Hepatic stellate cells regulate hepatic progenitor cells differentiation via the TGF-ß1/Jagged1 signaling axis.

Hepatic stellate cells (HSCs) play an important microenvironmental role in hepatic progenitor cells (HPCs) differentiation fate. To reveal the specific mechanism of HSCs induced by transforming growth factor ß1 (TGF-ß1) signaling in HPCs differentiation process, we used Knockin and knockdown technologies induced by lentivirus to upregulate or downregulate TGF-ß1 level in mouse HSCs (mHSCs) (mHSCs-TGF-ß1 or mHSCs-TGF-ßR1sih3). Primary mouse HPCs (mHPCs) were isolated and were cocultured with mHSCs-TGF-ß1 and mHSCs-TGF-ßR1sih3 for 7 days. Differentiation of mHPCs was detected by quantitative reverse transcriptase polymerase chain reaction analysis and immunofluorence in vitro. mHPCs-E14.5 cell lines and differently treated mHSCs were cotransplanted into mice spleens immediately after establishment of acute liver injury model for animal studies. Engraftment and differentiation of transplanted cells as well as liver function recovery were measured at the seventh day via different methods. mHSCs-TGF-ß1 were transformed into myofibroblasts and highly expressed Jagged1, but that expression was reduced after blockage of TGF-ß1 signaling. mHPCs highly expressed downstream markers of Jagged1/Notch signaling and cholangiocyte markers (CK19, SOX9, and Hes1) after coculture with mHSCs-TGF-ß1 in vitro. In contrast, mature hepatocyte marker (ALB) was upregulated in mHPCs in coculture conditions with mHSCs-TGF-ßR1sih3. At the seventh day of cell transplantation assay, mHPCs-E 14.5 engrafted and differentiated into cholangiocytes after cotransplanting with TGF-ß1-knockin mHSCs, but the cells had a tendency to differentiate into hepatocytes when transplanted with TGF-ßR1-knockdown mHSCs, which corresponded to in vitro studies. HSCs play an important role in regulating HPCs differentiation into cholangiocytes via the TGF-ß1/Jagged1 signaling axis. However, HPCs have a tendency to differentiate into hepatocytes after blockage of TGF-ß1 signaling in HSCs.

Hepatic stellate cells promote angiogenesis via the TGF-ß1-Jagged1/VEGFA axis.

Hepatic stellate cells (HSCs) are activated by transforming growth factor (TGF)-ß1 and function as mesenchymal cells in liver regeneration. Activated HSCs also have proangiogenic ability in vivo. In this study, knockin of the TGF-ß1 gene caused mHSCs to transform into myofibroblasts (MFs) highly expressing Jagged1 and vascular endothelial growth factor A (VEGFA). These MFs promoted formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs) in vitro, which was much reduced after blocking TGF-ß1 signaling. Transplantation of TGF-ß1-knockin mHSCs was followed by efficient engraftment into livers and accompanied by increased vascular organization and expression of Jagged1 and VEGFA compared with controls. Less hepatic angiogenesis and lower Jagged1 and VEGFA expression, was found in livers engrafted with TGF-ß-R1-knockdown mHSCs. Increased vascularization improved liver function. The findings showed that mHSCs were regulated by TGF-ß1 signaling to express Jagged1 and VEGFA, which were associated with hepatic angiogenesis, a novel mechanism of mHSC promotion of new vascular structures.

TGFß signaling controls intrahepatic bile duct development may through regulating the Jagged1-Notch-Sox9 signaling axis.

Due to the inherent limitations of the mouse models, the molecular mechanism of TGFß signaling involved in the development of intrahepatic bile ducts (IHBDs) has been investigated little. Here, we investigated the role of TGFß signaling and its regulatory mechanism in IHBDs development. We demonstrate that TGFß signaling pathway activity is essential for IHBDs development. When blocking TGFß signaling at E10.5, the number of bile ducts in hilum was reduced more than two fold and number of CK19 positive chlangiocytes in periphery was reduced more than 3.5-fold compared with controls. We also show that alpha-smooth muscle actin (α-SMA)-immunoreactive cells are located in the portal vein mesenchyme (PVM) adjacent to the bile ducts during IHBDs development and identify the α-SMA positive cells expressing the Notch ligand Jagged1 in the periportal area. Importantly, after blocking TGFß signaling, the expression of Jagged1 was selectively decreased in the PVM but not in biliary epithelial cells (BECs), which is associated with the transformation of portal mesenchyme cells (PMCs) into portal myofibroblasts (PMFs). In addition, Sox9, which is downstream of Notch, is decreased after blocking the TGFß signaling pathway in the liver. Our findings uncover a novel mechanism of TGFß signaling in controlling the development of IHBDs may through regulating the Jagged1-Notch-Sox9 signaling axis.

Direct and indirect coculture of mouse hepatic progenitor cells with mouse embryonic fibroblasts for the generation of hepatocytes and cholangiocytes.

The widespread use of hepatocytes and cholangiocytes for regenerative medicine and tissue engineering is restricted by the limited number of hepatocytes and cholangiocytes; a simple and effective method for the expansion and differentiation of the hepatic progenitor cells (HPCs) is required. Recent studies demonstrated that mouse embryonic fibroblasts (MEFs) play an important role in supporting the proliferation of the mouse hepatic progenitor cells (mHPCs). However, the effect of direct and indirect coculture of MEFs with mHPCs on the differentiation of mHPCs is poorly studied. Herein, we show that mHPCs rapidly proliferate and form colonies in direct or indirect contact coculture with MEFs in the serum-free medium. Importantly, after direct contact coculture of the mHPCs with MEFs for 6 days, mHPCs expressed the hepatic marker albumin (ALB) and did not express the cholangiocyte marker CK19, indicating their differentiation into hepatocytes. In contrast, after indirect contact coculture of the mHPCs with MEFs for 6 days, mHPCs expressed the cholangiocyte marker CK19 and did not express the hepatic marker ALB, indicating their differentiation into cholangiocytes. These results indicate that direct and indirect contact cocultures of the mHPCs with MEFs are useful for rapidly producing hepatocytes and cholangiocytes.

TGF-ß1 signaling activates hepatic stellate cells through Notch pathway.

Hepatic stellate cells (HSCs), as the most important stromal cells in the liver microenvironment, play crucial roles in hepatic fibrosis, hepatocellular carcinoma, liver regeneration and fetal liver development after transdifferentiating into myofibroblasts (MFs). Transforming growth factor ß1 (TGF-ß1), as an important polyergic cytokine, is involved in HSCs activation process. However, the specific mechanisms of HSCs transdifferentiation process are not clearly demonstrated. Here we added exogenous recombinant TGF-ß1 protein and transforming growth factor ß receptor 1 (TGF-ßR1) inhibitor SB431542 into mouse HSCs to detect the detailed impact of TGF-ß1 signaling on HSCs activation. TGF-ß1 signaling significantly increased phosphorylated (P)-Smad2/3 level and promoted Smad2/3 translocation from the cytoplasm to the nucleus, which also caused transdifferentiation of HSCs into MFs. Importantly, TGF-ß1 signaling also resulted in high expression of Notch pathway markers Notch1, Jagged1, Hes1 in HSCs. In contrast, expression of those above markers in mouse HSCs were obviously decreased after hampering TGF-ß1 signaling via TGF-ßR1 inhibitor SB431542. To further examine the effect of Notch pathway on HSCs activation process, TGF-ß1-stimulated HSCs and control HSCs were treated with or without LY450139, a specific inhibitor of Notch pathway. LY450139 evidently decreased the expression of Notch1 and MFs marker α-smooth muscle actin (α-SMA) expression in HSCs. These above results may provide a novel insight that TGF-ß1 signaling controls HSCs activation process through regulating the expression of Notch pathway markers.

TGF-ß1 signaling regulates mouse hepatic stellate cell differentiation via the Jagged1/Notch pathway.

AIMS: We tested whether transforming growth factor ß1 (TGF-ß1) signaling plays an important role in hepatic stellate cell differentiation fate and investigated the role of Jagged1/Notch in this process. MATERIALS AND METHODS: TGF-ß1 was overexpressed and transforming growth factor receptor 1 (TGF-ß-R1) was knocked down by a lentiviral vector in mouse hepatic stellate cells (mHSCs). Transfection efficiency was assessed with immunofluorescence, quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and western blotting. The downstream genes alpha-smooth muscle actin (α-SMA), Jagged1 and the differentiation markers alpha-fetoprotein (AFP), albumin (ALB), cytokeratin19 (CK19), SRY (sex determining region Y)-box 9 (SOX9), and hairy and enhancer of split-1 (Hes1) were measured with qRT-PCR and western blotting. KEY FINDINGS: SpHBLV-CMVIE-TGF-ß1, pHBLV-CMVIE-GFP, pHBLV-U6-TGF-ß-R1 shRNA, and pHBLV-U6-RFP were successfully transfected. Over-expression of the TGF-ß1 gene caused mHSCs to transform into myofibroblasts (MFs) and expression of Jagged1 and cholangiocyte markers (CK19, SOX9, Hes1) were significantly upregulated (P<0.01). Importantly, after blocking TGF-ß1 signaling via gene silencing, expression of Jagged1 was much reduced, but the mature hepatocyte marker (ALB) was obviously increased. In addition, AFP, a hepatic stem cell marker, was expressed at the highest level in the control groups. SIGNIFICANCE: Our findings emphasize that the TGF-ß1 signaling pathway regulates expression of Jagged1 in mHSCs which is associated with transformation of mHSCs into MFs, thus demonstrating a novel mechanism via which TGF-ß1 signaling controls the differentiation fate of mHSCs through regulation of the Jagged1/Notch pathway.