Differential MYC Phosphorylation Drives the Divergent Cholangiocyte Response to Stress

Abstract

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 (p16_prom-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."

Keywords: Ductular Reaction; Primary Sclerosing Cholangitis; Proliferation; Senescence.

Graphical abstract

Figure 1

Senescent and proliferating cholangiocytes distribute distinctly in tissue from patients with PSC. (A) Quantitation of combined fluorescent in situ hybridization CDKN2A (ie, p16) and immunofluorescence for KRT7 and PCNA in PSC patient tissue. Both senescence (p16) and proliferation (PCNA) were increased in tissue samples from patients with PSC. (B and C) p16 (green) and PCNA (red) positivity, by average pixel intensity, within regions of interest of tissue from patients with PSC (ie, portal tracts [areas localized near portal vein and artery]) vs KRT7 positive cholangiocytes within the parenchyma (ie, ductular reactive cholangiocytes). Senescent cholangiocytes accumulate in the portal tracts, whereas proliferative cholangiocytes were abundant in the parenchyma. ∗P < .05; ∗∗∗P < .001. n = 5 tissues from patients with late-stage PSC.

Figure 2

FAC sorting separates sen-sen from sen-res cholangiocytes. (A) FACS of NHC transfected with a p16-promoter driven GFP reporter. Approximately 40% of the cholangiocytes exposed to cellular stressors (LPS shown) were positive for the p16-GFP reporter. SA-b-gal assay supported increased senescence in LPS treated cholangiocytes. (B) qPCR of sorted sen and sen-res population. The senescent markers p16 and p21 were increased, whereas the proliferation marker KI67 was decreased in senescent (Sen) compared with FAC-sorted GFP negative (GFP−) senescent resistant (sen-res) NHCs treated with cellular stressors LPS. (C) Multiplex antibody array to detect secreted proinflammatory/fibrogenic mediators in conditioned media. Densitometric analysis of analyte spots revealed overlapping, yet unique profiles in sen vs sen-res populations. (D) Fluorescent SA-β-gal detection of senescence in NHCs vs PSC primary cells. NHCs exhibited ∼5% SA-b-gal-positive cholangiocytes, whereas PSC patient-derived cholangiocytes exhibited ∼35% SA-b-gal-positive cholangiocytes. SA-b-gal colorimetric assay of cells in culture confirmed an increase in senescence. (E) qPCR of sorted sen and sen-res PSC patient cholangiocytes. The senescent markers p16 and p21 were increased, whereas the proliferation marker KI67 decreased in SA-b-gal-positive PSC patient-derived cholangiocytes compared with FAC-sorted PSC patient-derived SA-b-gal-negative cholangiocytes. (F) Fluorescent SA-β-gal detection of senescence in freshly isolated WT and Mdr2-/- mouse cholangiocytes. WT mice exhibited ∼ 5% SA-β-gal-positive cholangiocytes, whereas Mdr2-/- mice exhibited ∼ 30% SA-β-gal-positive cholangiocytes. (G) qPCR of sorted sen-res and sen Mdr2-/- cholangiocytes. The senescent markers p16 and p21 were increased, whereas the proliferation marker Ki67 decreased in SA-β-gal-positive Mdr2-/- mouse cholangiocytes compared with FAC-sorted Mdr2-/- mouse-derived SA-β-gal-negative cholangiocytes.

Figure 3

Characteristics of FAC-sorted senescent cells. (A) H₂O₂ and IR induced senescence in ∼40% of the NHCs. (B) qPCR demonstrated increased senescence and decreased proliferation markers in IR and H₂0₂-induced NHC senescence. (C) qPCR for IL8, IL6, and the anti-apoptosis mediator, Bcl-xL, demonstrating increased expression in LPS-, IR-, and H₂O₂-induced NHC senescence. (D) Western blot demonstrates increased p21 expression in LPS induced senescent NHCs. (E) Phenotypic characteristics of LPS-induced senescent cholangiocytes. (F) The FAC-sorted senescent resistant NHCs continued to grow in the extended presence (24 days) of LPS or H₂O₂.

Figure 4

Clonal isolation of NHCs results in senescence-sensitive and -resistant cholangiocyte populations. (A) Schema of clonal isolation of cholangiocytes transfected with the senescence reporter p16 promoter - GFP. (B) Clones were either resistant (eg, clone 28) or sensitive (eg, clone 80) to H₂O₂- and LPS-driven senescence as demonstrated by p16-GFP positivity. Western blotting for p21 confirmed resistance (clone 28) or susceptibility (clone 80) to LPS-induced senescence. (C) qPCR of senescence and proliferation markers. Sen-sen clone 80, but not sen-res clone 28 exhibited increased p16 and p21 mRNA, and decreased KI67 when cultured in the presence of LPS.

Figure 5

Luminescence of implanted sen and sen-res cholangiocytes. Clonally derived sen-sensitive and sen-resistant cholangiocytes were stably transfected with a luciferase reporter and exposed to LPS for 10 days. The treated cells were injected into explanted mouse liver lobes maintained in growth media for 5 days and exposed to luciferin. The sen-sen clone showed no luminescence, whereas the sen-res clone exhibited increased luminescence, indicating continued growth. Luminescence readings were performed using IVIS Spectrum imaging.

Figure 6

Bulk RNA-seq demonstrates that the sen-res cholangiocytes are characterized by a MYC-driven gene expression profile. (A) Volcano plots showing that sen-sen cholangiocytes exhibited 745 genes that were significantly reduced (Log2FC ≤−1; −Log10FDR >1.3) and 1877 genes that were significantly increased compared with sen-res cholangiocytes. (B) GSEA revealed that MYC targets are upregulated, whereas IFNA response genes are downregulated in the sen-res compared with the sen population (nominal P-value < .01, FDR <0.25) and in NHCs vs primary PSC patient cholangiocytes. (C) IPA revealed the upstream activators that define sen and sen-res. Inhibition of the upstream activator, MYC, was the strongest signal defining downregulated genes in the sen vs the sen-res cholangiocytes (ie, upregulated in sen-res, Z score <−6.0, P-value of overlap < 8.0E-23). Conversely, activation of the upstream factors, INFG and IFNA, were the strongest signal defining upregulated genes in sen vs the sen-res cholangiocytes (Z scores >8.0 and P-value of overlap < 5.0E-54).

Figure 7

CRISPR/Cas9 deletion of MYC in NHC. (A) qPCR demonstrates that MYC is depleted. MYC mRNA is increased in induced senescent control NHCs but not in MYC depleted cells (DMYCs). (B) qPCR demonstrates that MYC depletion promotes senescence in NHCs. (C) qPCR showing that depletion of MYC suppresses MYC target gene expression. ∗P < .05.

Figure 8

The MYC inhibitor, MYCi975, diminishes cholangiocyte proliferation in mouse models of PSC. (A) Immunofluorescence for portal vs parenchymal cholangiocyte MYC expression in livers from DDC-fed and Mdr2-/- mice. MYC expression is increased ∼2-fold in the parenchymal cholangiocytes compared with cholangiocytes localized to the portal tracts in both animal models. (B) Representative images of H&E-stained liver sections showing parenchyma and portal tracts of vehicle-treated WT control (Veh Ctrl; n = 8), DDC-fed plus vehicle (Veh; n = 8), DDC-fed Myc inhibitor-treated (MYCi; n = 8), Mdr2-/- (n = 6), and Mdr2-/- MYC inhibitor-treated (n = 6) mice (left panels) and Picrosirius-red-stained liver sections (magnification: 4×) showing deposition of collagen (right panels). (C) Quantitation of Picrosirius reveals a significant reduction in fibrosis in MYC inhibitor-treated DDC-fed mice, and a trend towards significance (P = .133) in MYC inhibitor-treated Mdr2-/- mice. Data is presented as the percentage of Picrosirius-positive/total image area. (D) Immunofluorescence of KRT7 and PCNA in DDC-fed mice. KRT7 (KRT7-positive/total image area) and percentage of cholangiocytes positive for PCNA were reduced in mice treated with the MYC inhibitor. (E) qPCR for total liver Krt7, Pcna, and p16 in WT C57BL6, DDC-fed, and DDC-fed MYCi-treated mice. Total liver Krt7 and Pcna mRNA are reduced, whereas total liver p16 is increased in MYCi treated DDC-fed mice vs DDC-fed vehicle mice. (F) Immunofluorescence of KRT7 and PCNA in Mdr2-/- mice. Total KRT7 trended towards significance, and percentage of cholangiocytes positive for PCNA was reduced in Mdr2-/- mice treated with the MYC inhibitor. (G) qPCR for total liver Krt7, Pcna, and p16 in WT C57BL6, Mdr2-/-, and Mdr2-/- MYCi treated mice. Total liver Krt7 and Pcna mRNA are reduced, whereas total liver p16 is increased in MYCi treated Mdr2-/- vs vehicle-treated Mdr2-/- mice. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.

Figure 9

Serum biochemistries in the presence and absence of MYC inhibitor, MYCi975. (A) DDC-fed mice exhibited increased serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), total bilirubin (TBILI), total bile acids (BA). albumin (ALB), and cholesterol (CHOL). DDC-fed mice treated with the MYCi975 (Myci) exhibited a decrease in ALP, ALT, TBILI, BA, and CHOL. (B) Mdr2-/- mice treated with MYCi exhibited a trend towards decreased ALP, and significantly reduced TBILI.

Figure 10

Senescent and senescent-resistant cholangiocytes exhibit differential MYC phosphorylation. (A) RNAseq and qPCR show that MYC gene expression is elevated in sen-res vs sen cholangiocytes. (B and C) Immunoblots of total and phospho-MYC in FACs isolated sen and sen-res cholangiocytes. Total MYC protein is increased in sen-res vs sen cholangiocytes. MYC S62 phosphorylation is increased in FACS isolated sen-res vs sen cholangiocytes, and, conversely, that T58 phosphorylation is increased in the sen vs sen-res population (quantitation in C). (D and E) Immunoblot of total and phospho-MYC in clonal isolates. Sen-res clone (clone 28), but not the sen-sen clone (clone 80), exhibited increased S62 phosphorylation (∼1.5-fold increase), whereas clone 80, but not clone 28, exhibited increased T58 phosphorylation (∼2-fold increase) following LPS-mediated stress (quantitation in E). (F) Immunoblots for MYC, phospho-MYC, and p21 in NHCs and cholangiocytes depleted of MYC (DMYC). DMYC cholangiocytes (∼70% reduction of MYC), cultured in the absence of LPS insult, exhibited increased p21 expression (∼3-fold) compared with WT NHCs cultured in the absence of LPS. (G) SA-b-gal-positive cholangiocytes increased ∼5-fold, and proliferation, as measured by MTS assay, was reduced by 50% in DMYC vs NHC. (H and I) MYC, phospho-MYC, and p21 immunoblots in DMYC cholangiocytes reconstituted with WT MYC, or phospho mutants S62A MYC, or T58 MYC. MYC S62A exhibited increased LPS-induced p21 expression, an increased percentage of SA-b-gal-positive cells (∼5-fold), and diminished proliferation (50%) compared with DMYC cholangiocytes reconstituted with WT MYC. Conversely, MYC T58A exhibited decreased LPS-induced p21 expression, (∼50% and 75%) and decreased SA-b-gal positivity (∼50% and 75%) compared with experimentally induced senescent MYC WT and MYC S62A cholangiocytes.

Figure 11

MYC inhibition promotes senescence in the senescecnt resistant clone, clone 28. MYC inhibition (MYCi), in the presence or absence of LPS, promotes: p16 promoter-driven expression of the GFP reporter (A); increased mRNA expression of the senescence markers, p16 and p21 (B); increased expression of the SASP markers, IL6 and IL8 (C); and decreased expression of the proliferation markers, KI67 and PCNA (D).

Figure 12

GSK3B decreases MYC expression and promotes cholangiocyte senescence. (A and B) RNAseq demonstrates that GSK3B mRNA is increased in FACS-isolated senescent cholangiocytes and immunoblot confirms increased GSK3B protein expression. (C) Immunoblots showing that LPS-mediated stress in NHC reduced phosphorylation of GSK3B Serine 9 (pS9-CSK3B, inhibitory phospho-site). The GSK3B inhibitor, CHIR, in the presence of LPS, blocked the reduced Serine 9 phosphorylation, prevented loss of MYC S62 phosphorylation, and promoted loss of MYC T58 phosphorylation. Phosphorylation patterns and senescence detection in the sen-res clone, Clone 28, in the presence of LPS were unaffected by the addition of the GSK3B inhibitor. The GSK3B inhibitor in the senescent-sensitive clone, Clone 80, blocked the LPS-induced loss of inhibitory pS9-GSK3B, reduced the induction of LPS-induced p21 expression. (D) Quantitation of immunoblot p21 expression and detection of induced p16-GFP expression (marker of senescence). The GSK3B inhibitor suppressed LPS stress-associated p21 expression and reduced detection of p16-GFP in NHC and Clone 80.

Figure 13

The GSK3B Inhibitor, CHIR, promotes cholangiocyte proliferation in mouse models of PSC. (A) Immunofluorescence of T58-MYC in portal vs parenchymal cholangiocyte in livers from patients with PSC and DDC-fed and Mdr2-/- mice. T58-MYC expression is increased in portal compared with parenchymal cholangiocytes in livers from patients with PSC (∼4-fold) and both mouse models (∼2-fold). (B) Representative images of H&E-stained liver sections showing parenchyma and portal tracts of vehicle-treated WT control (Veh Ctrl; n = 6), DDC-fed plus vehicle (n = 6), and DDC-fed GSK3B inhibitor (CHIR) treated mice (n = 6; left panels) and Picrosirius red-stained liver sections showing deposition of collagen (right panels). (C) Quantitation of Picrosirius red reveals a significant increase in fibrosis in CHIR-treated DDC-fed mice and a trend towards significance (P = .087) in CHIR treated Mdr2-/- mice. Data is presented as percentage of Picrosirius positive/total image area. (D) Immunofluorescence of KRT7 and PCNA in DDC-fed mice. KRT7 (KRT7 positive/total image area) and percentage of cholangiocytes positive for PCNA were increased in mice treated with the GSK3B inhibitor, CHIR. (E) qPCR for total liver Krt7, Pcna, and p16 in WT C57BL6, DDC-fed, and DDC-fed CHIR-treated mice. Total liver Krt7 and Pcna mRNA are increased, whereas total liver p16 is decreased in CHIR-treated DDC-fed vs DDC-fed vehicle-treated mice. (F) Immunofluorescence of KRT7 and PCNA in Mdr2-/- mice. Total KRT7 trended towards a significant increase (P = .164), and the percentage of cholangiocytes positive for PCNA was increased in Mdr2-/- mice treated with CHIR. (G) qPCR for total liver Krt7, Pcna, and p16 in WT C57BL6, Mdr2-/-, and Mdr2-/- CHIR-treated mice. Total liver Krt7 and Pcna mRNA are increased, whereas total liver p16 is decreased in CHIR treated Mdr2-/- vs veh-treated Mdr2-/- mice.

Figure 14

Serum biochemistries in the presence and absence of the GSK3B inhibitor, CHIR99021. (A) DDC-fed mice treated with CHIR did not show significant alteration in serum biochemistries compared with DDC-fed vehicle-treated mice. (B) Mdr2-/- mice treated with CHIR exhibited elevated ALP, ALT, ALB, and CHOL compared with vehicle-treated Mdr2-/- mice.