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. 2021 Mar 23;118(13):e2001611118. doi: 10.1073/pnas.2001611118

Fig. 4.

Fig. 4.

Identification of IL-1α–induced signal transduction that results in increased PIEZO1 gene expression by delimited screening in porcine articular chondrocytes. (A) Inhibitors of p38 MAP-kinase significantly attenuated IL-1α–induced PIEZO1-mRNA increase in primary chondrocytes (IL-1α 1 ng/mL for 3 d, for all panels) but not inhibitors of MAP-kinases, JNK, MEK-ERK, and PI3K. (B) Western blot analysis of p38 phosphorylation indicates a significant increase of phospho-p38; total p38 does not change. (C) Starting with 96 TFs, their activation was assessed in response to IL-1α treatment; increased x-fold over baseline, ranked, and top 25 are shown here. Note the green dotted line at 4×, with 19 of the top 25 TFs ≥4. Below the bar diagram, labeled “MULAN”, note binary score (+/−) indicating whether the respective TF was found to have a predicted binding site in the proximal PIEZO1 promoter (see E, also SI Appendix, Fig. S6) using MULAN TF-binding prediction program. Indicated below, labeled “Inhibitor,” is whether the respective TF can be inhibited with a well-established selective small-molecule inhibitor compound. The two red arrows point toward the two TFs that fulfill all criteria, ATF2 and HNF4. (D) TFs that impact IL-1α–induced PIEZO1-mRNA increase were identified using selective compounds. HNF4 and ATF2/CREBP (the latter having the same DNA binding site as ATF2, known to form TF complexes) were confirmed as relevant; HIF, NFAT, and NFkB were not found to be involved. Inhibitors of ATF2/CREBP1 and HNF4 significantly attenuated PIEZO1-mRNA increase. The dose–response relationship of ATF2/CREBP1 inhibitor CBP30 showed a Pearson correlation coefficient = −0.85, P = 0.0008, indicating a significant correlation. The respective metrics for HNF4 inhibitor BI6015 were Pearson correlation coefficient = −0.86, P = 0.0014, also indicative of a signification correlation. (E) A schematic of predicted binding sites of HNF4 and ATF2/CREBP1 TFs in the proximal PIEZO1 promoter, as revealed by the bioinformatics platform MULAN. Please see also SI Appendix, Fig. S6. (F) Direct CREBP1 binding to the PIEZO1 promoter was assessed by chromatin immunoprecipitation followed by qPCR. Note fivefold increased abundance with IL-1α signaling versus control, indicative of direct CREBP1 binding to the PIEZO1 proximal promoter site indicated in E (yellow bar, between 0 and +500 PIEZO1 promoter). For A, B, D, and F, the numbers in bars indicate number of independent isolations of primary chondrocytes. The bars represent mean ± SEM; for group comparison B and F, t test, *P < 0.05; for group comparisons A and D, one-way ANOVA, Tukey’s post hoc test; *comparison IL-1α versus control, #comparison IL-1α plus treatment versus IL-1α. */#P < 0.05.