Transcriptional Activation of the Cholecystokinin Gene by DJ-1 through Interaction of DJ-1 with RREB1 and the Effect of DJ-1 on the Cholecystokinin Level in Mice

DJ-1 is an oncogene and also causative gene for familial Parkinson’s disease. DJ-1 has multiple functions, including transcriptional regulation. DJ-1 acts as a coactivator that binds to various transcription factors, resulting in stimulation or repression of the expression of their target genes. In this study, we found that the cholecystokinin (CCK) gene is a transcriptional target gene for DJ-1. CCK is a peptide hormone and plays roles in contraction of the gallbladder and in promotion of secretion of pancreatic fluid. CCK is co-localized with dopamine in the substantia nigra to regulate release of dopamine. Reduced expression of CCK mRNA was observed in DJ-1-knockdown cells. The Ras-responsive element (RRE) and Sp1 site were essential for promoter activity, and DJ-1 stimulated promoter activity by binding to RRE-binding protein 1 (RREBP1). The complex of DJ-1 with RREB1 but not with Sp1 bound to the RRE. Furthermore, the reduced CCK level in the serum from DJ-1-knockout mice compared to that from wild-type mice was observed. This is the first report showing that DJ-1 participates in peptide hormone synthesis

Nucleotide sequences of primers and PCR conditions used for RT-PCR and real-time PCR.

<p>R: RT-PCR; Q: Real-time PCR.</p

Reduced CCK level in the serum from DJ-1 knockout mice.

<p>Serum was isolated from wild-type and DJ-1-knockout mice at 23 weeks of age and the amount of CCK in serum was measured using an LC-MS as described in Materials and methods. Average amounts of CCK in wild-type and DJ-1-knockout mice are 21.9 and 14.6 µM, respectively. n = 4.</p

Reduction of <i>CCK</i> gene expression in DJ-1-knockdown cells.

<p>A and C. Relative mRNA levels of CCK and DJ-1 were examined by semi-quantitative RT-PCR in NIH3T3 and its DJ-1-knockdown D2 cells (A) and in SH-SY5Y and its DJ-1-knockdown KD4 cells (C). Actin mRNA was also amplified by PCR as loading controls. Reverse images of black and white staining are shown. B and D. Relative mRNA levels of CCK and DJ-1 were examined by quantitative RT-PCR (real-time PCR) in NIH3T3 and its DJ-1-knockdown D2 cells (B) and in SH-SY5Y and its DJ-1-knockdown KD4 cells (D). Actin mRNA was also amplified by real-time PCR as loading controls. All of the experiments were carried out 4 times (n = 4).</p

Association of DJ-1 with RREB1 on the LDLR promoter.

<p>A. Chromatin immunoprecipitation assays were carried out using chromatin prepared from SH-SY5Y cells. Chromatin was immunoprecipitated with anti-DJ-1, anti-RREB1 and anti-Sp1 antibodies or non-specific IgG. After extraction of DNA from precipitated chromatin, two regions spanning −1579 to −1291 and spanning −302 to −14 were amplified by PCR with specific primers and with amplified DNA and were separated on agarose gels as described in Materials and methods. n = 3. B. Proteins in SH-SY5Y nuclear extracts were immunoprecipitated with anti-RREB1, anti-DJ-1 and anti-Sp1 antibody or IgG. Immunoprecipitates were then analyzed by Western blotting with anti-RREB1, anti-DJ-1 and anti-Sp1 antibodies. n = 3. C. Nucleotide sequences used for labeled probe and competitors are shown (C-a). EMSA was carried out using SH-SY5Y cell nuclear extracts, IRDye800-labeled wild-type prove and non-labeled wild-type or mutated RRE competitors as described in Materials and methods (C-b). n = 3. MSA was carried out using SH-SY5Y cell nuclear extracts and 100 ng of recombinant DJ-1 on IRDye800-labeled wild-type prove (C-c). D. SH-SY5Y cell nuclear extract was first incubated with the IRDye800-conjugated probe and then incubated with 2 µg of anti-DJ-1, anti-RREB1 and anti-Sp1antibodies or non-specific IgG for 30 min at 4°C and subjected to EMSA. n = 3. E. Schematic model of DJ-1, RREB1 and Sp1 on the CCK promoter. DJ-1 complexed with RREB1 and Sp1 bind to the RRE and Sp1 site, respectively, to activate the CCK promoter.</p

Stimulation of CCK promoter activity by DJ-1 along with RREB1.

<p>A–C. D2 cells were transfected with various amounts of expression vectors for DJ-1-HA (A), FLAG-RREB1 (B) and T7-Sp1 (C) along with pGL3-CCK-1615. Forty-eight hrs after transfection, cell extracts were prepared and their luciferase activity was measured as described in Materials and methods. n = 4. D and E. D2 cells were transfected with various amounts of expression vectors for DJ-1-HA along with fixed amount of FLAG-RREB1 (D) or T7-Sp1 (E) and pGL3-CCK-1615. Forty-eight hrs after transfection, cell extracts were prepared and their luciferase activity was measured as described in Materials and methods. n = 4. Statistical analyses were performed using the Tukey-Kramer test.</p

Stimulation of promoter activity of the <i>CCK</i> gene by DJ-1.

<p>A. Various deletion constructs of the CCK promoter linked to the <i>luciferase</i> gene were constructed and transfected into D2 cells together with pEF or pEF-DJ-1-HA. Forty-four hrs after transfection, cell extracts were prepared and their luciferase activity was measured as described in Materials and methods. n = 6. B. pGL3-CCK-1615 (WT) and reporter constructs of pGL3-CCK-1615 with deletion of recognition sites of either RREB1 (del- RREB1) or Sp1 (del-Sp1) and of both RREB1 and Sp1 (del-Sp1/RREB1) were transfected into NIH3T3 and D2 cells. Forty-four hrs after transfection, cell extracts were prepared and their luciferase activity was measured as described in Materials and methods. n = 4. C. NIH3T3 cells were transfected with RREB1 siRNA or with control siRNA. a) At 48 hrs after transfection, proteins were extracted from cells and analyzed by Western blotting with anti-RREB1 and anti-actin antibodies. b) At 48 hrs after transfection, total RNAs were extracted from cells, and the expression levels of <i>CCK</i> and <i>actin</i> mRNA were examined by real-time PCR, and relative expression of CCK versus actin is shown. c) At 24 hrs after siRNA transfection, pGL3-CCK-1615 (WT) was transfected into siRNA-transfected cells and their luciferase activities were measured at 48 hrs after transfection of pGL3-CCK-1615 (WT). n = 3. D. D2 cells in a 6-well dish were transfected with 0.75 µg of pSVP-Luc (Empty), pSVP-4xRREB1-wt (4xRREB1-wt) and pSVP-4xRREB1-mut (4xRREB1-mut) together with 0.25, 0.75 and 1.0 µg of pEF-DJ-1-HA. Forty-four hrs after transfection, cell extracts were prepared and their luciferase activity was measured. The expression level of DJ-1-HA in cell extracts was analyzed by Western blotting with an anti-HA antibody. n = 3.</p