Differential MYC Phosphorylation Drives the Divergent Cholangiocyte Response to Stress.

BACKGROUND & AIMS: In primary sclerosing cholangitis (PSC), some cholangiocytes undergo cell cycle arrest (senescence), whereas others proliferate (ductular reaction). Our aim was to determine the mechanisms driving this divergent response.

METHODS

We analyzed PSC and control liver tissue by immunofluorescence for proliferative and senescent (sen) cholangiocytes. We used LPS to stress normal human cholangiocytes (NHCs) transfected with a senescence reporter (p16prom-GFP) and fluorescence-activated cell sorting (FACS)-sorted sen (GFP+) or senescent-resistant (sen-res, GFP-) fractions. We performed RNA sequencing and quantitative polymerase chain reaction (qPCR) for senescence markers and immunoblots for phospho-(p)T58- MYC and pS62-MYC, and the kinase, GSK3B. Non-phosphorylatable MYC mutant NHCs were generated, and MYC or GSK3B were depleted or inhibited to assess effects on cell fate. MYC and GSK3B inhibitors were tested in 2 PSC mouse models (DDC and Mdr2-/-).

RESULTS

PSC tissue showed an overall increase in sen (∼2×), and proliferative (∼10×) cholangiocytes compared with controls, with senescence enriched in portal tracts and proliferation in parenchyma. RNA sequencing showed enrichment of MYC responsive genes in sen-res cholangiocytes (P < .001). Sen-res cholangiocytes showed increased total and pS62-MYC protein (∼3×), increased mRNA of the proliferation marker, KI67 (>2.5×), and decreased p16/p21 mRNA (∼75%). MYC inhibition in sen-res cholangiocytes promoted senescence (∼15×), whereas T58-MYC mutation reduced senescence and enhanced proliferation (∼3×). Sen cholangiocytes exhibited increased GSK3B (∼2×); GSK3B inhibition or depletion in sen-sensitive cholangiocytes reduced pT58-MYC and senescence (∼50%). In mouse models, MYC inhibition reduced, whereas GSK3B inhibition increased, cholangiocyte proliferation and fibrosis.

CONCLUSION

MYC phosphorylation promotes either cholangiocyte proliferation or senescence. The results reveal kinase mediators of cholangiocyte fate and identify MYC as a stress-responsive "molecular switch."