Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis

Abstract

Primary sclerosing cholangitis (PSC) is an incurable cholangiopathy of unknown etiopathogenesis. Here we tested the hypothesis that cholangiocyte senescence is a pathophysiologically important phenotype in PSC. We assessed markers of cellular senescence and senescence-associated secretory phenotype (SASP) in livers of patients with PSC, primary biliary cirrhosis, hepatitis C, and in normals by fluorescent in situ hybridization (FISH) and immunofluorescence microscopy (IFM). We tested whether endogenous and exogenous biliary constituents affect senescence and SASP in cultured human cholangiocytes. We determined in coculture whether senescent cholangiocytes induce senescence in bystander cholangiocytes. Finally, we explored signaling mechanisms involved in cholangiocyte senescence and SASP. In vivo, PSC cholangiocytes expressed significantly more senescence-associated p16INK4a and γH2A.x compared to the other three conditions; expression of profibroinflammatory SASP components (i.e., IL-6, IL-8, CCL2, PAI-1) was also highest in PSC cholangiocytes. In vitro, several biologically relevant endogenous (e.g., cholestane 3,5,6 oxysterol) and exogenous (e.g., lipopolysaccharide) molecules normally present in bile induced cholangiocyte senescence and SASP. Furthermore, experimentally induced senescent human cholangiocytes caused senescence in bystander cholangiocytes. N-Ras, a known inducer of senescence, was increased in PSC cholangiocytes and in experimentally induced senescent cultured cholangiocytes; inhibition of Ras abrogated experimentally induced senescence and SASP. Conclusion Cholangiocyte senescence induced by biliary constituents by way of N-Ras activation is an important pathogenic mechanism in PSC. Pharmacologic inhibition of N-Ras with a resultant reduction in cholangiocyte senescence and SASP is a new therapeutic approach for PSC.

Autophagy and senescence in fibrosing cholangiopathies

“…For example, we have shown that CCL2 and CX3CL1, which are components of the SASPs of senescent cholangiocytes, induced cellular senescence in cholangiocytes [45,49,65]. This effect may lead to the accumulation of senescent cholangiocytes in the affected ducts and also senescent mesenchymal cells around the bile ducts in fibrosing cholangiopathies.…”

“…Collectively, the resultant periductal cytokine networks that are formed, contribute to biliary mucosal defense, and also the pathogenesis of fibrous cholangiopathy, and the subsequent acquired immunity [5,22,77,78,84,85]. A signaling pathway initiated by TLRs also involves activation of Ras, mediating the production of proinflammatory cytokines and chemokines by cholangiocytes such as IL6 followed by cholangiocyte proliferation [90], and also inducing cellular senescence, as described below [49]. These aspects of biliary innate immunity are shown in Fig.…”

“…Senescent cells display several distinctive characteristics such as unique histological changes [41,42], increased expression of p16 INK4 and p21 WAF1/Cip , and increased activity of senescence-associated b-galactosidase (SA-b-gal) [43]. As well as progression to a senescence-associated secretory phenotype (SASP), which is a persistent hypersecretory state characterized by increased expression and secretion of inflammatory molecules such as cytokines, chemokines, growth factors and profibrotic factors [5,37,39,[44][45][46][47][48][49][50][51].…”

“…Recent studies [45,49,65] suggest that cytokines/chemokines produced by senescent cholangiocytes could also facilitate cellular senescence in bystander cholangiocytes and other types of cells in the periductal tissue. For example, we have shown that CCL2 and CX3CL1, which are components of the SASPs of senescent cholangiocytes, induced cellular senescence in cholangiocytes [45,49,65].…”

“…Recent studies using cultured cholangiocytes showed interesting findings connecting innate immunity to oncogene-induced cellular senescence [9,48,49,90]. N-Ras is the predominant Ras isoform expressed in cholangiocytes.…”