Background & Aim:
In liver diseases with severe parenchymal loss, e.g. acute-on-chronic liver failure (ACLF), liver progenitor cells (LPC) are the main cell source to replenish lost hepatocytes through cell reprogramming. The molecular mechanisms underlying LPC towards hepatocytes differentiation in ACLF largely remain unknown to date. Activation of LPC is morphologically demonstrated as ductular reaction (DR). The similarity between DR and ductal plate (DP) of embryonic liver implies that LPC/DR differentiation towards hepatocytes might exploit similar mechanisms stem cells adopted in embryonic development. Tripartite motif protein (TRIM) 33 is a crucial transcription factor for differentiation of embryonic stem cells through the formation of transcription factor complexes with phosphorylated Smad2 and Smad3, the downstream substrates of activated TGF-β signaling. This transcription factor complex replaces heterochromatin protein 1, a main inhibitor of master regulators of cell differentiation, and thus opens binding sites at promoters for the additional transcription factor complexes, such as Smad4-pSmad2/3-FoxH1. The binding of the latter complexes leads to expression of master regulators of differentiation, e.g. goosecoid (GSC). The current study investigated the role of TRIM33 in LPC differentiation towards hepatocytes in ACLF.
Methods:
We enrolled 113 liver tissue specimens from patients with compensated cirrhosis, 20 ACLF with liver transplantation and 3 ACLF spontaneously recovered. Expression of TRIM33, FoxH1, GSC and presence of p-Smad2 was measured by immunohistochemistry. In vitro, BMOL cells, a murine LPC line, were used to establish an LPC towards hepatocyte differentiation model.
Results:
Given that more than 70% hepatocyte buds derive from LPC in cirrhotic liver, patients with cirrhosis are ideal candidates to observe successful LPC differentiation towards hepatocytes. In 90 HBV-associated compensated cirrhotic patients, most buds showed strong immune positivity for p-Smad2, TRIM33, FoxH1 and GSC in the same hepatocytes. However, in 20 ACLF patients, who received liver transplantation, expression of TRIM33, FoxH1 and GSC were undetectable. In contrast to these patients, 3 spontaneously recovered ACLF patients showed strong nuclear FoxH1 and p-Smad2 staining in LPC and hepatocyte-like cells. In vitro, BMOL cells differentiated into hepatocyte-like cells, expressing hepatocyte specific proteins, e.g. albumin and transferrin, synthesizing glycogen, and losing cholangiocyte phenotype as evident from reduced CK19 and SOX9 expression over time. In this transdifferentiation process, phosphorylation of Smad2 and Smad3, and expression of TRIM33 and FoxH1 were induced and maintained at high levels. Immunoprecipitation analysis revealed the presence of TRIM33/p-Smad2/3 complexes. Inhibition of TGF-β signaling, knockdown of TRIM33 or FoxH1 significantly reduced expression of albumin and transferrin. Interestingly, administration of TGF-β inhibited expression of TRIM33 and forced BMOL cells to differentiate into fibroblasts, but not hepatocytes. Furthermore, TRIM33 expression was upregulated by EGF, whereas blocking the EGF receptor reduced expression of TRIM33, albumin and transferrin.
Conclusions:
Transcription factor complexes TRIM33/p-Smad2/3 mediate differentiation of LPC towards hepatocyte in liver cirrhosis and ACLF.
