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Hepatic SATB1 induces paracrine activation of hepatic stellate cells and is upregulated by HBx

View Article: PubMed Central - PubMed

ABSTRACT

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver diseases, but its involvement in hepatic fibrogenesis remains unclear. Special AT-rich binding protein 1 (SATB1) has been implicated in reprogramming chromatin organization and transcription profiles in many cancers and non-cancer-related conditions. We found that hepatic SATB1 expression was significantly up-regulated in fibrotic tissues from chronic hepatitis B virus (HBV)-infected patients and HBV transgenic (HBV-Tg) mouse model. Knockdown of SATB1 in the liver significantly alleviated CCl4-induced fibrosis in HBV-Tg mouse model. Moreover, we suggested HBV encoded x protein (HBx) induced SATB1 expression through activation of JNK and ERK pathways. Enforced expression of SATB1 in hepatocytes promoted the activation and proliferation of hepatic stellate cells (HSCs) by secretion of connective tissue growth factor (CTGF), Interleukin-6 (IL-6) and platelet derived growth factor-A (PDGF-AA). Our findings demonstrated that HBx upregulated hepatic SATB1 which exerted pro-fibrotic effects by paracrine activation of stellate cells in HBV-related fibrosis.

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SATB1 activates LX-2 through IL-6, PDGF-AA and CTGF.(a) A cytokine profiling was used for the detection of 120 inflammatory factors. Cell supernatant was obtained from L02 cells transduced with lenti-SATB1 and lenti-ctrl for 72 h. (b,c) Blocking antibodies against IL-6 (1 ug/ml) and PDGF-AA (1 ug/ml) suppressed L02-SATB1 CM-induced proliferation of LX-2. The results were detected by EdU assay (b) and CCK8 (c), respectively (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (d) LX-2 cells were cultured for 48 h with CM obtained from L02 expressing SATB1 (L02-SATB1) in the presence of blocking antibodies IL-6 or PDGF-AA. The mRNA expression of α-SMA, COL1A1, cyclinD1, cyclinE1 were determined by real-time PCR (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (e) CTGF levels were determined in culture supernatants of L02 and PHC cells infected with lenti-SATB1 or lenti-ctrl for 72 h. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.
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f6: SATB1 activates LX-2 through IL-6, PDGF-AA and CTGF.(a) A cytokine profiling was used for the detection of 120 inflammatory factors. Cell supernatant was obtained from L02 cells transduced with lenti-SATB1 and lenti-ctrl for 72 h. (b,c) Blocking antibodies against IL-6 (1 ug/ml) and PDGF-AA (1 ug/ml) suppressed L02-SATB1 CM-induced proliferation of LX-2. The results were detected by EdU assay (b) and CCK8 (c), respectively (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (d) LX-2 cells were cultured for 48 h with CM obtained from L02 expressing SATB1 (L02-SATB1) in the presence of blocking antibodies IL-6 or PDGF-AA. The mRNA expression of α-SMA, COL1A1, cyclinD1, cyclinE1 were determined by real-time PCR (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (e) CTGF levels were determined in culture supernatants of L02 and PHC cells infected with lenti-SATB1 or lenti-ctrl for 72 h. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.

Mentions: To identify the factors responsible for HSC activation and proliferation, we performed a cytokine profile of CM obtained from L02 cells overexpressing SATB1 and control group. TIMP1, TIMP2, IL-6, PDGF-AA were detected at high levels and significantly elevated in conditioned media of L02-SATB1 compared to the controls (Fig. 6a). Therefore, we incubated LX-2 with conditioned media from L02-SATB1 in the presence of blocking antibodies against IL-6, PDGF-AA. We found that incubation of HSCs with blocking antibody against IL-6 and PDGF-AA abolished the increase in the proliferation induced by CM from L02-SATB1, while neutralizing antibodies against IgG showed no effect on LX-2 proliferation (Fig. 6b,c). However, the blocking antibody against IL-6, PDGF-AA both showed no effect on the increase in α-SMA expression in LX-2 in the presence of supernatants from L02-SATB1 (Fig. 6d). It has been reported SATB1 expression induced a marked change in many genes associated with metastasis in cancer cells, transforming growth factor-β1 (TGFB1) and connective tissue growth factor (CTGF) included23. Since both of them are responsible for HSCs activation, we analyzed the secretion of TGFB1 and CTGF from conditioned medium of SATB1 expressing L02 and PHC. The results of cytokine profile showed no prominent difference of TGFB1 between L02-SATB1 and control group (Supplementary Table S3). Moreover, L02-SATB1 and PHC-SATB1 induced CTGF secretion, when compared to the control croup respectively (Fig. 6e). Altogether, these findings demonstrate that IL-6 and PDGF-AA secreted by L02-SATB1 are responsible for enhanced proliferation of LX-2, while CTGF may mediates the paracrine activation of HSCs by SATB1-expressing liver cells.


Hepatic SATB1 induces paracrine activation of hepatic stellate cells and is upregulated by HBx
SATB1 activates LX-2 through IL-6, PDGF-AA and CTGF.(a) A cytokine profiling was used for the detection of 120 inflammatory factors. Cell supernatant was obtained from L02 cells transduced with lenti-SATB1 and lenti-ctrl for 72 h. (b,c) Blocking antibodies against IL-6 (1 ug/ml) and PDGF-AA (1 ug/ml) suppressed L02-SATB1 CM-induced proliferation of LX-2. The results were detected by EdU assay (b) and CCK8 (c), respectively (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (d) LX-2 cells were cultured for 48 h with CM obtained from L02 expressing SATB1 (L02-SATB1) in the presence of blocking antibodies IL-6 or PDGF-AA. The mRNA expression of α-SMA, COL1A1, cyclinD1, cyclinE1 were determined by real-time PCR (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (e) CTGF levels were determined in culture supernatants of L02 and PHC cells infected with lenti-SATB1 or lenti-ctrl for 72 h. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.
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f6: SATB1 activates LX-2 through IL-6, PDGF-AA and CTGF.(a) A cytokine profiling was used for the detection of 120 inflammatory factors. Cell supernatant was obtained from L02 cells transduced with lenti-SATB1 and lenti-ctrl for 72 h. (b,c) Blocking antibodies against IL-6 (1 ug/ml) and PDGF-AA (1 ug/ml) suppressed L02-SATB1 CM-induced proliferation of LX-2. The results were detected by EdU assay (b) and CCK8 (c), respectively (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (d) LX-2 cells were cultured for 48 h with CM obtained from L02 expressing SATB1 (L02-SATB1) in the presence of blocking antibodies IL-6 or PDGF-AA. The mRNA expression of α-SMA, COL1A1, cyclinD1, cyclinE1 were determined by real-time PCR (n = 3). *P < 0.05, **P < 0.01 vs. L02-SATB1 IgG. (e) CTGF levels were determined in culture supernatants of L02 and PHC cells infected with lenti-SATB1 or lenti-ctrl for 72 h. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.
Mentions: To identify the factors responsible for HSC activation and proliferation, we performed a cytokine profile of CM obtained from L02 cells overexpressing SATB1 and control group. TIMP1, TIMP2, IL-6, PDGF-AA were detected at high levels and significantly elevated in conditioned media of L02-SATB1 compared to the controls (Fig. 6a). Therefore, we incubated LX-2 with conditioned media from L02-SATB1 in the presence of blocking antibodies against IL-6, PDGF-AA. We found that incubation of HSCs with blocking antibody against IL-6 and PDGF-AA abolished the increase in the proliferation induced by CM from L02-SATB1, while neutralizing antibodies against IgG showed no effect on LX-2 proliferation (Fig. 6b,c). However, the blocking antibody against IL-6, PDGF-AA both showed no effect on the increase in α-SMA expression in LX-2 in the presence of supernatants from L02-SATB1 (Fig. 6d). It has been reported SATB1 expression induced a marked change in many genes associated with metastasis in cancer cells, transforming growth factor-β1 (TGFB1) and connective tissue growth factor (CTGF) included23. Since both of them are responsible for HSCs activation, we analyzed the secretion of TGFB1 and CTGF from conditioned medium of SATB1 expressing L02 and PHC. The results of cytokine profile showed no prominent difference of TGFB1 between L02-SATB1 and control group (Supplementary Table S3). Moreover, L02-SATB1 and PHC-SATB1 induced CTGF secretion, when compared to the control croup respectively (Fig. 6e). Altogether, these findings demonstrate that IL-6 and PDGF-AA secreted by L02-SATB1 are responsible for enhanced proliferation of LX-2, while CTGF may mediates the paracrine activation of HSCs by SATB1-expressing liver cells.

View Article: PubMed Central - PubMed

ABSTRACT

Chronic hepatitis B virus (HBV) infection is a major cause of chronic liver diseases, but its involvement in hepatic fibrogenesis remains unclear. Special AT-rich binding protein 1 (SATB1) has been implicated in reprogramming chromatin organization and transcription profiles in many cancers and non-cancer-related conditions. We found that hepatic SATB1 expression was significantly up-regulated in fibrotic tissues from chronic hepatitis B virus (HBV)-infected patients and HBV transgenic (HBV-Tg) mouse model. Knockdown of SATB1 in the liver significantly alleviated CCl4-induced fibrosis in HBV-Tg mouse model. Moreover, we suggested HBV encoded x protein (HBx) induced SATB1 expression through activation of JNK and ERK pathways. Enforced expression of SATB1 in hepatocytes promoted the activation and proliferation of hepatic stellate cells (HSCs) by secretion of connective tissue growth factor (CTGF), Interleukin-6 (IL-6) and platelet derived growth factor-A (PDGF-AA). Our findings demonstrated that HBx upregulated hepatic SATB1 which exerted pro-fibrotic effects by paracrine activation of stellate cells in HBV-related fibrosis.

No MeSH data available.


Related in: MedlinePlus