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Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

Calabro SR, Maczurek AE, Morgan AJ, Tu T, Wen VW, Yee C, Mridha A, Lee M, d'Avigdor W, Locarnini SA, McCaughan GW, Warner FJ, McLennan SV, Shackel NA - PLoS ONE (2014)

Bottom Line: Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity.Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo.Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.

View Article: PubMed Central - PubMed

Affiliation: Liver Cell Biology, Centenary Institute, Sydney, NSW, Australia; Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.

ABSTRACT

Background: The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.

Methods: Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention.

Results: In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls.

Conclusion: We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.

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The Effect of Inhibition of CD147 on Matrix Metalloproteinase Expression and Activities in pH5CH8 Hepatocytes.Downregulation of MMPs with CD147 knockdown in a hepatocyte cell line in-vitro. pH5CH8 cells were transfected with siCtrl (C) or siCD147 (Si) oligonucleotides. Transfected cells and mock controls (M) were cultured in serum free conditions for 48 hrs. Shown in panel A are representative immunoblots of CD147 with the higher (HG CD147) and lower molecular weight (LG CD147) glycoforms, MMP-14 and GAPDH as loading control. Gelatin zymography of MMP-2 and -9 on the conditioned media from the same cells are shown in panel B. Densitometry was performed on the CD147 immunoblots and the results are shown as HG CD147 and LG CD147 forms normalised for GAPDH (n = 3, Panels C and D). Densitometric analysis of gelatin zymography of MMP-9 and MMP-2 are shown (Panels E and F). *p<0.05 using Mann-Whitney U t-test, compared to Mock (n = 3 for all groups).
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pone-0090571-g007: The Effect of Inhibition of CD147 on Matrix Metalloproteinase Expression and Activities in pH5CH8 Hepatocytes.Downregulation of MMPs with CD147 knockdown in a hepatocyte cell line in-vitro. pH5CH8 cells were transfected with siCtrl (C) or siCD147 (Si) oligonucleotides. Transfected cells and mock controls (M) were cultured in serum free conditions for 48 hrs. Shown in panel A are representative immunoblots of CD147 with the higher (HG CD147) and lower molecular weight (LG CD147) glycoforms, MMP-14 and GAPDH as loading control. Gelatin zymography of MMP-2 and -9 on the conditioned media from the same cells are shown in panel B. Densitometry was performed on the CD147 immunoblots and the results are shown as HG CD147 and LG CD147 forms normalised for GAPDH (n = 3, Panels C and D). Densitometric analysis of gelatin zymography of MMP-9 and MMP-2 are shown (Panels E and F). *p<0.05 using Mann-Whitney U t-test, compared to Mock (n = 3 for all groups).

Mentions: Consistent with the observed minimal MMP activity seen in non-diseased liver by in-situ zymography, pH 5CH8 hepatocytes do not produce significant amounts of MMPs without stimulation. However, on exposure to the inflammatory mediator TNF these cells significantly upregulate MMP expression. This effect is analogous to the known in-vivo situation in advanced fibrosis [41], [44] and the situation documented in Figure 7. Compared with scrambled siRNA (C or CTRL) and untransfected control cells (M or mock) the siRNA targeting CD147 (siCD147) reduced HG-CD147 and LG-CD147 protein expression significantly (representative gel Figure 7, Panel A with quantitation in Panels C and D). This decreased protein expression was accompanied by a significant reduction in MMP-9 and MMP-2 activity (Figure 7, Panel B with quantitation in Panels E and F). Immunoblot analysis of MMP-14 in these cells also showed a decrease in protein level with CD147 knockdown, however this failed to reach statistical significance (graph not shown). These in-vitro results suggest a role for CD147 in regulation of hepatocyte MMP expression and activity.


Hepatocyte produced matrix metalloproteinases are regulated by CD147 in liver fibrogenesis.

Calabro SR, Maczurek AE, Morgan AJ, Tu T, Wen VW, Yee C, Mridha A, Lee M, d'Avigdor W, Locarnini SA, McCaughan GW, Warner FJ, McLennan SV, Shackel NA - PLoS ONE (2014)

The Effect of Inhibition of CD147 on Matrix Metalloproteinase Expression and Activities in pH5CH8 Hepatocytes.Downregulation of MMPs with CD147 knockdown in a hepatocyte cell line in-vitro. pH5CH8 cells were transfected with siCtrl (C) or siCD147 (Si) oligonucleotides. Transfected cells and mock controls (M) were cultured in serum free conditions for 48 hrs. Shown in panel A are representative immunoblots of CD147 with the higher (HG CD147) and lower molecular weight (LG CD147) glycoforms, MMP-14 and GAPDH as loading control. Gelatin zymography of MMP-2 and -9 on the conditioned media from the same cells are shown in panel B. Densitometry was performed on the CD147 immunoblots and the results are shown as HG CD147 and LG CD147 forms normalised for GAPDH (n = 3, Panels C and D). Densitometric analysis of gelatin zymography of MMP-9 and MMP-2 are shown (Panels E and F). *p<0.05 using Mann-Whitney U t-test, compared to Mock (n = 3 for all groups).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4116334&req=5

pone-0090571-g007: The Effect of Inhibition of CD147 on Matrix Metalloproteinase Expression and Activities in pH5CH8 Hepatocytes.Downregulation of MMPs with CD147 knockdown in a hepatocyte cell line in-vitro. pH5CH8 cells were transfected with siCtrl (C) or siCD147 (Si) oligonucleotides. Transfected cells and mock controls (M) were cultured in serum free conditions for 48 hrs. Shown in panel A are representative immunoblots of CD147 with the higher (HG CD147) and lower molecular weight (LG CD147) glycoforms, MMP-14 and GAPDH as loading control. Gelatin zymography of MMP-2 and -9 on the conditioned media from the same cells are shown in panel B. Densitometry was performed on the CD147 immunoblots and the results are shown as HG CD147 and LG CD147 forms normalised for GAPDH (n = 3, Panels C and D). Densitometric analysis of gelatin zymography of MMP-9 and MMP-2 are shown (Panels E and F). *p<0.05 using Mann-Whitney U t-test, compared to Mock (n = 3 for all groups).
Mentions: Consistent with the observed minimal MMP activity seen in non-diseased liver by in-situ zymography, pH 5CH8 hepatocytes do not produce significant amounts of MMPs without stimulation. However, on exposure to the inflammatory mediator TNF these cells significantly upregulate MMP expression. This effect is analogous to the known in-vivo situation in advanced fibrosis [41], [44] and the situation documented in Figure 7. Compared with scrambled siRNA (C or CTRL) and untransfected control cells (M or mock) the siRNA targeting CD147 (siCD147) reduced HG-CD147 and LG-CD147 protein expression significantly (representative gel Figure 7, Panel A with quantitation in Panels C and D). This decreased protein expression was accompanied by a significant reduction in MMP-9 and MMP-2 activity (Figure 7, Panel B with quantitation in Panels E and F). Immunoblot analysis of MMP-14 in these cells also showed a decrease in protein level with CD147 knockdown, however this failed to reach statistical significance (graph not shown). These in-vitro results suggest a role for CD147 in regulation of hepatocyte MMP expression and activity.

Bottom Line: Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity.Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo.Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.

View Article: PubMed Central - PubMed

Affiliation: Liver Cell Biology, Centenary Institute, Sydney, NSW, Australia; Sydney Medical School, The University of Sydney, Sydney, NSW, Australia.

ABSTRACT

Background: The classical paradigm of liver injury asserts that hepatic stellate cells (HSC) produce, remodel and turnover the abnormal extracellular matrix (ECM) of fibrosis via matrix metalloproteinases (MMPs). In extrahepatic tissues MMP production is regulated by a number of mechanisms including expression of the glycoprotein CD147. Previously, we have shown that CD147 is expressed on hepatocytes but not within the fibrotic septa in cirrhosis [1]. Therefore, we investigated if hepatocytes produce MMPs, regulated by CD147, which are capable of remodelling fibrotic ECM independent of the HSC.

Methods: Non-diseased, fibrotic and cirrhotic livers were examined for MMP activity and markers of fibrosis in humans and mice. CD147 expression and MMP activity were co-localised by in-situ zymography. The role of CD147 was studied in-vitro with siRNA to CD147 in hepatocytes and in-vivo in mice with CCl4 induced liver injury using ãCD147 antibody intervention.

Results: In liver fibrosis in both human and mouse tissue MMP expression and activity (MMP-2, -9, -13 and -14) increased with progressive injury and localised to hepatocytes. Additionally, as expected, MMPs were abundantly expressed by activated HSC. Further, with progressive fibrosis there was expression of CD147, which localised to hepatocytes but not to HSC. Functionally significant in-vitro regulation of hepatocyte MMP production by CD147 was demonstrated using siRNA to CD147 that decreased hepatocyte MMP-2 and -9 expression/activity. Further, in-vivo α-CD147 antibody intervention decreased liver MMP-2, -9, -13, -14, TGF-β and α-SMA expression in CCl4 treated mice compared to controls.

Conclusion: We have shown that hepatocytes produce active MMPs and that the glycoprotein CD147 regulates hepatocyte MMP expression. Targeting CD147 regulates hepatocyte MMP production both in-vitro and in-vivo, with the net result being reduced fibrotic matrix turnover in-vivo. Therefore, CD147 regulation of hepatocyte MMP is a novel pathway that could be targeted by future anti-fibrogenic agents.

Show MeSH
Related in: MedlinePlus