after the institutional review board approved our protocol , selected patients received comprehensive information and provided written consent for inclusion . in compliance with the tenets of the declaration of helsinki , small human tenon 's capsule specimens were obtained during strabismus surgery , and primary human tenon 's fibroblasts were cultured from the explants . cells were incubated in dulbecco 's modified eagle 's medium ( dmem ; life technologies , carlsbad , ca , usa ) supplemented with 10% fetal bovine serum ( life technologies ) , 100 units / ml penicillin , and 100 g / ml streptomycin ( life technologies ) at 37 in 5% co2 . after 12 hours of incubation in serum - free media , the cells were treated with 5 ng / ml of recombinant human tgf-1 ( r&d systems inc . , minneapolis , mn , usa ) for up to 48 hours . for the control group , the same volume of dmem small interfering rna molecules targeting fak mrna ( sirnafak ) were purchased from santa cruz biotechnology inc . ( santa cruz , ca , usa ) and were delivered into cells according to the manufacturer 's instructions . briefly , the fibroblasts were incubated in media containing 50 nm of sirnafak and 2 g / ml of lipofectamine 2000 ( life technologies ) for 16 hours at 37 in a co2 incubator . total rna was extracted using an rneasy mini kit ( qiagen , venlo , netherlands ) , and cdnas were synthesized using the superscript iii first - strand synthesis system ( life technologies ) . real - time polymerase chain reaction ( pcr ) was performed with 50 ng cdna per reaction using 25 l of iq sybr green supermix ( bio - rad laboratories , hercules , ca , usa ) containing 500 nm of specific primers ( table 1 ) in the icycler iq real - time pcr detection system ( bio - rad laboratories ) . whole cellular proteins were extracted from the cells , and equal amounts of protein ( 10 g ) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis . the proteins were transferred to polyvinylidene fluoride membranes and probed overnight with primary antibodies against human collagen type i , fak , phospho - fak ( pfak ) , sma , and -actin ( santa cruz biotechnology ) . immunoreactive bands were detected with horseradish peroxidase - conjugated secondary antibodies ( santa cruz biotechnology ) and visualized with an enhanced chemiluminescent system . quantitative reverse transcription ( rt)-pcr data is expressed as mean  sem and compared with the kruskal - wallis one - way analysis of variance using the medcalc ver . 1.43u ( national institutes of health , bethesda , md , usa ) . a p - value less than 0.05 was considered statistically significant . after the institutional review board approved our protocol , selected patients received comprehensive information and provided written consent for inclusion . in compliance with the tenets of the declaration of helsinki , small human tenon 's capsule specimens were obtained during strabismus surgery , and primary human tenon 's fibroblasts were cultured from the explants . cells were incubated in dulbecco 's modified eagle 's medium ( dmem ; life technologies , carlsbad , ca , usa ) supplemented with 10% fetal bovine serum ( life technologies ) , 100 units / ml penicillin , and 100 g / ml streptomycin ( life technologies ) at 37 in 5% co2 . after 12 hours of incubation in serum - free media , the cells were treated with 5 ng / ml of recombinant human tgf-1 ( r&d systems inc . , minneapolis , mn , usa ) for up to 48 hours . for the control group , the same volume of dmem small interfering rna molecules targeting fak mrna ( sirnafak ) were purchased from santa cruz biotechnology inc . ( santa cruz , ca , usa ) and were delivered into cells according to the manufacturer 's instructions . briefly , the fibroblasts were incubated in media containing 50 nm of sirnafak and 2 g / ml of lipofectamine 2000 ( life technologies ) for 16 hours at 37 in a co2 incubator . total rna was extracted using an rneasy mini kit ( qiagen , venlo , netherlands ) , and cdnas were synthesized using the superscript iii first - strand synthesis system ( life technologies ) . real - time polymerase chain reaction ( pcr ) was performed with 50 ng cdna per reaction using 25 l of iq sybr green supermix ( bio - rad laboratories , hercules , ca , usa ) containing 500 nm of specific primers ( table 1 ) in the icycler iq real - time pcr detection system ( bio - rad laboratories ) . whole cellular proteins were extracted from the cells , and equal amounts of protein ( 10 g ) were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis . the proteins were transferred to polyvinylidene fluoride membranes and probed overnight with primary antibodies against human collagen type i , fak , phospho - fak ( pfak ) , sma , and -actin ( santa cruz biotechnology ) . immunoreactive bands were detected with horseradish peroxidase - conjugated secondary antibodies ( santa cruz biotechnology ) and visualized with an enhanced chemiluminescent system . quantitative reverse transcription ( rt)-pcr data is expressed as mean  sem and compared with the kruskal - wallis one - way analysis of variance using the medcalc ver . 1.43u ( national institutes of health , bethesda , md , usa ) . a p - value less than 0.05 was considered statistically significant . the quantitative data of rt - pcr for fak and sma are presented in fig . the 16-hour treatment with tgf-1 significantly increased the mrna levels of sma ( 5.11  1.37 times to control , p = 0.023 ) but not of fak ( 1.16  0.06 times to control , p = 0.204 ) . on the western immunoblots , the tgf-1 treatment increased the protein expression of fak as well as that of collagen type i. expression of pfak , an active form of fak , was also increased . representative immunobands for collagen type i , fak , pfak , and -actin are shown in fig . 2 . regarding sma , though serum starvation itself slightly induced the expression of sma , the exposure to tgf-1 for 48 hours stimulated sma expression in human tenon 's fibroblasts . when the sirnafak molecules were introduced into the cells , the tgf-1-induced sma expression was attenuated . representative immunobands for sma in the control group , tgf-1 only treatment group , and sirnafak / tgf-1 treatment group are shown in fig . fak , also known as protein tyrosine kinase 2 , participates in the focal adhesion between the cytoskeleton and extracellular matrix and elicits intracellular signal transductions associated with cell migration and survival [ 11,12,16 - 18 ] . it is activated by autophosphorylation via smad- and p38 mapk - dependent mechanisms [ 19 - 22 ] and plays a key role in cancer metastasis as well as normal development . even though fak is also linked to the myofibroblast transdifferentiation of fibroblasts in response to tgf-1 [ 13 - 15 ] , the precise role of the kinase has not been well studied in the fibrotic process in the subconjunctival space . excessive subconjunctival fibrosis causes ocular morbidity in not only patients with ocularcutaneous disorders , but also patients who have undergone strabismus surgery and/or glaucoma - filtering surgery . a better understanding and modulation of fak signaling in this process may result in a novel therapeutic strategy for those patients . using primary cultured human tenon 's fibroblasts , we investigated the role of fak in tgf--induced myofibroblast transdifferentiation in the present study and found that silencing of fak action using sirnafak duplex significantly attenuated the sma expression induced by tgf-1 in human tenon 's fibroblasts . myofibroblasts contain characteristics found in smooth muscle cells and fibroblasts in that they possess a contractile phenotype of sma and synthesize extracellular matrix proteins [ 1 - 3 ] . since they play a crucial role in most fibrotic responses , myofibroblast transdifferentiation from activated fibroblasts is an essential step in the fibrotic process . in the subconjunctival space , tgf- typically initiates this myofibroblast transdifferentiation of fibroblasts [ 4 - 6 ] . on quantitative rt - pcr , the mrna expression of sma on western immunoblots , the protein levels of both fak and pfak were minimally stimulated by tgf-1 treatment . the increase in fak seems to result from an increase in translation rather than transcription . currently , many researchers have tried to develop anti - tgf- neutralizing antibodies to modulate postoperative scarring in patients undergoing ocular surface surgery [ 23 - 25 ] . fak , as a participant in tgf--associated intracellular signaling of myofibroblast transdifferentiation from fibroblasts , might also be valuable as a novel therapeutic strategy for anti - fibrosis .
purposeto investigate the role of focal adhesion kinase ( fak ) in transforming growth factor ( tgf)--induced myofibroblast transdifferentiation of human tenon 's fibroblasts.methodsprimary cultured human tenon 's fibroblasts were exposed to tgf-1 for up to 48 hours . 
 the mrna levels of fak ,  smooth muscle actin ( sma ) , and -actin were determined by quantitative real time reverse transcription polymerase chain reaction . 
 the protein levels of collagen type i , fak , phospho - fak , sma , and -actin were determined by western immunoblots . 
 after the small interfering rna targeting fak ( sirnafak ) molecules were delivered into the cells , the expressions of sma proteins were determined by western immunoblots.resultsin human tenon 's fibroblasts , tgf-1 significantly increased the mrna and protein expressions of sma . however , when the action of fak was inhibited using sirnafak , the tgf-1-induced expression of sma was attenuated.conclusionsour data suggest that fak may be associated with the tgf-1-induced transdifferentiation of human tenon 's fibroblasts to myofibroblasts , which is the essential step of subconjunctival fibrosis .