The protein phosphatase PPM1A dephosphorylates and activates YAP to govern mammalian intestinal and liver regeneration
Fig 5
PPM1A is critical for proliferation of intestinal organoids.
(A, B) Double depletion of PPM1A and PPM1B resulted in an enhanced level of phospho-YAP (S127), when compared with depletion of PPM1A alone (A). Depletion of PPM1B by a CRISPR-based strategy in PPM1A KO HEK293 cells led to a complete loss of colony formation (B). (C, D) Intestinal crypts were isolated from WT and PPM1A KO mice at 6-week age and cultured in vitro (C). A dramatic lower number of de novo crypts (D, top panel) and the decreased size of organoids (D, bottom panel) were seen in organoids from PPM1A KO mice, with roughly 50 intestine organoids each group examined. (E) Edu integration, which mostly localized in the ends of crypts and represented the proliferating cells, was significantly lower in intestinal organoids without PPM1A. (F) Anti-YAP/TAZ immunofluorescence imaging of organoid sections detected a substantial subset of cells containing nucleo-YAP/TAZ in the WT crypts and with active proliferation (Edu staining), in a sharp distinction of Ppm1a−/− crypts, where YAP/TAZ were located in the cytoplasm even in some proliferating cells. (G) The mRNA levels of YAP/TAZ target genes were significantly lower in Ppm1a−/− intestinal organoids, examined at Day 3. (H) Lentiviral delivery into Ppm1a−/− intestinal organoids of the hYAP 2SA mutant (S109A/S127A), which was resistant to the PPM1A-mediated dephosphorylation, restored the mRNA levels of CTGF and CYR61. (I, J) The decreased number of de novo crypts (I) and size of organoids (J) in PPM1A KO intestinal organoids were failed to be recovered, when TGF-β signaling was inhibited by small molecule inhibitor SB431542 or when IFN-I signaling was blocked by the neutralizing antibody targeting IFNAR1, examined at Day 6, with roughly 20 intestine organoids each group. (K) The mRNA levels of YAP/TAZ target genes in Ppm1a−/− intestinal organoids were unable to be restored, under inhibition of TGF-β signaling or IFN-1 signaling, examined at Day 3. Unprocessed images of blots are shown in S1 Raw Images. Statistics source data are provided in S1 Data. IFN-I, type I interferon; KO, knockout; phospho-YAP, phosphorylating forms of YAP; PPM1A, protein phosphatase magnesium-dependent 1A; TAZ, transcriptional coactivator with PDZ-binding motif; TGF-β, transforming growth factor beta; WT, wild-type; YAP, Yes-associated protein.