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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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Hepatic SATB1 promotes activation of HSCs.(a) LX-2 cells and rat primary stellate cells (R-HSCs) were cultured for 48 h with conditioned medium (CM) from L02 and rat primary hepatic cells (PHC) transduced with lenti-SATB1 and lenti-ctrl virus. The mRNA extracts were used to analyze α-SMA, COL1A1, TGFB1, CTGF, TIMP1, TIMP2 expression. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. (b) Protein levels of α-SMA were analyzed from LX-2 and R-HSC cells after incubation with CM from lenti-SATB1 or lenti-ctrl expressing L02 cells (L02-SATB1, L02-Vector) and PHC cells (PHC-SATB1, PHC-Vector). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. Full-length blots are included in the Supplementary Fig. S9. (c) R-HSCs were cultured for 48 h with CM obtained from hepatocytes expressing enhanced SATB1 or control, and α-SMA expression was determined by immunofluorescence analysis. (d) LX-2 cell proliferation was detected by EdU assay after incubation with CM from SATB1 expressing L02 cells for 48 h. LX-2 and R-HSCs cell proliferation were determined by cell counting kit-8 (CCK8) after incubation with CM from SATB1 expressing PHC (e) and L02 cells (f) for 48 h (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.
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f5: Hepatic SATB1 promotes activation of HSCs.(a) LX-2 cells and rat primary stellate cells (R-HSCs) were cultured for 48 h with conditioned medium (CM) from L02 and rat primary hepatic cells (PHC) transduced with lenti-SATB1 and lenti-ctrl virus. The mRNA extracts were used to analyze α-SMA, COL1A1, TGFB1, CTGF, TIMP1, TIMP2 expression. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. (b) Protein levels of α-SMA were analyzed from LX-2 and R-HSC cells after incubation with CM from lenti-SATB1 or lenti-ctrl expressing L02 cells (L02-SATB1, L02-Vector) and PHC cells (PHC-SATB1, PHC-Vector). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. Full-length blots are included in the Supplementary Fig. S9. (c) R-HSCs were cultured for 48 h with CM obtained from hepatocytes expressing enhanced SATB1 or control, and α-SMA expression was determined by immunofluorescence analysis. (d) LX-2 cell proliferation was detected by EdU assay after incubation with CM from SATB1 expressing L02 cells for 48 h. LX-2 and R-HSCs cell proliferation were determined by cell counting kit-8 (CCK8) after incubation with CM from SATB1 expressing PHC (e) and L02 cells (f) for 48 h (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.

Mentions: It was reported that HBx-expressing hepatocytes activated HSC by TGFB1 in a paracrine way15. Having demonstrated HBx upregulated SATB1 levels, we were determined to find out whether hepatic SATB1 could exert a paracrine effect on HSCs. L02 cells and rat primary hepatic cells (PHC) were transduced with lentivectors expressing SATB1 and vectors (Supplementary Fig. S3), culture medium were collected and used to culture human hepatic stellate cell lines LX-2 and rat primary hepatic stellate cells (R-HSCs) for 48 h. We observed that α-SMA was significantly increased in HSCs either incubation with L02-SATB1 or PHC-SATB1 conditioned medium (CM) (Fig. 5a–c). Besides, LX-2 and R-HSCs proliferation was assessed by CCK8 and the rate of incorporation of EdU into the DNA. Notably, SATB1 overexpression in PHC and L02 led to enhanced HSCs proliferation (Fig. 5e,f). In line with this result, the number of LX-2 incorporating EdU was increased when incubation with conditioned medium from L02-SATB1 compared to the control group (Fig. 5d). These observations indicate that SATB1 expressing in liver cells L02 and rat primary hepatic cells can promote the activation and proliferation of HSCs.


Hepatic SATB1 induces paracrine activation of hepatic stellate cells and is upregulated by HBx
Hepatic SATB1 promotes activation of HSCs.(a) LX-2 cells and rat primary stellate cells (R-HSCs) were cultured for 48 h with conditioned medium (CM) from L02 and rat primary hepatic cells (PHC) transduced with lenti-SATB1 and lenti-ctrl virus. The mRNA extracts were used to analyze α-SMA, COL1A1, TGFB1, CTGF, TIMP1, TIMP2 expression. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. (b) Protein levels of α-SMA were analyzed from LX-2 and R-HSC cells after incubation with CM from lenti-SATB1 or lenti-ctrl expressing L02 cells (L02-SATB1, L02-Vector) and PHC cells (PHC-SATB1, PHC-Vector). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. Full-length blots are included in the Supplementary Fig. S9. (c) R-HSCs were cultured for 48 h with CM obtained from hepatocytes expressing enhanced SATB1 or control, and α-SMA expression was determined by immunofluorescence analysis. (d) LX-2 cell proliferation was detected by EdU assay after incubation with CM from SATB1 expressing L02 cells for 48 h. LX-2 and R-HSCs cell proliferation were determined by cell counting kit-8 (CCK8) after incubation with CM from SATB1 expressing PHC (e) and L02 cells (f) for 48 h (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.
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f5: Hepatic SATB1 promotes activation of HSCs.(a) LX-2 cells and rat primary stellate cells (R-HSCs) were cultured for 48 h with conditioned medium (CM) from L02 and rat primary hepatic cells (PHC) transduced with lenti-SATB1 and lenti-ctrl virus. The mRNA extracts were used to analyze α-SMA, COL1A1, TGFB1, CTGF, TIMP1, TIMP2 expression. (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. (b) Protein levels of α-SMA were analyzed from LX-2 and R-HSC cells after incubation with CM from lenti-SATB1 or lenti-ctrl expressing L02 cells (L02-SATB1, L02-Vector) and PHC cells (PHC-SATB1, PHC-Vector). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector. Full-length blots are included in the Supplementary Fig. S9. (c) R-HSCs were cultured for 48 h with CM obtained from hepatocytes expressing enhanced SATB1 or control, and α-SMA expression was determined by immunofluorescence analysis. (d) LX-2 cell proliferation was detected by EdU assay after incubation with CM from SATB1 expressing L02 cells for 48 h. LX-2 and R-HSCs cell proliferation were determined by cell counting kit-8 (CCK8) after incubation with CM from SATB1 expressing PHC (e) and L02 cells (f) for 48 h (n = 3). *P < 0.05, **P < 0.01 vs. L02-Vector or PHC-Vector.
Mentions: It was reported that HBx-expressing hepatocytes activated HSC by TGFB1 in a paracrine way15. Having demonstrated HBx upregulated SATB1 levels, we were determined to find out whether hepatic SATB1 could exert a paracrine effect on HSCs. L02 cells and rat primary hepatic cells (PHC) were transduced with lentivectors expressing SATB1 and vectors (Supplementary Fig. S3), culture medium were collected and used to culture human hepatic stellate cell lines LX-2 and rat primary hepatic stellate cells (R-HSCs) for 48 h. We observed that α-SMA was significantly increased in HSCs either incubation with L02-SATB1 or PHC-SATB1 conditioned medium (CM) (Fig. 5a–c). Besides, LX-2 and R-HSCs proliferation was assessed by CCK8 and the rate of incorporation of EdU into the DNA. Notably, SATB1 overexpression in PHC and L02 led to enhanced HSCs proliferation (Fig. 5e,f). In line with this result, the number of LX-2 incorporating EdU was increased when incubation with conditioned medium from L02-SATB1 compared to the control group (Fig. 5d). These observations indicate that SATB1 expressing in liver cells L02 and rat primary hepatic cells can promote the activation and proliferation of HSCs.

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