Cell lysates were immunoprecipitated and subsequently immunoblotted with the indicated antibodies. and induces formation of the TBL1SUMO-TBLR1SUMO-NF-B complex, which ultimately leads to transcriptional activation of NF-B target genes. Therefore, this study suggests a regulatory mechanism for elevated NF-B-mediated inflammatory signaling in AIPCs via reversible SUMOylation of TBL1 and TBLR1. RESULTS TBL1 and TBLR1 SUMOylation and inflammatory cytokines are elevated in AIPC cells NF-B is usually constitutively activated in prostate tumors and cell lines [5]. Therefore, we first examined the inflammatory cytokine levels in prostate cancer cell lines by performing cDNA microarrays using the androgen-dependent prostate cancer (ADPC) cell line LNCaP and the AIPC cell line PC-3. In agreement with a previous report [24], we observed that this pro-inflammatory cytokines IL-8, IL-1, and IL-6 were strongly elevated in PC-3 cells compared with LNCaP cells (Physique ?(Figure1A).1A). Quantitative RT-PCR analysis verified the elevated cytokine levels in PC-3 cells (Physique ?(Figure1B1B). Open in a separate window Physique 1 SUMOylation of TBL1 and TBLR1 is usually strongly elevated in androgen-independent prostate cancer cells enriched with inflammatory cytokines(A) Inflammatory cytokine levels are higher in PC-3 cells than in LNCaP cells. PF-05241328 Changes in mRNA expression were evaluated by cDNA microarray analysis using the Illumina HumanRef-8 v3 Expression BeadChip. (B) Validation of cDNA microarray analysis by quantitative real-time PCR. Expression levels of each gene were analyzed by quantitative RT-PCR. Statistical significance was decided using Student’s < 0.01 LNCaP cell lines. (C) SUMOylation of TBL1 and TBLR1 levels are higher in PC-3 and C4-2B cells than in LNCaP cells. Immunoprecipitation analysis was performed using cell lysates, and immunoblotting was performed using the indicated antibodies. (D) Validation of TBL1 SUMOylation in LNCaP and PC-3 cells. Duo-link PLA analysis was performed as described in Materials and methods with the indicated antibodies. A recent study reported that this TBL1 corepressor acts as a cofactor for recruiting p65 to NF-B target gene promoters, which eventually leads to the transcriptional activation of inflammatory cytokines [23]. Therefore, we explored the possibility that TBL1 and TBLR1 are involved in cytokine elevation in AIPC cells. First, we assessed TBL1 and TBLR1 levels in prostate cancer cells by performing western blot analysis. Immunoprecipitation analysis revealed that the conversation between TBL1/TBLR1 and RelA in PC-3 cells was strongly increased compared with that in LNCaP cells, and the TBL1 and TBLR1 protein levels in PC-3 cells also were higher than those in LNCaP cells (Physique ?(Physique1C,1C, left panel). TBL1 and TBLR1 SUMOylation caused TBL1 and TBLR1 dissociation from the NCoR corepressor complex [21]. Therefore, we next examined the relative association of TBL1 and TBLR1 with NCoR/HDAC3 corepressor complexes in PC-3 and LNCaP cells. TBL1 and TBLR1 association with NCoR/HDAC3 corepressor complexes were significantly lower in PC-3 cells than in LNCaP cells (Physique ?(Physique1C,1C, left panel). To verify these results, we performed Duo-link proximity ligation assay (PLA) analysis, which enables the detection of protein interactions and modifications, and verified elevated SUMOylation levels of endogenous TBL1 in PC-3 cells (Physique ?(Figure1D1D). Due to their high metastatic potential resulting from their androgen-insensitive state, PC-3 cells have been less extensively studied than PF-05241328 LNCaP cells for investigating biochemical changes in advanced prostate cancer. PC-3 cell line was established from bone metastasis of prostate cancer. Therefore, we selected the bone metastasis subline C4-2B, which was generated from parental LNCaP cells, to confirm whether these comparable cell lines share the same biochemical features as PC-3 cells. The results showed that inflammatory cytokine levels were highly elevated in C4-2B cells compared with those in LNCaP cells, which was similar to the observed cytokine levels in PC-3 cells. The levels of TBL1 and TBLR1 SUMOylation and association of TBL1 and TBLR1 with RelA were higher in C4-2B cells than in PC-3 cells (Physique ?(Physique1C,1C, right panel). Collectively, these results suggest that constitutive activation of inflammatory signaling in AIPC cells correlates with TBL1 and TBLR1 SUMOylation. Inflammatory stimulation promotes TBL1 and TBLR1 SUMOylation Recent work showed that SUMO modification acts as a molecular switch that regulates corepressive and coactive functions of TBL1 PF-05241328 and TBLR1 during Wnt signaling activation [21]. Therefore, we examined whether TBL1 and ERBB TBLR1 SUMOylation increases in response to inflammatory activation. Myc-TBL1 or Myc-TBLR1 was co-transfected with Flag-SUMO1 into PC-3 cells, the cells were treated with TNF-, and immunoprecipitation assays were performed. In response to treatment, SUMOylation of.