Figure 3. CDK4 and CDK6 phosphorylate EZH2 to induce STAT3 activation.

(A) STAT3 activity was detected by analyzing the phosphorylation state at tyrosine 705 (Y705) and threonine 727 (T727) of STAT3 in primary human keratinocytes. After overnight starvation, cells were stimulated for 1 hour with IL-36α or IL-17A/TNF-α in the presence or absence of abemaciclib (Abe). (B) STAT3 activity in CDK4- and CDK6-depleted keratinocytes. Stimulation as in A. (C) Immunoblot detection of phosphorylated STAT3 (Y705) in IL-36α–stimulated keratinocytes, in which EZH2 function was suppressed by the EZH2 inhibitor EPZ6438 (EPZ, 10 μM) or shRNA-mediated knockdown. Detection of H3K27me3 controlled effective EZH2 inhibition or depletion. (D) Immunoblot detection of phosphorylated EZH2 at threonine 345 (T345) and threonine 487 (T487) in abemaciclib-treated or CDK4/6-depleted keratinocytes following stimulation with IL-36α. (E) Coimmunoprecipitation of EZH2 and STAT3 in HaCaT cells treated for 30 minutes with IL-36α in the presence or absence of abemaciclib. An EZH2-specific antibody or IgG was used for pull down of protein complexes. STAT3 and pEZH2 (T345) were detected by immunoblotting. (F) Luciferase activity assay of the NFKBIZ promoter in HEK293T cells, which transiently overexpress CDK6, WT EZH2 (wt), mutant EZH2 (T345A), or STAT3, alone or in combination. Equal protein expression was detected by immunoblotting. n = 3 ± SD. (G) Gene expression in IL-36α– and abemaciclib-treated, primary keratinocytes following transient expression of a phospho-mimicking EZH2 (T345D) mutant. Input controls (left). mRNA levels of NFKBIZ and its target genes were normalized to RPL37A (right). n = 3 ± SD. (H) Overexpression of IκBζ overrides the inhibitory effects of EPZ6438 (EPZ) on IL-36α–stimulated gene expression in primary keratinocytes. n = 3 ± SD. Significance was calculated using a 1-way ANOVA for multiple groups and a 2-tailed Student’s t test for comparing 2 groups: *P < 0.05; **P < 0.01; ***P < 0.001.