assays were performed with the −88 ApaI Dmp1 promoter in MEFs with downregulated AP-1 proteins. Dmp1 promoter activation by RasV12 was completely attenuated in cells where c-Jun or JunB proteins were knocked down, whereas c-Fos, Fra-1, Fra-2, or JunD proteins were dispensable for Dmp1 promoter activation by Ras. (D) Restoration of the Ras responsiveness of the Dmp1 promoter in Jun knock-down cells. Reporter assays were performed in c-Jun or JunB knock-down (K.D.) MEFs with c-Jun or JunB and RasV12 expression vectors. The responsiveness of the Dmp1 promoter to RasV12 was completely restored by transfection of c-Jun or JunB knock-down cells with a c-Jun or JunB expression vector, respectively.
- FIG. 6.
Dmp1 plays a key role in Arf promoter activation by RasV12. (A) Mapping of the RasV12-responsive element on the Arf promoter. (Left panel) Reporter assay performed with either wild-type or mutant murine Arf promoter to study the importance of the Dmp1/Ets site in response to oncogenic Ras activation. Arf promoter activation was almost completely abolished by mutating the Dmp1/Ets binding site (17). (Middle panel) Reporter assay performed with wild-type Arf promoter in both wild-type and Dmp1-null MEFs. Oncogenic Ras does not activate the Arf promoter in Dmp1-null cells. (Right panel) Restoration of the Ras responsiveness of the Arf promoter in Dmp1-null cells. The responsiveness of the Arf promoter to RasV12 was completely recovered by transfection of Dmp1-null cells with the Dmp1-expression vector. (B) ChIP assay on the Arf promoter. Endogenous Dmp1 protein was found on the Arf promoter in response to oncogenic Raf signaling (lanes 1 and 2). Lanes 3 and 4, no antibodies were used for the immunoprecipitation; lanes 5 and 6, signals from total chromatin samples. (C) Cyclin D1 does not inhibit the Dmp1 activity on the Arf promoter. A reporter assay was performed in NIH 3T3 cells with expression vectors for Dmp1 and cyclins. Dmp1 and D-type cyclins additively activated the Arf promoter, while other cyclins (cyclin A and cyclin H) had little effect on Arf promoter activation by Dmp1. The K114E cyclin D1 mutant (D1 KE) that does not interact with Cdk4 did not influence Dmp1's activity on the Arf promoter, suggesting that the collaborative effect was dependent on Cdk4 activation. (D) Detection of cyclins in transfected cells. The expression of cyclins was confirmed by Western blotting of lysates from the luciferase assay with specific antibodies. Lane 1, pFLEX1 vector only; lane 2, Flag-tagged cyclin D1; lane 3, cyclin D1 KE mutant; lane 4, cyclin D2; lane 5, cyclin D3; lane 6, cyclin A; lane 7, cyclin H.
- FIG. 7.
The novel Jun-Dmp1 pathway. Dmp1 is a key molecule linking Ras-Raf-MEK-ERK oncogenic signaling and the Arf-Mdm2-p53 tumor suppressor pathway. The pathway mediated by Dmp1 directly links oncogenic Ras-Raf signaling and p19Arf through activation of c-Jun/JunB proteins. Oncogenic Ras activates both the Jun-Dmp1 pathway and the classical cyclin D1/Cdk4-Rb-E2F pathway to activate Arf gene expression to achieve premature senescence. Since the Ras-Raf pathway activates the Mdm2 promoter as well, the levels of p53 are delicately regulated by the activity of the Dmp1-Arf and AP-1/Ets-Mdm2 pathways.
