Suppression of cell-cycle progression by Jun dimerization protein-2 (JDP2) involves downregulation of cyclin-A2

Abstract

We report here a novel role for Jun dimerization protein-2 (JDP2) as a regulator of the progression of normal cells through the cell cycle. To determine the role of JDP2 in vivo, we generated Jdp2-knockout (Jdp2KO) mice by targeting exon-1 to disrupt the site of initiation of transcription. The epidermal thickening of skin from the Jdp2KO mice after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) proceeded more rapidly than that of control mice, and more proliferating cells were found at the epidermis. Fibroblasts derived from embryos of Jdp2KO mice proliferated faster and formed more colonies than fibroblasts from wild-type mice. JDP2 was recruited to the promoter of the gene for cyclin-A2 (ccna2) at the AP-1 site. Cells lacking Jdp2 had elevated levels of cyclin-A2 mRNA. Furthermore, reintroduction of JDP2 resulted in the repression of transcription of ccna2 and of cell-cycle progression. Thus, transcription of the gene for cyclin-A2 appears to be a direct target of JDP2 in the suppression of cell proliferation.

Oncogenic function of the homeobox A13-long noncoding RNA HOTTIP-insulin growth factor-binding protein 3 axis in human gastric cancer

“…After cultivation for 3 days, cells were injected into SCID mice (5 × 10 6 cells/spot). For siRNA-mediated gene knockdown, cells were transfected with negative control siRNA (Thermo Fisher Scientific, D-001810-10-05) or the following specific siRNA-like IGFBP-3-targeting siRNA (Ambion-Thermo Fischer, s7227, s7228, s7229), HoxA13-targeting siRNA (Ambion-Thermo Fisher; s106130, s6785, 6886, 6787), HOTTIP-targeting siRNA (Sigma-Aldrich, LQ-011052-00-0002) or c-JUN-targeting siRNA (Ambion-Thermo Fischer: s7658) using Lipofectamine RNA/MAX reagents (Thermo Fisher) [43–45]. All sequences were run on BLAST, to exclude sequences that would suppress undesired genes and to ensure specificity.…”

Transcription factor TLX1 controls retinoic acid signaling to ensure spleen development

“…In brief, cells were harvested using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology). The preparation of cell lysates, SDS-PAGE (8% or 10% gel), and Western blotting were performed as described elsewhere (80). In the case of sequential immunoprecipitation and Western blot analysis, 293T cells were transfected with pcDNA4-JDP2 or pcDNA3-TLX1 by Lipofectamine 2000 (Invitrogen), and after 48 hours, the cell lysates of each transformant were prepared for the sequential immunoprecipitation and Western blotting as described elsewhere (80).…”

“…Binding of HRP-labeled anti-rabbit or -mouse secondary antibodies (NA934 or NA931, respectively; GE Healthcare) was detected with the SuperSignal West Pico Chemiluminescent kit (Thermo Scientific). Immunoprecipitation was performed as previously described (80,81). In brief, cells were harvested using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology).…”

“…The preparation of cell lysates, SDS-PAGE (8% or 10% gel), and Western blotting were performed as described elsewhere (80). In the case of sequential immunoprecipitation and Western blot analysis, 293T cells were transfected with pcDNA4-JDP2 or pcDNA3-TLX1 by Lipofectamine 2000 (Invitrogen), and after 48 hours, the cell lysates of each transformant were prepared for the sequential immunoprecipitation and Western blotting as described elsewhere (80). Each supernatant was precleared with protein A/G beads (Millipore), and incubated with antibodies specific for JDP2 (2) or TLX1 (1:550 stock 200 μl/ml; SC-880; Santa Cruz Biotechnology Inc.) at 4°C for 16 hours.…”

G protein-coupled receptor kinase 4-induced cellular senescence and its senescence-associated gene expression profiling

“…The abnormal expression of these CDKs and cyclins has been linked to cell cycle arrest and cellular senescence. For example, the activity of CDK2 alone or cooperatively with CDK4 or CDK6 was required for cell cycle G1/S transition and down-regulating expression of CDKs or cyclin D1/A2 or both resulted in cellular senescence or cell cycle G1/G0 phase arrest [25–30]. We analyzed differential expression profile of 78 senescence-related genes in the sorted HEK293-GRK4(+) cells that had demonstrated G1 arrest and senescence and found that the expressions of cyclin A2, cyclin D1, CDK2, CDK4 and CDK6 were down-regulated.…”