Migration Of Human Hepatic Stellate Cells Research Articles

Hepatitis B virus X protein activates human hepatic stellate cells through upregulating TGFβ1.

We investigated the effects of the hepatitis B virus X gene (HBV X) on the activation of human hepatic stellate cells (HSCs) and the possible mechanisms underlying the pathway. Recombinant plasmid pHBV-X-IRES2-EGFP was constructed and transfected into HL-7702 cells using a lipid-mediated method. Transfected cells were screened by G418, which detected stable expression of the X gene by reverse transcription (RT)-PCR and Western blot analysis, and named L02/x. Cells not subjected to G418-selection were analyzed to confirm the transient expression of the X gene and named L02/48x. Subsequently, L02/x and L02/48x, together with non-HBx-expressing cells, were co-cultured with HSCs in a non-contact transwell system. After 36 h of co-culture, the proliferation and migration of HSCs was detected using different cell counting methods. Finally, the mRNA and protein levels of α-SMA, Col I, and TGFβ1 in HSCs were detected by real-time PCR and western blot analysis. RT-PCR and Western blot analysis showed that L02/x and L02/48x cells can express HBV X gene mRNA and protein. Additionally, HSCs co-cultured with L02/x or L02/48x cells showed significantly higher proliferation and migration levels than control groups. Real-time PCR and Western blot analysis showed that the mRNA and protein expressions of α-SMA, Col I, and TGFβ1 in HSCs co-cultured with HBx-expressing liver cells were higher than those in control groups. HBx protein activated HSCs in vitro, leading to increased proliferation and migration of HSCs and upregulation of α-SMA and Col I. The TGFβ1 gene may be involved in this pathway.

Depletion of Thymosin β4 Promotes the Proliferation, Migration, and Activation of Human Hepatic Stellate Cells

Background & Aims: It has recently been reported that thymosin beta-4 (Tβ4) has anti-fibrogenic effects in human hepatic stellate cells (HSCs) in vitro, but the mechanisms underlying these effects remain unclear. The aim of this study was to investigate the roles of Tβ4 in the proliferation, migration, and activation of HSCs. Methods: Enzyme-linked immunosorbent assays (ELISA), immunohistochemistry, and western blot assays were utilized to determine the expression levels of Tβ4 in serum, liver tissues, and LX-2 cells. Tβ4 was depleted in LX-2 cells using small interfering RNAs (siRNAs). Cell proliferation was analyzed using cell counting kit-8 (CCK-8) viability assays, and cell migration was investigated using wound-healing and transwell migration assays. Results: The expression of Tβ4 was significantly reduced during the progression of liver fibrosis. The depletion of Tβ4 significantly promoted the proliferation and migration of LX-2 cells via the activation of the PI3K/Akt signaling pathway. The pro-migratory and pro-proliferative effects of Tβ4 depletion in LX-2 cells can be counteracted by treatment with the Akt inhibitor MK-2206. In addition, Tβ4 depletion was also associated with the activation of HSCs via the enhanced expression of α-smooth muscle actin (α-SMA) and vimentin. Conclusions: Our results suggest that Tβ4 participates in liver fibrosis by inhibiting the migration, proliferation, and activation of HSCs and that Tβ4 may be an effective target in the treatment of liver fibrosis.

Essential roles of sphingosine 1‐phosphate receptor types 1 and 3 in human hepatic stellate cells motility and activation

The biological roles of sphingosine 1-phosphate (S1P) and S1P receptors (S1PRs) have been broadly investigated. However, at present pathophysiological roles of S1P/S1PRs axis in liver fibrosis are not well defined. Here, we investigated the functions of S1P/S1PRs axis in human hepatic stellate cells (HSC) line, LX-2 cells. We found that S1PR types 1, 2 and 3 (S1PR1-3) are clearly detected in LX-2 cells, as determined by RT-PCR, Western blot and immunocytochemistry analysis. S1P exerted a powerful migratory action on LX-2 cells, as determined in Boyden chambers, and stimulated fibrogenic activity of LX-2 cells, as demonstrated by increase of expression of smooth muscle α-actin, procollagen α1(I) and α1(III) and total hydroxyproline content. Moreover, the effects of S1P were mimicked by S1PR1 agonist SEW2871, and abrogated by W146 (S1PR1 antagonist) and/or silencing S1PR1, three expression with small interfering RNA, suggesting the main roles of S1PR1 and 3. However, studies with S1PR2 antagonist JTE-013 and silencing S1PR2 expression indicated that S1PR2 negatively regulated S1P-induced cell migration. Interestingly, exogenously added S1P induced significant up-regulation of sphingosine kinase-1 and the synthesis of additional S1P, and expression of S1PR1,3, but not S1PR2. In conclusion, our data have identified an additional function regulated by S1P/S1PR1,3 axis involving migration and fibrogenic activation of HSCs. These results suggest that selective modulation of S1PR activity may represent a new antifibrotic strategy.

Adenosine monophosphate–activated protein kinase modulates the activated phenotype of hepatic stellate cells

Adiponectin limits the development of liver fibrosis and activates adenosine monophosphate-activated protein kinase (AMPK). AMPK is a sensor of the cellular energy status, but its possible modulation of the fibrogenic properties of hepatic stellate cells (HSCs) has not been established. In this study, we investigated the role of AMPK activation in the biology of activated human HSCs. A time-dependent activation of AMPK was observed in response to a number of stimuli, including globular adiponectin, 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR), or metformin. All these compounds significantly inhibited platelet-derived growth factor (PDGF)-stimulated proliferation and migration of human HSCs and reduced the secretion of monocyte chemoattractant protein-1. In addition, AICAR limited the secretion of type I procollagen. Knockdown of AMPK by gene silencing increased the mitogenic effects of PDGF, confirming the negative modulation exerted by this pathway on HSCs. AMPK activation did not reduce PDGF-dependent activation of extracellular signal-regulated kinase (ERK) or Akt at early time points, whereas a marked inhibition was observed 24 hours after addition of PDGF, reflecting a block in cell cycle progression. In contrast, AICAR blocked short-term phosphorylation of ribosomal S6 kinase (p70(S6K)) and 4E binding protein-1 (4EBP1), 2 downstream effectors of the mammalian target of rapamycin (mTOR) pathway, by PDGF. The ability of interleukin-a (IL-1) to activate nuclear factor kappa B (NF-kappaB) was also reduced by AICAR. Activation of AMPK negatively modulates the activated phenotype of HSCs.