Endocytosis of collagen by hepatic stellate cells regulates extracellular matrix dynamics

Abstract

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.

A new front in cell invasion: The invadopodial membrane

“…Signaling from endosomes that originate from endocytic events at invadopodia might allow rapid feedback and adaption during invasion. Consistent with this possibility, endocytosis of collagen has been observed in macrophages, fibroblasts, and hepatic stellate cells (a liver cell that maintains extracellular matrix homeostasis during liver damage) (Bi et al, 2014). Further, in fibrosarcoma cells fibronectin is internalized into the endolysosome, the same compartment as MT1-MMP (Sung et al, 2011).…”

“…Further, in fibrosarcoma cells fibronectin is internalized into the endolysosome, the same compartment as MT1-MMP (Sung et al, 2011). Intriguingly, collagen endocytosis in hepatic stellate cells leads to the transcriptional upregulation of MMP-9, consistent with a signaling function of endocytosed collagen (Bi et al, 2014). It is thus possible that the invadopodial membrane has numerous essential functions in invadopodia formation, function, and adaptation to the microenvironment.…”

Akt and SGK protein kinases are required for efficient feeding by macropinocytosis

“…Akt inhibition does not depress macropinosome formation in macrophages, although these studies did not rule out smaller macropinosomes and reduced fluid uptake (Pacitto et al, 2017; Yoshida et al, 2015). Macropinocytic uptake of collagen by hepatic stellate cells, measured quantitatively by flow cytometry, is reduced by Akt inhibition (Bi et al, 2014). Whether this is due to smaller macropinosomes was not investigated but is a tantalising possibility.…”

Hepatic Stellate Cell Selective Disruption of Dynamin-2 GTPase Increases Murine Fibrogenesis through Up-Regulation of Sphingosine-1 Phosphate–Induced Cell Migration

“…In HSCs, Dyn2K44A is reported to induce accumulation of RTKs on HSC surface, and enhanced RTK induced signaling; however, additional studies show that Dyn2K44A overexpression in HSCs blocks collagen internalization and matrix metalloproteinase production. 29 In the present study in HSCs, the Dyn2K44A mechanism of action appeared through transcriptional regulation rather than through membrane receptor trafficking, as discussed below.…”