Background: Contraction and migration of hepatic stellate cells (HSC) during hepatic injury is essential for wound-healing, liver fibrosis and portal hypertension. Aims: To investigate the role of nonmuscle myosin II (NMMHC-II) isoforms in mouse HSC. Methods: First all, we identified which NMMHC-II(s) is expressed in HSC by QRT-PCR and Western blot. Then their subcellular localization was visualized by immunofluorescence and/or confocal microscopy after immunofluorescence staining. The interaction with actin isoforms was explored by dual-immunostaining and further conformed by co-immunopericiptation. Under the condition of isoform-specific siRNA gene silencing or treatment with chemical inhibitors, morphological and functional assays were performed. Changes were visualized with phase contrast microscopy. ET-1-induced contractile force generation was examined by collagen gel contraction and silicone wrinkle formation assay. Intracellular Ca2+ increase induced by ET-1stimulation was measured using Fluo-4. Cell migration was evaluated using the 'wound healing' assay. Results: NMMHC-IIA and -IIB are expressed in cultured HSC at both mRNA and protein level. NMMHC-IIA mRNA and protein levels were roughly constant. It was located in the subcortical area of quiescent HSC and colocalized with ?-SMA in stress fibers in activated HSC. Knockdown of NMMHC-IIA by siRNA decreased cell size, stress fibers and vinculin-containing focal adhesion. Knockdown of NMMHC-IIA completely impaired wrinkle formation on silicone substrate, and also blocked intracellular [Ca2+] release response to ET-1, but accelerated wound-induced cell migration. NMMHC-IIB was upregulated at both mRNA and protein levels. It was present in cytoplasmic processes of quiescent HSC, lamellipodia and cytoplasm in activated HSC. Colocalization of NMMHC-IIB and ?-actin was observed in the leading edge of lamellipodia. We also found expansion of ?-tubulin in lamellipodia (but not the leading edge) following spreading of the leading edge. Knockdown of NMMHC-IIB by siRNA decreased the formation of lamellipodia, impaired expansion of ?-tubulin in lamellipodia, and slowed down cell migration. We did not observe a significant effect of NMMHC-IIB on stress-fiber formation, cell contraction and intracellular Ca2+ release. Conclusions: myosin IIA and IIB are differentially expressed and localized in mHSC. They play distinct and complementary roles: myosin IIA controls cell body, focal adhesions and cell contraction by regulating stress-fiber formation which contain myosin IIA and ?-SMA. Myosin IIB mediates lamellipodia spreading and cell migration by distribution of ?-actin in the leading edge and by inducing ?-tubulin-expansion in lamellipodia.
Original languageEnglish
Title of host publicationThe Belgian Week of Gastroenterology
Publication statusPublished - Feb 2009

    Research areas

  • Myosin II isoforms, contraction and migration, HSC

ID: 1841275