Rho-associated kinases are crucial for myofibroblast differentiation and production of extracellular matrix in scleroderma fibroblasts.
OBJECTIVE
Rho-associated kinases (Rock) are the major cellular mediators of Rho GTPases and play an important role in the organization of the actin cytoskeleton. Inhibitors of Rock are currently being evaluated for the treatment of pulmonary arterial hypertension. This study was undertaken to analyze the role of Rock in the activation of fibroblasts in systemic sclerosis (SSc).


METHODS
Rock signaling was inhibited using chemical inhibitors and small interfering RNA. The expression of extracellular matrix (ECM) proteins and alpha-smooth muscle actin was analyzed by real-time polymerase chain reaction, Western blotting, and SirCol assay. Metabolic activity was quantified by MTT assay. Cell viability was assessed by staining with annexin V and propidium iodide. The role of MAP kinases was investigated using selective inhibitors and Western blotting.


RESULTS
Inhibition of Rock strongly reduced the synthesis of the major ECM proteins at the messenger RNA level as well as the protein level. Counterregulatory changes in the expression of tissue inhibitors of metalloproteinases and matrix metalloproteinases were not observed. Inhibition of Rock prevented myofibroblast differentiation. Transforming growth factor beta activated ERK in a Rock-dependent manner, and ERK mediated in part the stimulatory effects of Rock on myofibroblast differentiation. Toxic adverse effects of the inhibition of Rock were not observed.


CONCLUSIONS
Our findings demonstrate that Rock potently stimulates the differentiation of resting fibroblasts into myofibroblasts and the production of ECM at biologically relevant concentrations without cell toxicity. These findings, along with the beneficial effects of Rock inhibition on vascular disease, indicate that inhibition of Rock might be an interesting novel therapeutic approach for the treatment of SSc.
Akhmetshina
Alfiya
A
University of Erlangen-Nuremberg, Erlangen, Germany.
Dees
Clara
C
Pileckyte
Margarita
M
Szucs
Gabriella
G
Spriewald
Bernd M
BM
Zwerina
Jochen
J
Distler
Oliver
O
Schett
Georg
G
Distler
Jörg H W
JH
D000328:Adult; D000368:Aged; D000577:Amides / Q000494:pharmacology; D016022:Case-Control Studies; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology*; D006801:Humans; D008297:Male; D020782:Matrix Metalloproteinases / Q000378:metabolism; D008875:Middle Aged; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012595:Scleroderma, Systemic / Q000201:enzymology* / Q000473:pathology*; D012867:Skin / Q000201:enzymology / Q000473:pathology; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000577:Amides; D004791:Enzyme Inhibitors; D010982:Platelet-Derived Growth Factor; D011725:Pyridines; D019714:Tissue Inhibitor of Metalloproteinases; D016212:Transforming Growth Factor beta; C108830:Y 27632; D054460:rho-Associated Kinases; D020782:Matrix Metalloproteinases