| Background:Myofibroblasts is characterized by the expression of a-smooth muscle actin(?-SMA)and production of collagen,which is key contributors to cardiac fibrosis.Fibrillar type I collagen plays a pivotal role in the proliferation of normal fibroblasts in the extracellular matrix(ECM).?2?1 integrin,cell surface receptor,is a major receptor of fibrillar type I collagen.However,the mechanism of how ?2?1 integrin regulates the differentiation and proliferation of myofibroblasts in cardiac fibrosis through fibrillar collagen(FC)remains uncertain.We established FC mimicked the 3-dimensional extracellular matrix(ECM)of fibroblasts from post-myocardial infarction(MI)patients in vivo.This allowed us to figure out the differentiation and proliferation of cardiac fibroblasts on FC.In this paper,we will address whether ?2?1 integrin can lead to the differentiation and proliferation of myofibroblasts and identify the downstream targets of ?2?1 integrin in myofibroblasts in cardiac fibrosis.ObjectiveTo figure out the mechanism of how ?2?1 integrin regulates the differentiation and proliferation of myofibroblasts in cardiac fibrosis through fibrillar collagen(FC)Materials and Methods1.Isolation and Culture of Cardiac Fibroblasts Cardiac fibroblasts were isolated from 3-month old B6 mice as previously described.The experiments were performed only on cells at passage 32.FC:The FC solution was prepared with 1.25 ml type I collagen solution,0.9 ml 5x DMEM and diluted to 5 ml with lx DMEM with 1%FBS.3.Cell lineGD25 was a ?2?1 integrin-null fibroblast,and GD25 ?2?1 integrin cells were GD25 cells reconstituted with ?2?1 integrin in fibroblasts.4.Cell Migration AssayCell migration was performed by Electric Cell-substrate Impedance Sensing(ECIS)(Applied BioPhysics,Troy,NY,USA),which was an impedance based method to study cell activities in tissue culture in real-time.The migration was assessed by continuing impendent measurements at 20 hours.5.Pulse-Chase AssayThe PTEN labeled with 35S-methionine was quantified by phosphorimaging and normalized to the amount of beta actin present as loading control by Western blot6.PP2A Phosphatase Activity AssayPP2A activity was assessed by dephosphorylation of the phosphopeptide K-R-pT-I-R-R according to the manufacturer's instructions(Millipore,Temecula,CA,USA).7.Proliferation AssayCells were seeded in DMEM medium with 10%FBS on day 1.On day 4,cells were harvested by trypsin digestion and counted.8.Data Analysis All experiments were repeated a minimum of three times.Data were expressed as mean ± SD.Data from Western blots were measured by densitometry with BioSpectrum Imaging System(UVP,Upland,CA,USA).All results were analyzed using a two-sided,unpaired t-test.A p value of<0.05 was deemed as significant.Results1.FC Promoted the Differentiation and Proliferation of Cardiac Fibroblasts.To explore the relationship between FC and the differentiation and proliferation of myofibroblasts,the cells were cultured on FC for five days.We observed that more spindle-shaped and reticular shaped fibers appeared in cells on FC compared to the cells on plastic tissue culture dishes(TC).Meanwhile,a-SMA expression was increased in fibroblasts on FC in comparison to those from TC.Cell proliferation was increased on FC(increase of 34%)compared to those that grew on TC(,p<0.001).To further investigate the differentiation of myofibroblasts regulated by FC compared to other factors,the cells were treated with FC,TGF ?1,okadaic acid(OA,a PP2A inhibitor),or the combination of these factors.The findings support that FC strongly enhanced fibroblast differentiation.2.FC Induced Low Expressions of ?2?1 Integrin and PTENWe found that ?2?1 integrin expression was lower on FC compared to on TC.Meanwhile,PTEN expression was also decreased,and levels of phosphorylated AKT(p-AKT)and a-SMA were elevated in response to TC in a time-dependent manner by Western blot.Next,we examined ?1 integrin expression in mouse fibroblasts isolated from lesions of MI or normal tissues by immunofluorescence.We found that ?1 integrin expression was reduced significantly when compared with control.To further explore how ?2?1 integrin and PTEN expressions were regulated,we examined the transcription level of ?2,?1 integrin,and PTEN by semi-quantitative RT-PCR.we found that mRNA levels of both a2 and ?1 integrin were decreased,but mRNA of PTEN did not change on FC,compared to the ones on TC.PTEN stability was further investigated by the Pulse-Chase assay.The result showed that PTEN protein level in the cells was rapidly degraded on FC compared to the one on TC.3.FC Enhanced the Differentiation and Proliferation of Myofibroblasts by Lowering ?2?1 Integrin expression.We found that knocking-down ?1 integrin increased proliferation of fibroblasts on FC compared to that of the control by Western blot(p<0.01).In addition,the PTEN level decreased.As a result,the p-AKT and a-SMA levels were elevated.We further studied myofibroblast proliferation via gain and loss of function of PTEN.The results showed that knocking down PTEN by shRNA promoted cell proliferation(p<0.001).As expected,?2 and ?1 integrin expression were unchanged,but p-AKT was markedly elevated.4.PP2 A Activity Was Regulated by ?2?1 Integrin on FC.We investigated whether low ?2?1 integrin resulted in altered PP2A activity in fibroblasts.We first examined PP2A activity was significantly decreased on FC,compared to that on TC(p<0.002).To further confirm whether ?1 integrin expression was correlated to PP2A activity,we knocked-down ?1 integrin by shRNA in fibroblasts.We found that PP2A activity was markedly reduced in fibroblasts with?1 integrin shRNA on FC,compared to control-shRNA(p<0.004).Furthermore we examined PP2A activity in both GD25 ?2?1 integrin-null fibroblasts and GD25 fibroblasts reconstructed with a2pl integrin cultured on FC.Notably,GD25 cells could not attach well on FC.Similar to the results of knock-down ?1 integrin,GD25 fibroblasts had very low PP2A activity.In contrast,GD25 fibroblasts with reconstituted ?2?1 integrin had higher PP2A activity on FC(5-fold).5.PP2A Regulated the Differentiation and Proliferation of Myofibroblasts on FC.The fibroblasts were treated by PP2A inhibitor OA(10 nM)for 4 days,or PP2A was knocked down by shRNA.First,we found that proliferation was increased by both OA treatment and PP2Ac knock-down.Secondly,PTEN expression was only changed mildly when PP2Ac expression was knocked down by 79%.Interestingly,p-AKT level was markedly elevated(up 3.98-fold)and a-SMA expression was also increased in the cells with shRNA-PP2Ac,compared to the cells with control-shRNA.The cells were cultured on FC without or with OA as a function of time.Again,we found that PTEN expression was reduced on FC.But PTEN expression had a little change,compared to the cells with or without OA.However,the expressions of p-AKT and ?-SMA were significantly increased(about 3.93-fold or 5.7-fold,respectively).This result indicated that PP2A mainly targeted AKT,rather than PTEN.We also observed that phosphorylated ERK in fibroblasts was increased on FC.To further investigate how AKT or ERK signaling contributed to the regulation of fibroblast proliferation,the fibroblasts were treated with wortmannin(an AKT inhibitor),or U0126(an ERK inhibitor)or both for 4 days.The results indicated that ERK alone might have a minor role in regulating the differentiation and proliferation of myofibroblast.ConclusionsIn this study,we demonstrated that FC induced the differentiation and proliferation of myofibroblasts in cardiac fibrosis via pathologically low ?2?1 integrin expression.Abnormal activation of AKT was due to the inappropriately low activity of PTEN and PP2A.The integrin,PTEN,and PP2A pathways may provide a potential therapeutic target for cardiac fibrosis. |
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