All-trans retinoic acid modulates mitogen-activated protein kinase pathway activation in human scleral fibroblasts through retinoic acid receptor beta.
OBJECTIVE
All-trans retinoic acid (ATRA) is known to inhibit the proliferation of human scleral fibroblasts (HSFs) and to modulate the scleral intercellular matrix composition, and may therefore serve as a mediator for controlling eye growth. Cell proliferation is regulated by the mitogen-activated protein kinase (MAPK) pathway. The aim of the current study was to investigate whether changed activation of the MAPK pathway could be involved in the response of HSFs exposed to ATRA.


METHODS
HSFs were cultured in Dulbecco Modified Eagle's Medium/F12 (DMEM/F12) and exposed to 1 μmol/l ATRA for 10 min, 30 min, 1 h, 8 h, or 24 h. The activation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK) in HSFs was assessed with western blot analysis and immunocytofluorescence.


RESULTS
After exposure to ATRA for 24 h, the HSFs appeared shrunken and thinner than the control cells. The intercellular spaces were wider, and the HSFs appeared less numerous than in the control culture. Western blot showed decreased activation of ERK 1/2 in the HSFs from 30 min (p=0.01) to 24 h (p<0.01) after the start of exposure to ATRA, and increased activation of the JNK protein from 10 to 30 min (p<0.01) after the start of exposure to ATRA. Indirect immunofluorescence confirmed changes in activation of ERK 1/2 and JNK in HSFs exposed to ATRA. No change in activation of p38 in HSFs was observed after exposure to ATRA. Pretreatment of the HSFs with LE135, an antagonist of retinoic acid receptor beta (RARβ), abolished the ATRA-induced changes inactivation of ERK 1/2 and JNK.


CONCLUSIONS
ATRA inhibits HSF proliferation by a mechanism associated with modulation of ERK 1/2 and JNK activation and depends on stimulation of retinoic acid receptor beta.
State Key Laboratory of Ophthalmology,Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, P. R. China.
Lijun
L
Huo
Dongmei
D
Cui
Xiao
X
Yang
Zhenya
Z
Gao
Klaus
K
Trier
Junwen
J
Zeng
D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D003984:Dibenzazepines / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D018168:Receptors, Retinoic Acid / Q000378:metabolism*; D012590:Sclera / Q000166:cytology*; D014212:Tretinoin / Q000494:pharmacology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D003984:Dibenzazepines; C104182:LE 135; D018168:Receptors, Retinoic Acid; C082925:retinoic acid receptor beta; D014212:Tretinoin; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases