Abstract
ABSTRACTThe actin cytoskeleton of hepatic stellate cells (HSCs) is reorganized when they are cultured in 3D collagen matrices. Here, we investigated the molecular mechanism of actin cytoskeleton reorganization in HSCs cultured in 3D floating collagen matrices (FCM) compared to those on 2D polystyrene surfaces (PS). First, we found that the generation of dendritic cellular processes was controlled by Rac1. Next, we examined the differential gene expression of HSCs cultured on 2D PS and in 3D FCM by RNA-Seq and focused on the changes of actin cytoskeleton reorganization-related molecular components and guanine nucleotide exchange factors (GEFs). The results showed that the expression of genes associated with actin cytoskeleton reorganization-related cellular components, filopodia and lamellipodia, were significantly decreased, but podosome-related genes was significantly increased in 3D FCM. Furthermore, we found that a Rac1-specific GEF, ARHGEF4, played roles in morphological changes, migration and podosome-related gene expression in HSCs cultured in 3D FCM.Abbreviations: 2D PS: 2-dimensional polystyrene surface; 3D FCM: 3-dimensional floating collagen matrices; ARHGEF4: Rho guanine nucleotide exchange factor 4; ARHGEF6: Rho guanine nucleotide exchange factor 6; GEF: guanine nucleotide exchange factor; HSC: hepatic stellate cell
Highlights
Hepatic stellate cells (HSCs), located in the sinusoidal space of Disse between endothelial cells and parenchymal cells, have been found to form many long cellular processes that surround the hepatic sinusoids in vivo [1]
Hepatic stellate cells cultured in 3D floating collagen matrices (FCM) exhibited stellate or dendritic shapes and long, slender cellular processes (Figure 1(b)). This morphology is closer to the in situ morphology, as HSCs are located in the space of Disse and possess cytoplasmic processes that extend along the sinusoids in vivo
In situ immunofluorescence staining of GFAP in mouse liver revealed that the GFAPpositive cells, HSCs, had extended cellular processes along the sinusoids, which is similar to the phenotype of HSCs cultured in 3D FCM (Figure 1(c))
Summary
Hepatic stellate cells (HSCs), located in the sinusoidal space of Disse between endothelial cells and parenchymal cells, have been found to form many long cellular processes that surround the hepatic sinusoids in vivo [1]. HSCs show a flattened, fibroblast-like shape with well-developed lamellipodia and actin stress fibers when cultured on a 2D polystyrene surface (PS) or on a type I collagen-coated surface, suggesting the regulation of HSC morphology and function by extracellular matrix (ECM) components in the perisinusoidal space of Disse [2–4]. HSCs exhibit an in vivo morphology with long cellular processes when cultured in 3D collagen matrices and do not form clear lamellipodia and actin stress fibers [3,5–7]. Small G proteins ( known as GTPases), such as Rac and RhoA, are important in actin cytoskeleton reorganization [12,13]. These GTPases are regulated by guanine nucleotide exchange factors (GEFs), which activate GTPases by facilitating the exchange of GDP for GTP [14]. The morphological change of HSCs cultured in 3D collagen matrices has been known for over a decade, the underlying mechanism of the HSC morphological change and the molecular mechanism of actin cytoskeleton reorganization in 3D culture remain to be elucidated
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