Downregulation of p16 Decreases Biliary Damage and Liver Fibrosis in the Mdr2^/ Mouse Model of Primary Sclerosing Cholangitis

Abstract

Biliary senescence and hepatic fibrosis are hallmarks of cholangiopathies including primary sclerosing cholangitis (PSC). Senescent cholangiocytes display senescence-associated secretory phenotypes (SASPs, e. g., TGF-β1) that further increase biliary senescence (by an autocrine loop) and trigger liver fibrosis by paracrine mechanisms. To determine the effect of p16 inhibition and role of the TGF-β1/miR-34a/SIRT1 axis in biliary damage and liver fibrosis in the Mdr2-/- mouse model of PSC. We treated (i) in vivo male wild-type (WT) and Mdr2-/- mice with p16 Vivo-Morpholino or controls before measuring biliary mass (IBDM) and senescence, biliary SASPs levels and liver fibrosis; and (ii) in vitro intrahepatic murine cholangiocyte lines (IMCLs) with siRNA against p16 before measuring the mRNA expression of proliferation, senescence and fibrosis markers. p16 and miR-34a increased but SIRT1 decreased in Mdr2-/- mice and PSC human liver samples compared to controls. P16 immunoreactivity and biliary senescence and SASPs levels increased in Mdr2-/- mice but decreased in Mdr2-/- mice treated with p16 Vivo-Morpholino. The increase in IBDM and hepatic fibrosis (observed in Mdr2-/- mice) returned to normal values in Mdr2-/- mice treated with p16 Vivo-Morpholino. TGF-β1 immunoreactivity and biliary SASPs levels were higher in Mdr2-/- compared to those of WT mice, but returned to normal values in Mdr2-/- mice treated with p16 Vivo-Morpholino. The expression of fibrosis/senescence markers decreased in cholangiocytes from Mdr2-/- mice treated with p16 Vivo-Morpholino (compared to Mdr2-/- mice) and in IMCLs (after p16 silencing) compared to controls. Modulation of the TGF-β1/miR-34a/SIRT1 axis may be important in the management of PSC phenotype.

Genetic or pharmacological reduction of cholangiocyte senescence improves inflammation and fibrosis in the Mdr2 mouse

“…As predicted, the absence of p16 INK4a in the liver decreased inflammation and fibrosis at two months. In accordance with our results, previous studies have demonstrated that removal of senescent cells reversed fibrosis and inflammation in atherosclerosis, pulmonary fibrosis, steatosis, and sclerosing cholangitis (17)(18)(19)31). Numerous studies have now discovered compounds that mimic the effects of these transgenic murine models.…”

“…In accordance with these results, previous studies demonstrated that removal of senescent cells reversed fibrosis and inflammation in atherosclerosis, pulmonary fibrosis, steatosis, and sclerosing cholangitis. [17] , [18] , [19] , 31 Numerous studies have now discovered compounds that mimic the effects of these transgenic murine models. The term ‘Senolytics’ has come to be used to refer to compounds that selectively kill senescent cells.…”

Loss of p16: A Bouncer of the Immunological Surveillance?

“…Additionally, we found that low CDKN2A expression in tumors of 6 different types, including melanoma and pancreatic adenocarcinoma, correlated with a decreased SASP signature [ 18 ], further demonstrating the role of p16 in regulation of the SASP. Consistent with our observation, other articles have found decreased expression of SASP factors upon suppression of p16 in a murine model of intervertebral disc regeneration [ 95 ] and liver fibrosis [ 96 ]. Interestingly, induction of senescence through p16 overexpression does not increase SASP gene expression [ 50 ], suggesting that p16 is necessary but not sufficient to induce the SASP.…”

Cholangiocyte senescence in primary sclerosing cholangitis is associated with disease severity and prognosis

“…However, data regarding cellular senescence in PSC are scant, mainly from end stage disease, and nothing is known about its clinical meaning. Interestingly, recent data on mouse models of chronic cholestatic liver disease showed CS involvement in cholestatic damage and fibrosis, which can ameliorate after specific interventions targeting CS [14,15]. Therefore, the aim of our study was to evaluate the hepatic tissue expression of CS markers in liver samples from PSC patients in different histological stages, and analyze its correlation with clinical-pathological features and patients' prognosis.…”

“…Interestingly, recent data on mouse models of chronic cholestatic liver disease showed CS involvement in cholestatic damage and fibrosis, which can ameliorate after specific interventions targeting CS. 14 , 15 …”