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T. Sun et al.
tive to those in the controls. Among the kinases that were affected by LV-shWDR4 infection, GSK3β, AKT, ERK, and CREB are known to be upstream of IL-6 (Figure 7A). We next validated the results by Western blotting. Phosphorylation levels of GSK3β (S9), AKT1/2/3 (S472/S473/S474), ERK1/2 (T202/Y204, T185/Y187), and CREB (S133) significantly decreased with WDR4 knock- down in HD MSC and increased with WDR4-overexpres- sion in ET MSC (Figure 7B).
To determine which kinases are involved in WDR4- mediated IL-6 upregulation, we used kinase inhibitors specific for AKT1/2/3 (GDC-0068), ERK1/2 (SCH772984), and GSK3β (SB216763), and an siRNA specific for CREB in ET MSC infected with LV-WDR4. We thereby found that inhibition of ERK1/2, GSK3β, or CREB could at least par- tially suppress WDR4-induced IL-6 upregulation, while
inhibition of AKT1, -2, or -3 caused no significant effect as assessed with qPCR (Figure 7D), Western blotting (Figure 7E), and ELISA (Figure 7F). These findings indicate that WDR4 promotes IL-6 expression and secretion via the ERK–GSK3β–CREB signaling pathway in BM-MSC.
Neuropathy and aberrant expression of IL-1β in the BM of patients with JAK2V617F-positive ET
Markedly lower numbers of sympathetic nerve fibers and insheathing Schwann cells were found in the BM of patients with JAK2V617F-positive ET (Figure 8A). Norepinephrine, which is mainly secreted by sympathetic nerve fibers, was also significantly downregulated in ET BM (Figure 8C). Additionally, B3AR, a norepinephrine receptor, and IL-1β levels were upregulated in the BM of patients with JAK2V617F-positive ET (Figure 8B-D).
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B
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Figure 8. Neuropathy and aberrant expression of IL-1β in the bone marrow (BM) of patients with JAK2V617F-positive essential thrombocythemia (ET). A. Sympathetic nerve fibers quantitated with the help of an anti-TH antibody (control, n=9; ET, n=42), and Schwann cells visualized using an anti-GFAP antibody (control, n=9; ET, n=40) decreased in the BM of patients with JAK2V617F-positive ET relative to those in the HD, as determined by immunohistochemistry. B. Increased expres- sion of B3AR in the BM of patients with JAK2V617F-positive ET (n=4) relative to the HD (n=4) as determined by Western blotting. C–D. Lower NE levels (C) (control, n=20; ET, n=20) and higher IL-1β levels (D) (control, n=20; ET, n=20) in the BM of patients with JAK2V617F-positive ET relative to the control, as measured by ELISA on the supernatant of the BM aspirates. E. A model illustrating BM hematopoietic dysfunction in JAK2V617F-positive ET. *P<0.05; **P<0.01, ***P<0.001; ****P<0.0001. Data are presented as the mean ± SEM. ET: essential thrombocythemia; HD: healthy donors; n: number of unique donors in each group; TH: tyrosine hydroxylase; GFAP: glial fibrillary acidic protein; NE: norepinephrine; B3AR: β3 adrenoceptor; SEM: standard error of mean.
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haematologica | 2020; 105(3)