The Effects Of Transforming Growth Factor-β1 On The Differentiation Of BALB/c 3T3 Fibroblast Into Myofibroblast And The Expression Of Early Growth Response Gene-1

prefaceAlthough pulmonary fibrosis has diverse etiologies, there is a common feature characteristic of this process, namely the abnormal deposition of extracellular matrix that effaces the normal lung tissue architecture.A important cellular source of this matrix is the myofibroblast,which is the key cell that give rise to the pulmonary fibrosis.The origin of these cells is controversial,but most studies suggest that the myofibroblast in pulmonary fibrosis is derived from preexisting peribronchial and perivascular adventitial fibroblast. Furthermore,cultured fibroblasts in vitro can be induced to differentiate into myofibroblasts by treatment with cytokines,such as TGF-(?)1．Egr-1 is a zinc finger transcription factor that has been implicated in commitments to proliferation, differentiation, and the activation of cell death pathways.Recently studies demonstrated a very close correlation between the pulmonary fibrosis and the induction of Egr-1 .Chun Geun Lee et al targeted bioactive TGF-(?) 1 to the murine lung and TGF-(?) 1 produced a trasient wave of epithelial apoptosis that was followed by mononuclear rich inflammation,tissue fibrosis,myofibroblast and myocyte hyperplasia,and septal rupture with honeycombing.They also demonstrated that a null mutation of Egr-1 ameliorated TGF-(?) 1-induced tissue response. Therefore,Egr-1 probably mediate the TGF-(?) 1-induced structural cell apoptosis and pulmonary f韇rosis.The role of Egr-1 in the phenotypic alteration of interstitial fibroblasts into myof韇roblasts is not been enough investigated. To characterize the change of Egr-1 expression in the TGF-(?) 1-induced phenotypic alternation of myof韇roblast,we use the BALB/c 3T3 fibroblasts and lay the foundation of further research.Materials and methods1 .materials(1) BALB/c 3T3 mouse fibroblasts(2) human recombinant TGF-(?) 1(3) anti-(?)-SMA monoclonal antibody(4) anti-Egr-1 polyclonal antibody(5) horsedish peroxidase-conjugated secondary antibody(6) fitc-conjugated secongary antibody(7) fetal calf serum(8) Dulbecco's modified Eagle's medium2．methods(1) cell cultures (2) flow cytometry assays (3) immunocytochemistry assays (4) Western blot assaysResultsThe percentage of (?)-SMA positive cells was (6．65(?)0．48) % when cells were treated with 10ng/ml TGF-(?) 1 for 24h, and the control group was (5．53(?)0．62) %, which showed no statistical significance (P>0．05) .However the percentage was up to (28．38(?)3．60) % after treated with TGF-(?) 1 for 48h, and the control group was (9．49(?)0．21) % (P<0．05) . After treated for 72h, The percentage was (36．04(?)0．73) % and the control group was (15．23(?)0．33) % ,and the difference had statistical significance (P<0．01) . Immunocytochemistry showed that Egr-1 protein positive signals were brown in color, mostly located in nucleus in TGF-(?) 1 group at 60min and 90min.The color was slight in control group.The absorbency of the control group was 37．66(?)18．00 measured by Western blot.After treated with 10ng/ml TGF-(?) 1 for l5min, the absorbency was up to 55．30(?)26．03, which had no statistical significance (p>0．05) compared to the conrol group. The absorbency was increased by continued incubation.It was 114．68(?)14．38 at 60min and reached their peak 128．68(?)22．08 at 90min. They were significantly higher than the control group (p<0．01) .The absorbency was decreased after 90min and reached their neap at 240min. which was 38．99(?)6．80,and the difference had no statistical significance (p>0．05) compared to the conrol group.DiscussionTGF-(?) 1 is a multifunctional cytokine that has been implicated in the pathogenesis of diverse biologic processes including cell growth and survival,cell and tissue differentiation,tissue remodeling and repair. TGF-(?) 1 administration in vivo results in the formation of granulation tissue in which (?)-SMA expressing myofibroblasts are abundant.Furthermore, TGF-(?) 1 has been shown to induce (?)-SMA expression in cultured dermal fibroblasts,suggesting that TGF-(?) 1 plays an important role in myofibroblast differentiation during cutaneous would healing and fibrosis.Our study also suggests that TGF-(?) 1 can induce BALB/c 3T3 fibroblast-myofibroblast differentiation.Egr-1 is the product of an immediate-early gene and a prototype member of the zinc finger superfamily of transcription factors.The gene encodes a protein with three tandem cys2-his2 zinc finger motifs.Egr-1 is an important mediator of tissue inflammation and remodeling.Recently studies demonstrated a very close correlation between the pulmonary f韇rosis and the induction of Egr-1．We hypothesized that Egr-1 is a critical mediator of fibroblast-myofibroblast differentiation.To test this hypothesis we observe the Egr-1 expression with the immunocytochemistry assays and Western blot assays in the course of differentiation of f韇roblast into myofibroblast induced by TGF-(?) 1 .Our studies indicate that the peak expression of Egr-1 at 90min,but the obvious expression of (?)-SMA at 72h.It demonstrated that the expression of Egr-1 prior to the (?)-SMA and the enhanced expression of Egr-1 occurs during the early phase of myofibroblast differentiation.If the Egr-1 direct mediates the (?)-SMA expression still need further observation.ConclusionTGF-(?) 1 can induce BALB/c 3T3 f韇roblast-myofibroblast differentiation. The expression of Egr-1 prior to the (?)-SMA and the enhanced expression of Egr-1 occurs during the early phase of myofibroblasts differentiation.If the Egr-1 direct mediates the (?)-SMA expression still need further observation.