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, 49 (6), 2055-67

Hepatic Stellate Cells Express Functional CXCR4: Role in Stromal Cell-Derived factor-1alpha-mediated Stellate Cell Activation

Affiliations

Hepatic Stellate Cells Express Functional CXCR4: Role in Stromal Cell-Derived factor-1alpha-mediated Stellate Cell Activation

Feng Hong et al. Hepatology.

Abstract

Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell-derived factor-1alpha (SDF-1alpha), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF-1alpha/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF-1alpha is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture-induced activation. Treatment of stellate cells with recombinant SDF-1alpha increases expression of alpha-smooth muscle actin and collagen I and stimulates a dose-dependent increase in HSC proliferation. Inhibitor studies suggest that SDF-1alpha/CXCR4-dependent extracellular signal-regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation.

Conclusion: HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF-1alpha is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase pathways mediate SDF-1alpha-induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches.

Conflict of interest statement

Potential conflict of interest: Nothing to report.

Figures

Fig. 1
Fig. 1
Increased hepatic CXCR4 and SDF-1α protein expression in patients with HCV cirrhosis and expression of CXCR4 by stellate cells in vivo. (A) To compare expression of CXCR4 and SDF-1α in HCV cirrhotic livers versus normal livers, whole liver homogenate was prepared from either the explanted liver of patients undergoing liver transplantation for HCV (n = 7) or normal liver tissue margin in patients with no underlying liver disease undergoing resection for isolated colon cancer metastasis (n = 5). Fifty micrograms protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and membrane-probed for CXCR4 and SDF-1α. β-actin was used as a loading control. Representative western blots of three samples from each group are shown. (B) Densitometry was performed on all samples to compare relative expression normalized to β-actin and is graphically represented. Protein expression of CXCR4 was an average 1.8-fold higher and SDF-1α protein expression 2.4-fold higher (**P < 0.005) in patients with HCV cirrhosis. (C) To determine whether activated stellate cells express CXCR4 in vivo, coimmunostaining for α-SMA (green) and CXCR4 (red) performed on frozen sections obtained from three explanted HCV cirrhotic livers. Representative images are shown (magnification ×100). Arrows denote positive-stain stellate cells expressing CXCR4 and α-SMA. 4′,6-Diamidino-2-phenylindole was used to stain nuclei blue. (D) Immunohistochemistry for CXCR4 on HCV cirrhotic livers confirms CXCR4+ cells with stellate cell morphology (black arrows) as well as staining of bile duct epithelial cells (green arrow) and lymphocytes (orange arrow) within the portal tract. Representative images are shown (magnification ×200 and ×400).
Fig. 2
Fig. 2
Human HSCs express CXCR4. (A) Western blot analysis demonstrates CXCR4 protein expression in both a human HSC line (LX-2) and primary HSCs (passage 3). Jurkat T cell lysate was used as a positive control. (B) Immunofluorescent staining on primary HSCs reflects cytoplasmic and cell surface expression of CXCR4 in red. Nuclei were stained blue with 4′,6-diamidino-2-phenylindole. (C) To further examine baseline cell surface and total CXCR4 receptor expression in human HSCs, FACS analysis was performed on LX-2 cells. Cells were fixed (4% paraformaldehyde) to examine cell surface expression and fixed and permeabilized (4% paraformaldehyde and 0.1% saponin) to examine total receptor expression and incubated with either PE-immunoglobulin G2a isotype control or PE-conjugated anti-CXCR4 for 1 hour at room temperature. Cells were washed and analyzed using a FACS instrument (FACScan, Becton Dickinson). Representative FACS analyses from three independent experiments are shown.
Fig. 3
Fig. 3
CXCR4 expression in primary human and murine stellate cells increases with culture-induced activation. (A) Normal liver tissue from patients undergoing hepatic resection was obtained, and primary human HSCs were isolated by cannulating portal vessels for in situ digestion with pronase and collagenase. Stellate cells were isolated via density centrifugation and plated on plastic. Phase-contrast microscopy depicts phenotypic changes that occur during progressive activation in culture. HSCs were stained with PE-conjugated anti-CXCR4 or isotype control at progressive stages of activation (day 3, day 21, and passage 3). Representative FACS analyses from three independent isolations are shown demonstrating increased cell surface and total CXCR4 expression during culture-induced activation. (B) Increased cell surface expression of CXCR4 with progressive activation is represented graphically. (C) As a proof of concept, murine primary stellate cells were isolated via portal vein cannulation, in situ digestion, and density centrifugation. Pooled cells (six mice per isolation) were plated on plastic and FACS was performed at day 2 and 12 in culture. An average 22% increase (n = 3 independent isolations; **P < 0.024) in CXCR4 receptor expression was noted during progressive culture-induced activation.
Fig. 4
Fig. 4
LX-2 and primary HSCs secrete SDF-1α and exogenous SDF-1α induces a dose-dependent increase in α-SMA. (A) Enzyme-linked immunosorbent assay for SDF-1α performed on 72-hour conditioned media from LX-2 and passage 1 primary HSCs reveals significant secretion of SDF-1α (2,500–3,000 pg/mL) by both cell types. Serum-free media and PBS were used as a negative control. Purified recombinant human SDF-1α was used to generate a standard curve. (B) LX-2 cells treated with increasing concentrations of SDF-1α (100–750 ng/mL) and protein expression of α-SMA and GAPDH as a loading control were assessed via western blotting. (C) Maximal induction was noted at 750 ng/mL and resulted in an average three-fold increase in α-SMA protein expression via densitometry normalized to GAPDH (n = 3 independent experiments; **P < 0.05). (D) LX-2 cells were transfected with either control shRNA (shControl) or CXCR4-targeted shRNA (shCXCR4), and western blotting was performed to determine the efficiency of knockdown. Reduction in CXCR4 protein expression was noted 72 hours after transfection. (E) Seventy-two hours after transfection, cells were treated with SDF-1α (500 ng/mL), and western blotting was performed for α-SMA, demonstrating a 50% reduction in α-SMA expression in the presence of shCXCR4.
Fig. 5
Fig. 5
SDF-1α induces proliferation in immortalized human stellate cells that is mediated by both the ERK 1/2 and PI3K-Akt pathways. (A) LX-2 cells were plated at a density of 20,000 cells per well, serum-starved for 48 hours, and treated with increasing concentrations of SDF-1α (50, 200, and 500 ng/mL) for 24 to 72 hours, and proliferation was assessed via [3H]-thymidine incorporation. A dose-dependent effect of SDF-1α on stellate cell proliferation was noted at all time points. (B) LX-2 cells were transfected with either shControl or shCXCR4 and then treated with SDF-1α (500 ng/mL) for 24 to 72 hours, and proliferation was assessed via [3H]-thymidine incorporation. A 50% (**P < 0.007) and 45% (*P < 0.01) reduction in proliferation was noted in the presence of shCXCR4 compared with shControl at 48 and 72 hours. (C) To determine whether SDF-1α activates ERK 1/2 or PI3K-Akt pathways, LX-2 cells were treated with control (C) or SDF-1α (S) at a dose of 500 ng/mL for 15, 30, and 60 minutes, and western blot analysis was performed for phospho-ERK 1/2, total ERK, and α-tubulin as a loading control. (D) LX-2 cells were treated with control or SDF-1α for 30, 60, and 120 minutes, and western blot analysis was performed for p-Akt, total Akt, and β-actin. Both increased phospho-ERK1/2 and p-Akt were noted in response to SDF-1α. (E) Pretreatment of cells with either the ERK inhibitor (UO126; 10 nM) or PI3K inhibitor (LY294002; 25 μM) 30 minutes prior to SDF-1α treatment resulted in a significant decrease in SDF-1α–induced stellate cell proliferation by over 50% (***P < 0.001). All data are expressed as the mean ± standard deviation of three independent experiments. Synergistic effects were noted in the presence of both inhibitors.
Fig. 6
Fig. 6
SDF-1α induces proliferative response in primary human HSCs that is mediated by both ERK 1/2 and PI3K-AKt pathways. (A) Passage 3 primary HSCs were plated at a density of 2 × 104, serum-starved for 48 hours, and then treated with SDF-1α (500 ng/mL) for 24 to 72 hours, and proliferative response was measured using 3H-thymidine incorporation. SDF-1α induced a significant proliferative response at all time points. **P < 0.05, ***P < 0.005 versus control. (B) The ability to achieve CXCR4 knockdown in primary human HSCs was confirmed via western blot analysis. (C) Primary HSCs transfected with either shControl or shCXCR4 were subsequently treated with SDF-1α for 24 to 72 hours. A reduction in SDF-1α induced proliferation by 20% at 24 hours (*P < 0.01), 38% at 48 hours (**P < 0.005), and 36% at 72 hours (**P < 0.005). (D) Both ERK1/2 (U0126; 10 nM) and PI3K (LY294002; 25 μM) resulted in early and complete abrogation of the SDF-1α–induced proliferative response. All data are expressed as the mean ± standard deviation of three independent experiments.
Fig. 7
Fig. 7
SDF-1α promotes collagen I expression predominantly via PI3K-Akt pathways. (A,B) LX-2 cells were treated with SDF-1α (500 ng/mL) for 2, 6, and 12 hours, and RNA was extracted for RT-PCR analysis (A) and protein extracted for western blot analysis (B) to examine levels of collagen I α1 mRNA and collagen I protein, respectively. (C) LX-2 cells were transfected with shControl and shCXCR4 followed by treatment with SDF-1α for 12 hours, and western blot analysis for collagen I was performed. A significanct reduction in SDF-1α–induced collagen I expression was observed in shCXCR4-treated cells. (D) LX-2 cells were preincubated with either ERK1/2 inhibitor (UO126) or PI3K inhibitor (LY294002) for 30 minutes followed by SDF-1α (500 ng/mL) for 6 hours. Both inhibitors resulted in a reduction in collagen I protein expression on western blotting, though the effect was more pronounced in the presence of the PI3K inhibitor. Representative western blots from three independent experiments are shown.
Fig. 8
Fig. 8
SDF-1α effects on stellate cell proliferation and collagen I expression are not mediated by CXCR7. (A) FACS was performed to determine cell surface expression of CXCR7 (range, 21%-33%). (B) LX2 cells were transfected with nontargeting shRNA (shControl) or CXCR7-targeted shRNA (shCXCR7), and western blot analysis performed on cell lysate revealed a 50% reduction in CXCR7 expression. (C,D) LX-2 cells were serum-starved, and 72 hours after transfection with either shControl versus shCXCR7 were treated with SDF-1α (500 mg/mL). Proliferation was assessed via thymidine incorporation (C) and collagen I expression was assessed via western blot analysis (D). No reduction in SDF-1α induced proliferation or collagen I expression was noted in the presence of shCXCR7. All experiments performed in triplicate.

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