Hepatocyte Growth Factor-induced Differentiation of BMSCs toward Hepatocyte-like Cells via the NF-κB and P38MAPK Signaling Pathway

Author(s): Yingying Li, Ling Li, Yi WANG, Kelong Wu, Linlin WANG, Yu WANG, Bingyan LIU, Xiangmin YU

Bone marrow mesenchymal stem cells(BMSCs) have great potential ability of multi-directional differentiation and reproductive activity. Recent studys have demonstrated that BMSCs can be induced into hepatocyte-like cells. However, the molecular mechanism of hepatocellular differentiation remains unknown. In this study, we investigated the nexus between p38 MAPK and NF-κB signaling pathway in the process of the hepatocellular differentiation. We isolated BMSCs from femurs and tibias of rats. The third generation were divided into three main groups: induction group, inhibition group and negative control group. Hepatic differentiation was induced by 10% fetal bovine serum with hepatocyte growth factor(HGF). The inhibitors of p38 (SB203580) and NF-κB(BAY 11-7082) were added to the differentiation medium for inhibition of signaling molecular activities. Morphological characteristics and transferring function of the differentiated cells were examined by indocyanine green (ICG) uptake assay. Immunohistochemical staining was used to evaluate the protein expression position of NF-кB. And western blot analysis was used to detect the protein expression of several markers, including the specifc markers of hepatocytes(AAT), phosphorylated-p38(p-p38) and NF-кB. NF-кB were observed transferred into nuclear in the induction group. The respective inhibitors inhibited the expressions of NF-кB and p-p38 effectively. Compared to the induction group, expressions of specifc marker, AAT, were decreased visibly in the p38 and NF-κB inhibitor-treated groups. Notably, expression of NF-κB were significantly lowered in the p38 inhibitor-treated group. These data suggest both NF-κB pathway and p38MAPK pathway participate in the hepatocellular differentiation of BMSCs, p38MAPK can affect the regulation of NF-κB to this process of differentiation.

Bone marrow stem cells, Hepatocyte, Differentiation, Hepatocyte growth factor, Nuclear factor-kappa B, p38MAPK

1. Prockop D J. Marrow stromal cells as stem cells for nonhematopoietic tissues[J]. Science, 1997,276(5309):71-74.
2. Peng S, Zhou G, Luk K D, et al. Strontium promotes osteogenic differentiation of mesenchymal stem cells through the Ras/MAPK signaling pathway[J]. Cell Physiol Biochem, 2009,23(1-3):165-174.
3. Zhang A, Wang Y, Ye Z, et al. Mechanism of TNF-alpha-induced migration and hepatocyte growth factor production in human mesenchymal stem cells[J]. J Cell Biochem, 2010,111(2):469-475.
4. Davis R J. Signal transduction by the JNK group of MAP kinases[J]. Cell, 2000,103(2):239-252.
5. Lu T, Yang C, Sun H, et al. FGF4 and HGF promote differentiation of mouse bone marrow mesenchymal stem cells into hepatocytes via the MAPK pathway[J]. Genet Mol Res, 2014,13(1):415-424.
6. Lee F S, Peters R T, Dang L C, et al. MEKK1 activates both IkappaB kinase alpha and IkappaB kinase beta[J]. Proc Natl Acad Sci U S A, 1998,95(16):9319-9324.
7. Je J H, Lee J Y, Jung K J, et al. NF-kappaB activation mechanism of 4-hydroxyhexenal via NIK/IKK and p38 MAPK pathway[J]. FEBS Lett, 2004,566(1-3):183-189.
8. Pittenger M F, Mackay A M, Beck S C, et al. Multilineage potential of adult human mesenchymal stem cells[J]. Science, 1999,284(5411):143-147.
9. Jiang Y, Jahagirdar B N, Reinhardt R L, et al. Pluripotency of mesenchymal stem cells derived from adult marrow[J]. Nature, 2002,418(6893):41-49.
10. Buxton A N, Bahney C S, Yoo J U, et al. Temporal exposure to chondrogenic factors modulates human mesenchymal stem cell chondrogenesis in hydrogels[J]. Tissue Eng Part A, 2011,17(3-4):371-380.
11. Chen Y, Dong X J, Zhang G R, et al. Transdifferentiation of mouse BM cells into hepatocyte-like cells[J]. Cytotherapy, 2006,8(4):381-389.
12. Bianco P, Riminucci M, Gronthos S, et al. Bone marrow stromal stem cells: nature, biology, and potential applications[J]. Stem Cells, 2001,19(3):180-192.
13. Forte G, Minieri M, Cossa P, et al. Hepatocyte growth factor effects on mesenchymal stem cells: proliferation, migration, and differentiation[J]. Stem Cells, 2006,24(1):23-33.
14. Pan R L, Chen Y, Xiang L X, et al. Fetal liver-conditioned medium induces hepatic specification from mouse bone marrow mesenchymal stromal cells: a novel strategy for hepatic transdifferentiation[J]. Cytotherapy, 2008,10(7):668-675.
15. Yamada T, Yoshikawa M, Kanda S, et al. In vitro differentiation of embryonic stem cells into hepatocyte-like cells identified by cellular uptake of indocyanine green[J]. Stem Cells, 2002,20(2):146-154.
16. Obaid R, Wani S E, Azfer A, et al. Optineurin Negatively Regulates Osteoclast Differentiation by Modulating NF-kappaB and Interferon Signaling: Implications for Paget's Disease[J]. Cell Rep, 2015,13(6):1096-1102.
17. Han D, Wu G, Chang C, et al. Disulfiram inhibits TGF-beta-induced epithelial-mesenchymal transition and stem-like features in breast cancer via ERK/NF-kappaB/Snail pathway[J]. Oncotarget, 2015,6(38):40907-40919.
18. Grethe S, Ares M P, Andersson T, et al. p38 MAPK mediates TNF-induced apoptosis in endothelial cells via phosphorylation and downregulation of Bcl-x(L)[J]. Exp Cell Res, 2004,298(2):632-642.
19. Li J, Zhao Z, Liu J, et al. MEK/ERK and p38 MAPK regulate chondrogenesis of rat bone marrow mesenchymal stem cells through delicate interaction with TGF-beta1/Smads pathway[J]. Cell Prolif, 2010,43(4):333-343.
20. Davis R J. Signal transduction by the JNK group of MAP kinases[J]. Cell, 2000,103(2):239-252.
21. Chun J S. Expression, activity, and regulation of MAP kinases in cultured chondrocytes[J]. Methods Mol Med, 2004,100:291-306.