Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics

Hepatic stellate cells (HSCs) generate matrix, which in turn may also regulate HSCs function during liver fibrosis. We hypothesized that HSCs may endocytose matrix proteins to sense and respond to changes in microenvironment. Primary human HSCs, LX2, or mouse embryonic fibroblasts (MEFs) [wild-type; c-abl^-/-; or Yes, Src, and Fyn knockout mice (YSF^-/-)] were incubated with fluorescent-labeled collagen or gelatin. Fluorescence-activated cell sorting analysis and confocal microscopy were used for measuring cellular internalization of matrix proteins. Targeted PCR array and quantitative real-time PCR were used to evaluate gene expression changes. HSCs and LX2 cells endocytose collagens in a concentration- and time-dependent manner. Endocytosed collagen colocalized with Dextran 10K, a marker of macropinocytosis, and 5-ethylisopropyl amiloride, an inhibitor of macropinocytosis, reduced collagen internalization by 46%. Cytochalasin D and ML7 blocked collagen internalization by 47% and 45%, respectively, indicating that actin and myosin are critical for collagen endocytosis. Wortmannin and AKT inhibitor blocked collagen internalization by 70% and 89%, respectively, indicating that matrix macropinocytosis requires phosphoinositide-3-kinase (PI3K)/AKT signaling. Overexpression of dominant-negative dynamin-2 K44A blocked matrix internalization by 77%, indicating a role for dynamin-2 in matrix macropinocytosis. Whereas c-abl^-/- MEF showed impaired matrix endocytosis, YSF^-/- MEF surprisingly showed increased matrix endocytosis. It was also associated with complex gene regulations that related with matrix dynamics, including increased matrix metalloproteinase 9 (MMP-9) mRNA levels and zymographic activity. HSCs endocytose matrix proteins through macropinocytosis that requires a signaling network composed of PI3K/AKT, dynamin-2, and c-abl. Interaction with extracellular matrix regulates matrix dynamics through modulating multiple gene expressions including MMP-9.