Syndecan-1 Promotes Hepatocyte-Like Differentiation of Hepatoma Cells Targeting Ets-1 and AP-1

Abstract

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which is indispensable in the structural and functional integrity of epithelia. Normal hepatocytes display strong cell surface expression of syndecan-1; however, upon malignant transformation, they may lose it from their cell surfaces. In this study, we demonstrate that re-expression of full-length or ectodomain-deleted syndecan-1 in hepatocellular carcinoma cells downregulates phosphorylation of ERK1/2 and p38, with the truncated form exerting an even stronger effect than the full-length protein. Furthermore, overexpression of syndecan-1 in hepatoma cells is associated with a shift of heparan sulfate structure toward a highly sulfated type specific for normal liver. As a result, cell proliferation and proteolytic shedding of syndecan-1 from the cell surface are restrained, which facilitates redifferentiation of hepatoma cells to a more hepatocyte-like phenotype. Our results highlight the importance of syndecan-1 in the formation and maintenance of differentiated epithelial characteristics in hepatocytes partly via the HGF/ERK/Ets-1 signal transduction pathway. Downregulation of Ets-1 expression alone, however, was not sufficient to replicate the phenotype of syndecan-1 overexpressing cells, indicating the need for additional molecular mechanisms. Accordingly, a reporter gene assay revealed the inhibition of Ets-1 as well as AP-1 transcription factor-induced promoter activation, presumably an effect of the heparan sulfate switch.

Keywords: AP-1; Ets-1; MMP-7; differentiation; epithelium; heparan sulfate; liver cancer; shedding; syndecan-1.

Figure 1

Immunofluorescence images of HepG2 and Hep3B cells after transfection with empty enhanced green fluorescent protein (EGFP) vector, as well as full-length and truncated syndecan-1 EGFP constructs. A diffuse green signal was seen after transfection with the empty vector. Upon transfection with the full-length syndecan-1 construct, the fluorescent signal localized to the cell membrane. Although truncated syndecan-1 partly localized to the cytoplasm, the signal was enriched at the plasma membrane, indicating that truncated syndecan-1, lacking the extracellular domain, was sorted successfully. Representative images are at 1000× magnification (scale bar 15 μm).

Figure 2

Hepatocyte-like differentiation of HepG2 and Hep3B cells upon overexpression of full-length or truncated syndecan-1. (A) H&E stained empty vector transfected control cells were oval or spindle shape with prominent nuclei and high nucleus-to-cytoplasm ratio, and featured frequent cell divisions. In the syndecan-1-transfected cell lines, the numbers of poorly differentiated and dividing cells decreased, and groups of cells with a more hepatocyte-like morphology appeared. Cells showing signs of differentiation were characterized by a lower nucleus-to-cytoplasm ratio; (B) As a sign of cell differentiation, desmoplakin immunocytochemistry showed relocalization of the protein to the cell surface in syndecan-1-transfected cells. In the native hepatoma cell lines, desmoplakin mainly localized to the cytoplasm. Red, desmoplakin and blue, nuclei; (C) Numerous albumin-positive cells were detected among the truncated syndecan-1 transfectants. Green, albumin and red, nuclei. Representative images are at 1000× magnification (scale bar 15 μm), 600× magnification (scale bar 30 μm), and 200× magnification (scale bar 100 μm).

Figure 2

Hepatocyte-like differentiation of HepG2 and Hep3B cells upon overexpression of full-length or truncated syndecan-1. (A) H&E stained empty vector transfected control cells were oval or spindle shape with prominent nuclei and high nucleus-to-cytoplasm ratio, and featured frequent cell divisions. In the syndecan-1-transfected cell lines, the numbers of poorly differentiated and dividing cells decreased, and groups of cells with a more hepatocyte-like morphology appeared. Cells showing signs of differentiation were characterized by a lower nucleus-to-cytoplasm ratio; (B) As a sign of cell differentiation, desmoplakin immunocytochemistry showed relocalization of the protein to the cell surface in syndecan-1-transfected cells. In the native hepatoma cell lines, desmoplakin mainly localized to the cytoplasm. Red, desmoplakin and blue, nuclei; (C) Numerous albumin-positive cells were detected among the truncated syndecan-1 transfectants. Green, albumin and red, nuclei. Representative images are at 1000× magnification (scale bar 15 μm), 600× magnification (scale bar 30 μm), and 200× magnification (scale bar 100 μm).

Figure 3

Syndecan-1 mRNA and protein levels in the empty vector and syndecan-1 transfected HepG2 and Hep3B cell lines. (A) Transcripts coding for the extracellular domain (ED) and the cytoplasmic domain (CD) of syndecan-1 were elevated in the full-length transfectants, while the expression of the CD only was increased in the truncated transfectants, whereas that of the ectodomain-coding transcript remained unchanged; (B) High amounts of full-length (ectodomain-containing) syndecan-1 protein were detected on the surface of both full-length and truncated syndecan-1 transfectants by immunofluorescence using an ectodomain-specific antibody; (C) ELISA confirmed elevation of full-length syndecan-1 in both transfectants; (D) A comparison with the empty vector-transfected cells, shows that the abundance of AO4B08-reactive (internal 2-O-sulfated/6-O-sulfated) HS epitopes are decreased, whereas the amount of HS4C3-reactive (highly sulfated, 3-O-sulfated) epitopes are increased in both Hep3B cell lines containing syndecan-1 constructs. Data points represent the mean ± standard deviation (SD), n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001 versus the empty vector control. Representative images are at 600× magnification (scale bar 30 μm) and 400× magnification (scale bar 50 μm).

Figure 4

Shedding of syndecan-1 ectodomain. The amount of shed (soluble) syndecan-1 ectodomain was measured from cell culture media (CCM) by ectodomain-specific antibody. Shedding of the ectodomain was increased in full-length transfectants, whereas it remained unchanged in truncated transfectants in spite of the overall increased abundance of endogenous syndecan-1 (see Figure 3B). Data points represent the mean ± SD, n = 2; * p < 0.05; *** p < 0.001 versus the empty vector transfectant; ^# p < 0.05; ^## p < 0.01 versus the full-length sdc-1 transfectant.

Figure 5

The effect of syndecan-1 overexpression on the proliferation of HepG2 and Hep3B cell lines. (A) Transfection with the truncated syndecan-1 construct significantly slowed down the proliferation of both HepG2 and Hep3B cell lines. A moderate decrease in the proliferation of HepG2 full-length transfectant was also observed. Notably, overexpression of full-length syndecan-1 in Hep3B cells led to a decrease in double time from 33.89 to 25.27 h (by 25.4%); (B) The log phase of growth curves; (C) Doubling time calculated from the log phase of growth curves. Data points represent the mean ± SD, n = 8; *** p < 0.001 versus the empty vector transfectant.

Figure 6

The effect of syndecan-1 constructs on MMP-7 expression. (A) Except for HepG2 transfected with full-length syndecan-1, all other syndecan-1 transfectants displayed decreased MMP7 mRNA expression and (B) protein levels as compared with the empty vector transfected cells. Red, MMP-7 and blue, nuclei. Data points represent the mean ± SD, n = 3; * p < 0.05 versus the empty vector transfectant. Representative images are at 1000× magnification (scale bar 15 μm) and 600× magnification (scale bar 30 μm).

Figure 7

Decreased activity of Ets-1 and AP-1 upon syndecan-1 overexpression. (A) Intensive nuclear Ets-1 immunopositivity was present in the empty vector-transfected hepatoma cell lines. A modest decrease of the staining intensity was found in the full-length syndecan-1 transfected cells, whereas nuclei of truncated syndecan-1 transfectants showed hardly any Ets-1 immunoreaction. The protein was partially (full-length) or totally (truncated) sequestered in the cytoplasm; (B) In HepG2, decreased Ets-1 response element-driven promoter activity was found in the truncated transfectants, whereas AP-1 response element-driven promoter activity was suppressed in both full-length and truncated syndecan-1 cell lines. In Hep3B, both syndecan-1 constructs interfered with Ets-1 and AP-1 response element-driven promoter activity; (C) Transfection with either syndecan-1 construct inhibited the activation of ERK1/2 and p38 MAP kinases. Data points represent the mean ± SD, (B) n = 3, (C) n = 2; * p < 0.05; ** p < 0.01; *** p < 0.001 versus the empty vector transfectant. Representative images are at 600× magnification (scale bar 30 μm) and 400× magnification (scale bar 50 μm).

Figure 8

Silencing of Ets-1 by RNA interference in HepG2 and Hep3B cells. (A) As indicated by the qRT-PCR results, knockdown of Ets-1 expression was successful in both cell lines; (B) The silencing constructs effectively hindered Ets-1 protein expression; (C) Except for miR-362, Western blots confirmed the downregulation of Ets-1; however, inactivation of ERK1/2 was mostly detected in Hep3B cell line. Data points represent the mean ± SD, (A) n = 3, (C) n = 2; * p < 0.05; ** p < 0.01; *** p < 0.001 versus miR-lacZ transfectant. Representative images are at 600× magnification (scale bar 30 μm).

Figure 9

Changes in cell morphology upon Ets-1 silencing. With regard to inhibition of MMP-7 and differentiation, miRNAs targeting Ets-1 failed to fully replicate the changes observed after syndecan-1 transfection. The miR-641 did not inhibit the expression of MMP-7 and only modest desmoplakin relocalization was detected. H&E staining showed very few differentiated cells throughout the various transfectants. Representative images are at 1000× magnification (scale bar 15 μm) and 200× magnification (scale bar 100 μm).

Figure 10

A schematic model to explain the effects of syndecan-1 overexpression. The central event is the accumulation of intact syndecan-1 molecules on the cell surface. This is achieved directly in cells transfected with the full-length construct and indirectly in cells transfected with the truncated syndecan-1 construct. The downstream effect is reduced Ets-1- and AP-1-mediated promoter activation with subsequent inhibition of cell proliferation and differentiation. RE, response element and GAG, glycosaminoglycan.