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.
Fukushima
Marie
M
Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Nakamuta
Makoto
M
Kohjima
Motoyuki
M
Kotoh
Kazuhiro
K
Enjoji
Munechika
M
Kobayashi
Naoya
N
Nawata
Hajime
H
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