SCG10 expression on activation of hepatic stellate cells promotes cell motility through interference with microtubules

Abstract

During liver fibrogenesis, quiescent hepatic stellate cells switch their phenotype toward a myofibroblastic-like pattern with a gain in motility. Here, we show that SCG10 (superior cervical ganglia 10) mRNA expression, a microtubule-destabilizing protein that favors cell growth and motility in neurons, both increases and correlates with the stage of fibrosis in patients with chronic hepatitis C. We also show the de novo expression of SCG10 mRNA in two rat models of liver fibrosis. We demonstrate that activated hepatic stellate cells appear to be the major cellular sources of SCG10 in the liver. Tracking of the SCG10 pathway in hepatic stellate cells shows that SCG10 initially accumulates in the perinuclear Golgi area then migrates in small vesicle-like structures along individual microtubules. Moreover, SCG10 vesicles cluster at the distal ends of microtubules in areas where tubules are spread and decompacted, suggesting their preferential association with destabilized and dynamic microtubules. Inhibition of SCG10 expression by gene-specific short interfering RNA in primary rat hepatic stellate cells is associated with a significant reduction in microtubule-dependent cellular functions, such as proliferation and migration. In conclusion, the de novo expression of SCG10 by hepatic stellate cells may play a major role in cellular mechanisms associated with HSC activation, namely cell motility and division, through interference with microtubules. SCG10 may represent a potential molecular target for anti-fibrosis therapies.

Figure 1

Quantitative SCG10 expression level assessed by real-time PCR. A: In liver biopsies of patients with hepatitis C and different stages of fibrosis according to METAVIR scoring system, SCG10 increases according to the score of fibrosis while SCLIP and RB3, other members of the family, do not change. B: In experimental models of rat liver fibrosis, chronic CCl4 intoxication, and bile duct ligation (BDL), SCG10 expression significantly increases in both models compared with sham-operated animals. Data are normalized to normal liver (A) or sham-operated rats (B). Mann–Whitney U-test, **P < 0.01.

Figure 2

Western blot analysis of SCG10 expression in HSCs on days 1, 4, and 7 after isolation and plating. PC12 were used as control cells.

Figure 3

SCG10 trafficking pathway in HSCs. HSCs were transfected with a SCG10-myc fusion protein after 2 days of plating and fixed with paraformaldehyde 48 hours later. Expression of exogenous SCG10 was detected using an anti-myc anti-serum. Microtubules were costained with anti–α-tubulin antibody. Transfected SCG10 concentrate mainly at the Golgi area, with small vesicles along the cytoplasmic processes.

Figure 4

Localization of endogenous SCG10 in HSC. Immunofluorescence microscopy after double labeling for SCG10 (green) and tubulin (red). A: After 4 days in culture, SCG10 is mainly located in the perinuclear network, suggestive of the Golgi area. B: In fully activated cells (7 days in culture), SCG10 labeling appears as small intracytoplasmic vesicle-like structures. Coimmunostaining of microtubules shows that SCG10 was organized in close contact and along individual microtubules.

Figure 5

Colocalization of SCG10 and tubulin at the distal end of microtubules in HSC after 7 days in culture. Double immunofluorescence of SCG10 (green) and tubulin (red) in activated HSCs. A: SCG10 vesicles are present along microtubules and accumulate at the end and in areas where microtubules are decompacted. B: Higher magnification showing SCG10 vesicles where microtubules are spread out.

Figure 6

Inhibition of SCG10 expression by gene-specific siRNAs in HSCs. Cells were transfected on day 4 after plating and tested after 2 days. A: Real-time PCR revealed a significant reduction in the SCG10 transcript level by siRNA (si#12, si#22) compared with untreated cells and nonsilencing control (sc#1). No effect was observed on gene expression of TGF-β1, collagen type I α 1, CTGF, or MMP2, *P < 0.001. B: Western blot with anti-SCG10 antibody. Control PC12 cells and hepatic stellate cells transfected with scRNA showed SCG10 expression whether complete disappearance of SCG10 was observed after siRNA transfection or not.

Figure 7

Functional effect of reduced SCG10 expression levels in HSCs. SiRNA-mediated inhibition (si#12, si#22) affects cell migration and cell proliferation compared with untreated cells used as controls or cells transfected with scRNA (sc#1, sc#2), *P < 0.01.