TY - JOUR
T1 - Collagen release by human hepatic stellate cells requires vitamin C and is efficiently blocked by hydroxylase inhibition
AU - Smith-Cortinez, Natalia
AU - Fagundes, Raphael R
AU - Gomez, Valentina
AU - Kong, Defu
AU - de Waart, Dirk R
AU - Heegsma, Janette
AU - Sydor, Svenja
AU - Olinga, Peter
AU - de Meijer, Vincent E
AU - Taylor, Cormac T
AU - Bank, Ruud
AU - Paulusma, Coen C
AU - Faber, Klaas Nico
N1 - Publisher Copyright:
© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology
PY - 2021/2
Y1 - 2021/2
N2 - Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.
AB - Liver fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly composed of collagen. Hepatic stellate cells (HSCs) mediate liver fibrosis by secreting collagen. Vitamin C (ascorbic acid) is a cofactor of prolyl-hydroxylases that modify newly synthesized collagen on the route for secretion. Unlike most animals, humans cannot synthesize ascorbic acid and its role in liver fibrosis remains unclear. Here, we determined the effect of ascorbic acid and prolyl-hydroxylase inhibition on collagen production and secretion by human HSCs. Primary human HSCs (p-hHSCs) and the human HSCscell line LX-2 were treated with ascorbic acid, transforming growth factor-beta (TGFβ) and/or the pan-hydroxylase inhibitor dimethyloxalylglycine (DMOG). Expression of collagen-I was analyzed by RT-qPCR (COL1A1), Western blotting, and immunofluorescence microscopy. Collagen secretion was determined in the medium by Western blotting for collagen-I and by HPLC for hydroxyproline concentrations. Expression of solute carrier family 23 members 1 and 2 (SLC23A1/SLC23A2), encoding sodium-dependent vitamin C transporters 1 and 2 (SVCT1/SVCT2) was quantified in healthy and cirrhotic human tissue. In the absence of ascorbic acid, collagen-I accumulated intracellularly in p-hHSCs and LX-2 cells, which was potentiated by TGFβ. Ascorbic acid co-treatment strongly promoted collagen-I excretion and enhanced extracellular hydroxyproline concentrations, without affecting collagen-I (COL1A1) mRNA levels. DMOG inhibited collagen-I release even in the presence of ascorbic acid and suppressed COL1A1 and alpha-smooth muscle actin (αSMA/ACTA2) mRNA levels, also under hypoxic conditions. Hepatocytes express both ascorbic acid transporters, while p-hHSCs and LX-2 express the only SVCT2, which is selectively enhanced in cirrhotic livers. Human HSCs rely on ascorbic acid for the efficient secretion of collagen-I, which can be effectively blocked by hydroxylase antagonists, revealing new therapeutic targets to treat liver fibrosis.
KW - Actins/genetics
KW - Amino Acids, Dicarboxylic/pharmacology
KW - Animals
KW - Ascorbic Acid/metabolism
KW - Cell Line
KW - Cells, Cultured
KW - Collagen Type I, alpha 1 Chain
KW - Collagen Type I/genetics
KW - Hepatic Stellate Cells/drug effects
KW - Humans
KW - Liver Cirrhosis/metabolism
KW - Prolyl-Hydroxylase Inhibitors/pharmacology
KW - Rats
KW - Sodium-Coupled Vitamin C Transporters/metabolism
KW - Transforming Growth Factor beta/pharmacology
KW - hypoxia
KW - ascorbic acid/vitamin C
KW - collagen-I
KW - myofibroblast
KW - liver fibrosis
UR - http://www.scopus.com/inward/record.url?scp=85096751472&partnerID=8YFLogxK
U2 - 10.1096/fj.202001564RR
DO - 10.1096/fj.202001564RR
M3 - Article
C2 - 33236467
SN - 0892-6638
VL - 35
SP - e21219
JO - FASEB journal : official publication of the Federation of American Societies for Experimental Biology
JF - FASEB journal : official publication of the Federation of American Societies for Experimental Biology
IS - 2
M1 - e21219
ER -