Page 51 - Haematologica July

haematologica | 2018; 103(7)
Mouse model of sickle cell disease
activated by ET-1.30,31 To confirm this relationship among ET-1, ETA receptors, and Nav1.8 channels in DRG neu- rons, we cultured HbAA DRG neurons in the presence of ET-1 peptide. ET-1 stimulation of HbAA DRG neurons for 24 hours resulted in increases in the levels of Nav1.8 pro- tein (Figure 8A) and phosphorylated p65 protein (an indi- cator of NF-κB activation32), a NF-κB subunit (Figure 8B). Co-incubation with ABT-627 or pyrrolidine dithiocarba- mate (PDTC), an NF-κB-specific inhibitor, prevented these increases (Figure 8A and 8B). Furthermore, we examined the expression of phosphorylated p65 in in vivo mice injected intraperitoneally (i.p) with vehicle or ABT-627 once daily for 4 days. Phosphorylated p65 levels in HbSS DRG significantly increased after vehicle injection, but did not significantly change after ABT-627 administration compared to that in the HbAA mice after vehicle injection (Figure 8C). Total p65 protein levels did not significantly differ among treatment groups (Figure 8C). These findings indicate that NF-κB activation and Nav1.8 upregulation in HbSS DRG are ETA receptor-dependent and may be involved in SCD-associated pain. This conclusion is fur-
Figure 4. Male HbSS mice with conditional genetic knockdown of DRG ETA receptors fail to develop pain hypersensitivity before and after hypoxia/reoxygenation exposure. Paw withdrawal frequency (PWF) in response to a 0.16 g low mechanical force (A) and a 0.4 g medium mechanical force (B) and paw withdrawal latency (PWL) in response to heat (C) and cold (D) stimuli on both left and right sides of ETAfl/fl mice and ETAcre/fl mice 2 months after BerkSS (SS) or BerkAA (AA) bone marrow transplantation (BMT). n = 6-8 mice/group. **P<0.01 vs. the corresponding baseline value. ##P<0.01 vs. the corresponding BMT + Normoxia value.
ther supported by our behavioral observations that single i.p administration of PDTC, a specific inhibitor of NF-κB activation, or A-803467, a specific antagonist of Nav1.8,33 alleviated mechanical allodynia and/or thermal hyperalge- sia in HbSS mice, without affecting the basal responses of HbAA mice (Figure 8D; Online Supplementary Figure S10).
We then examined whether activated NF-κB is directly linked to increased Nav1.8 expression in HbSS DRG. Using ChIP analyses, a fragment within the Scn10a pro- moter was amplified from a complex immunoprecipitated with an anti-p65 antibody (Figure 8E), indicating the bind- ing of p65 to the Scn10a gene promoter in the DRG. This binding activity increased in the HbSS DRG compared to HbAA DRG, as shown by a 7-fold increase in band densi- ty (Figure 8E). To examine whether NF-κB activation directly regulates Scn10a transcriptional activity, we per- formed a dual luciferase assay using a mouse neuronal cell line (CAD cells)34 transfected with a luciferase reporter vector containing the Scn10a gene promoter. Since phor- bol 12-myristate 13-acetate (PMA) can activate NF-κB through protein kinase C (PKC) activation,35 we applied
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