Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells.
OBJECTIVE
The Rho-ROCK signaling pathways play an important role in the activation of hepatic stellate cells (HSCs). We investigated the effects of fasudil hydrochloride hydrate (fasudil), a Rho-kinase (ROCK) inhibitor, on cell growth, collagen production, and collagenase activity in HSCs.


METHODS
Rat HSCs and human HSC-derived TWNT-4 cells were cultured for studies on stress fiber formation and alpha-smooth muscle actin (alpha-SMA) expression. Proliferation was measured by BrdU incorporation, and apoptosis by TUNEL assay. The phosphorylation states of the MAP kinases (MAPKs), extra cellular signal -regulated kinase 1/2 (ERK1/2), c-jun kinase (JNK), and p38 were evaluated by western blot analysis. Type I collagen, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) production and gene expression were evaluated by ELISA and real-time PCR, respectively. Collagenase activity (active MMP-1) was also evaluated.


RESULTS
Fasudil (100 microM) inhibited cell spreading, the formation of stress fibers, and expression of alpha-SMA with concomitant suppression of cell growth, although it did not induce apoptosis. Fasudil inhibited phosphorylation of ERK1/2, JNK, and p38. Treatment with fasudil suppressed the production and transcription of collagen and TIMP, stimulated the production and transcription of MMP-1, and enhanced collagenase activity.


CONCLUSIONS
These findings demonstrated that fasudil not only suppresses proliferation and collagen production but also increases collagenase activity.
Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Marie
M
Fukushima
Makoto
M
Nakamuta
Motoyuki
M
Kohjima
Kazuhiro
K
Kotoh
Munechika
M
Enjoji
Naoya
N
Kobayashi
Hajime
H
Nawata
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D017364:Collagenases / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007728:Kupffer Cells / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D008297:Male; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D051381:Rats; D017208:Rats, Wistar; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism; D054460:rho-Associated Kinases
D024042:Collagen Type I; D047908:Intracellular Signaling Peptides and Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; C015482:protein kinase modulator; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; C049347:fasudil