Fig. 6.
Tumor necrosis factor (TNF)-α induces hyperalgesia via bromodomain-containing protein 4 (Brd4)–cyclin-dependent kinase-9 (CDK9)–RNA polymerase II phosphorylation (pRNAPII)–voltage-gated sodium channel 1.7 (Nav1.7) signaling in dorsal root ganglia (DRG) neurons. (A) Reverse transcription polymerase chain reaction (RT-PCR) analyses demonstrating complete Freund’s adjuvant (CFA) enhanced TNF-α messenger RNA (mRNA) expression in DRG at day 1 after intraplantar injection. *P < 0.01 versus saline 1d; n = 6. (B) and (C) TNF-α–neutralizing antibody (CFA 1d + TNF-α Ab; 10 μl, 100 ng, intrathecally [i.t.]) inhibited the Nav1.7 promoter fragments immunoprecipitated by Brd4-, CDK9-, pRNAPII-, and H3-specific antibodies in the DRG of CFA-treated rats. **P < 0.01 versus CFA 1d; n = 6. (D) and (E) TNF-α–neutralizing antibody (CFA 1d + TNF-α Ab; 10 μl, 100 ng, i.t.) reversed the expression of Nav1.7 mRNA, the paw withdrawal latency in the DRG of CFA-treated rats. **P < 0.01 versus CFA 1d; n = 7. (F) TNF-α (10 μl, 1 pM, i.t.) increased the abundance Nav1.7 promoter fragments immunoprecipitated by the H3-specific antibody. **P < 0.01 versus naive; n = 6. (G) Intrathecal administering with Brd4-specific siRNA (Brd4 RNAi + TNF-α, 10 μl, 5 μg) and CDK9-specific siRNA (CDK9 RNAi + TNF-α, 10 μl, 5 μg) both inhibited TNF-α (10 μl, 1 pM, i.t.)–increased Nav1.7 promoter fragments immunoprecipitated by Brd4-, CDK9-, and pRNAPII-specific antibodies. RNAi = RNA interference. *P < 0.05, **P < 0.01 versus naive; #P < 0.05, ##P < 0.01 versus TNF-α; n = 6. (H) RT-PCR analysis demonstrating TNF-α (10 μl, 1 pM, i.t.)–enhanced Nav1.7 mRNA expression in DRG was attenuated by Brd4-specific siRNA (Brd4 RNAi + TNF-α, 10 μl, 5 μg) and CDK9-specific siRNA (CDK9 RNAi + TNF-α, 10 μl, 5 μg). *P < 0.05 versus naive; #P < 0.05 versus TNF-α; n = 6. (I) TNF-α (10 μl, 1 pM, i.t.)–decreased paw withdrawal latency was ameliorated by Brd4-specific siRNA (Brd4 RNAi + TNF-α, 10 μl, 5 μg) and CDK9-specific siRNA (CDK9 RNAi + TNF-α, 10 μl, 5 μg). **P < 0.01 versus naive; ##P < 0.01 versus TNF-α; n = 7. (J) Representative traces of total Nav currents and ProTx-II–resistant currents recorded from DRG neurons dissected from the naive, TNF-α–treated, and TNF-α–treated rats received daily administration with Brd4-targeting siRNA and CDK9-targeting siRNA. Peak currents were induced by voltage step from –60 to –20 mV before (black line) and after (gray line) bath application of ProTx-II (5 nM). Scale bar = 200 pA, 5 ms. The normalized peak values of the Nav1.7-dependent current densities (subtract ProTx-II from control; pA/pF) recorded from groups. **P < 0.01 versus naive; ##P < 0.01 versus TNF-α; n = 6.