Focal Adhesion Kinase Regulates Migration Of Human Lung Fibroblasts Via Regulation Of Integrin Beta-1

Objectives: To study the effects and mechanisms of focal adhesion kinase(FAK) on migration of lung fibroblasts.Methods:1.Cell experiments1.1 Human Lung Fibroblasts(HLFs) purchased from the American Type Culture Collection(ATCC, Manassas, VA) were maintained and propagated in Dulbecco ’s modified Eagle’s medium(DMEM) supplemented with 10% fetal bovine serum(FBS), 2 mM L-glutamine, and 100 units/ml of a cocktail of penicillin/streptomycin/gentamycin. Experiments were performed on cells at early passages(passage 7–9). After cells cultured on FN were suspended in DMEM and lysed,whole cell lysates were collected. Wound healing assay were also performed.1.2 FAK activation was examined by Western blot, association between FAK and integrin β 1 was examined by Co-immunoprecipitation(Co-IP) and Western blot(WB), migration of Human Lung Fibroblasts(HLFs) on fibronect was examined by Wound healing assay.2.Animal experiments2.1 All animal interventions were approved by our institutional IACUC. Mice(C57Bl6, 8-11 weeks old) were anesthetized and bleomycin(3 U/kg body weight in 50 μl saline) or saline(50 μl) as the vehicle control was slowly instilled through airway to the lungs of the animals using an intratracheal catheter. A group of the animals were daily injected via i.p. with 50mg/kg PF-573228, based on information provided by vendor and published data, while another group as the control was i.p injected with the vehicle. Each group contained 5 to 6 animals. The animals were euthanized and lung tissues were collected on day 21 after bleomycin treatment. The lung tissues were used for histological and biochemical analyses, including cell migration signaling analysis.2.2 In order to evaluate the degree of pulmonary fibrosis,Ashcroft score of lung pathology was observed by Hematoxylin-Eosin(HE) staining,whole lung hydroxyproline level was examined by the acidic hydrolysis.Further more Fibronectin(FN),Procollagen TppeⅠProtein(Pro-Col1),α-Smooth Muscle Actin(α-SMA)was examined by Western blot.In order to evaluate the degree of lung fibroblast migration in the lung tissue,the level of active Rac(GTP-bound form),FAK activation and S100A4 protein expression was detected by Western blot.3. Statistical AnalysisData were analyzed using the unpaired or paired t-test analysis(for comparisons between two groups), and were presented as means ± SE. Experiments were performed three or four times, each with duplicate. A p value < 0.05 was considered statistically significant.Results:1.FAK was associated with integrin β1 in response to PDGF-BB treatment in fibroblastsPDGF-BB induced FAK activation at two ranges of concentration, one at the range of 0.1-0.5 ng/ml and the other at the range of 1-5 ng/ml, whereas PDGF-BB at a concentration less than 0.01 ng/ml could not induce significant FAK activation in the human lung fibroblasts. FAK was activated as early as 6 minutes after PDGF-BB stimulation at a concentration of 2 ng/ml in the fibroblasts. An early but small FAK activation appeared at 30 minutes after PDGF-BB stimulation. FAK activation was further increased 5 hours after PDGF-BB treatment and peaked at 20 hours after PDGF-BB treatment.2.PDGF-BB-induced fibroblast migration was dose-dependent and timedependent on FN. Inhibition of FAK activation decreased PDGF-BB-induced fibroblast migration without blocking the association of FAK with FN receptor integrinβ 1.PDGF-BB induced fibroblast migration in a dose-dependent manner. PDGF-BB-stimulated migration rate, calculated as the wound covered area per 24 hours, was positively correlated with the concentration used, with the highest migration rate at the range of 1-5 ng/ml. PDGF-BB induced fibroblast migration also showed a time-dependent fashion, with significantly increases at 5 hours and then at 24 hours post PDGF-stimulation(p<0.001). PF-573228 inhibited, in a dose-dependent manner, the FAK activation(pY397 of FAK) and the fibroblast migration induced by PDGF-BB. Treatment with the FAK inhibitor could not block the association of FAK with integrin β 1, as evidenced by that PF-573228 had no effect on FAK- integrin β 1 interaction, even at the concentration(1 or 5 μM) that could effectively inhibit PDGF-BB- induced FAK activation and fibroblast migration.3.Integrins α5β1 and α4β1 were the main integrin receptors contributing to FAK-mediated fibroblast migration on FN and FAK activation.Compared with the control IgG, integrin β 1 blocking antibody reduced the migration of fibroblasts by about 85%(p<0.001). Integrin α 5- orα 4-blocking antibody inhibited the migration on FN by about 50%. Blocking integrin α vβ 3, α vβ 6, or α vβ 8 individually inhibited the migration on FN by about 5%. This was further supported by that blocking integrin α v inhibited fibroblast migration on FN by about 15%. Integrin β 1 blocking antibody significantly blocked PDGF-BB-stimulated FAK activation. Integrin β1 blocking antibody or control IgG had no effect on basal FAK activation in fibroblasts on FN.4.Effects and mechanisms of the FAK inhibitor on pulmonary fibrosis in a mouse modelMorphometric analysis of lung tissue sections revealed an approximately 1.2-fold decrease(p<0.01) in fibrotic score in the bleomycin-challenged mice treated with FAK inhibitor, when compared to that in bleomycin-challenged mice treated with the vehicle only. Using the quantifiable measure of hydroxyproline as a surrogate for the total collagen content in whole lung tissues, FAK inhibitor significantly decreased total collagen level in bleomycin-challenged mice when compared to that in vehicle treated mice(p< 0.01). When compared to the vehicle treated mice, FAK inhibitor significantly decreased the activation of FAK and Rac(p< 0.01), decreased S100A4 expression(p< 0.01), decreased the whole lung fibronectin and procollagen-1 levels(p< 0.01), and reduced α-SMA expression(p< 0.01) in bleomycin-challenged mice.Conclusion:1. FAK could be activated by PDGF-BB in a dose- and time-dependent manner, and FAK could be recruited by and directly associated with integrinβ 1 in response to PDGF-BB treatment in fibroblasts.2. PF-573228 inhibits PDGF-BB-induced FAK activation in a dose- and time-dependent manner and then inhibits PDGF-BB-induced migration of HLFs.3. Different integrin receptors have different functions during PDGF-BB induced migration of HLFs, and the integrin beta1 receptor plays a main role.4. FAK inhibitor alleviates the development of fibrotic lesions at least by inhibition of fibroblast migration, extracellular Matrix(ECM) protein production and myofibroblast differentiation.5. Our findings collectively suggest that targeting FAK signaling may be an effective therapeutic strategy for fibrosis.