Fig. 7: GPSM1 deficiency-induced TNFAIP3 upregulation is via the Gαi3/cAMP/PKA/CREB axis.
From: GPSM1 impairs metabolic homeostasis by controlling a pro-inflammatory pathway in macrophages
BMDMs from GPSM1f/f; Lyz2-cre and GPSM1f/f mice were treated with 200 ng/ml LPS or vehicle control for indicated times. a cAMP levels in BMDMs treated with LPS (n = 4 independent samples per group). b GPSM1f/f and GPSM1f/f; Lyz2-cre BMDMs were pre-treated with H-89 (50 μM) or vehicle for 30 min and then stimulated with LPS for additional indicated times. Immunoblots for p-PKA substrate, p-CREB, CREB and A20 in BMDMs. c ChIP-qPCR of A20 in BMDMs with or without LPS stimulation (n = 3 independent samples per condition). β-globin was as a control. d Luciferase activity of a WT TNFAIP3 promoter reporter (TNFAIP3-Luc) or of a mutant-TNFAIP3 reporter (TNFAIP3-Mut-Luc) containing a deletion in the CREB-responsive element in HEK293T cells transiently expressing either vector (pRL-TK) or CREB (n = 3 independent samples per condition). e IP of flag from BMDMs transfected with Ad-flag-GPSM1 or Ad-Con, followed by immunoblot analysis of the interaction of GPSM1 with Gαi3 under LPS stimulation or not. f Immunoblot analysis of p-P65, P65, GPSM1, and Gαi3 in BMDMs infected with Ad-GPSM1 or Ad-GFP, and Ad-shGαi3 or Ad-shNC for 72 h and treated with LPS for indicated times. Independent experiments were performed three times with similar results (b, e, and f). All data are presented as means ± SEM. P values are determined by two-way ANOVA with Sidak’s multiple-comparisons test (a), or two-tailed Student’s t-test (c, d).
