Downregulation of LIM kinase 1 suppresses ocular inflammation and fibrosis.
OBJECTIVE
The purpose of this study was to determine if downregulation of LIM kinase 1 (LIMK1) by genetic deletion or direct application of LIMK1-targeted siRNA could suppress TGF-beta mediated ocular inflammation and fibrosis.


METHODS
LIMK1 specific siRNAs designed from the human sequence were transfected into human corneal fibroblasts in culture. Immunofluorescence and immunoblotting were performed to examine the fibronectin assembly. The effects of LIMK1 downregulation on actin cytoskeleton organization and focal adhesion formation were studied. A wound closure assay was used to assess cell migration in in vitro fibroblast cultures. The in vivo effects of LIMK1 genetic deletion or downregulation by mouse siRNA were evaluated in a mouse model of ocular inflammation generated by subconjunctival injection of phosphate buffered saline and latex beads. Cellularity on tissue sections was examined after staining with hematoxylin and eosin. Anti-CD45 antibody was used for the leukocyte detection.


RESULTS
Downregulation of LIMK1 in cultured corneal fibroblasts impaired fibronectin secretion and assembly, diminished actin polymerization and focal adhesion formation, and retarded cell migration. In the mouse model of ocular inflammation, both genetic deletion and downregulation of LIMK1 by siRNA significantly reduced inflammatory response.


CONCLUSIONS
Downregulation of LIMK1 was efficacious to decrease the ocular inflammation. We disclose a possibility that LIMK1 may mediate TGF-beta-dependent signaling during ocular inflammation. A direct application of siRNA into eyes to downregulate LIMK1 expression may provide a novel therapy for suppression and prevention of ocular inflammation and fibrosis.
Gorovoy
Matvey
M
Department of Pharmacology, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA.
Koga
Takahisa
T
Shen
Xiang
X
Jia
Zhengping
Z
Yue
Beatrice Y
BY
Voyno-Yasenetskaya
Tatyana
T
D000199:Actins / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000818:Animals; D002465:Cell Movement; D004195:Disease Models, Animal; D015536:Down-Regulation; D005123:Eye / Q000201:enzymology* / Q000473:pathology*; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology; D005353:Fibronectins / Q000378:metabolism; D005355:Fibrosis; D022001:Focal Adhesions / Q000201:enzymology; D017353:Gene Deletion; D006801:Humans; D007249:Inflammation / Q000201:enzymology*; D054461:Lim Kinases / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008875:Middle Aged; D034741:RNA, Small Interfering / Q000378:metabolism
D000199:Actins; D005353:Fibronectins; D034741:RNA, Small Interfering; D054461:Lim Kinases