| Backgroud: Eukaryotic initiation factor 6(e IF6) is an evolutionary conserved protein, which functions in ribosomal biogenesis and translation control by regulating the binding of 40 S and 60 S ribosomal subunits. Previous evidence has shown that e IF6 has a dual function:(1) nucleolar e IF6 is necessary for the biogenesis of 60 S subunits both in yeast and mammals and(2) cytoplasmic e IF6 is required for insulin and growth factor-stimulated translation in mammals. The factor e IF6 is constitutively expressed in vitro, but its expression is variable in vivo. A high level of e IF6 expression has been found in rapidly proliferating cells. Dysregulation of e IF6 expression has proved to be relevant to some of pathological states. Moreover e IF6 overexpression has been reported to cause an aberrant eye development in Xenopus laevis. Furthermore, e IF6 overexpression has been found in selected cancer types, such as colorectal carcinomas, head and neck cancer, malignant mesothelioma. It has been proposed that e IF6 is a rate-limiting controller of the initiation of translation, able to affect tumorigenesis and tumor growth and that the impairment of e IF6 activity in cytoplasma restricts lymphomagenesis and tumor progression.We have previously demonstrated that e IF6 has been identified to interact with a neuronal protein 3.1(P311) promoting hypertrophic scar(HS) formation and contraction by enhancing the expression of transforming growth factor-β1(TGF-β1). The protein P311 was identified as the most intensely upregulated gene in fibrosis-associated myofibroblasts(MFBs), as revealed by gene expression profiling and comparative proteomic analysis. Myofibroblasts are key mediator of pathogenesis of all fibrotic diseases that significantly contribute to connective tissue remodeling by exerting traction forces and synthesizing extracellular matrix(ECM) components. Myofibroblasts are characterized by the expression of α-smooth muscle actin(α-SMA), which is a common key element for detection of myofibroblast differentiation and a major player in contractile force production. Profibrotic cytokine TGF-β1, along with the mechanical resistance of the extracellular matrix, is an amply documented primary stimulus for myofibroblast differentiation and persistence. Thus, the data suggests that e IF6 might be relevant with TGF-β1 expression and myofibroblast differentiation, which has yet to be examined.Objective: The aim of the present study is to determine whether e IF6 has a role in the myofibroblast differentiation? Whether this regulatory effect is associated with the expression of TGF-β1? And what is the potential mechanism governing the regulatory effect of e IF6 on TGF-β1 expression?Part One Isolation, culture and identifying the myofibroblast derived from dermis ofe IF6 deficiency heterozygote miceMethods:1. Isolate the primary dermal fibroblasts from the skin of the neonatal(3 day old)e IF6+/-and wild-type(WT) mice(e IF6 null embryos are lethal at preimplantation.). The cells between the first and the third passages(P1–P3) were used to perform the experiments. 2. Immunocytochemistry was carried out to detect the Vimentin and α-SMA expression in the isolated dermal cells. 3. The reverse-transcription PCR(RT-PCR) was applied to identify the genotype of the isolated dermal cells to distinguish the WT cells from the e IF6+/- cells. 4. The Real-Time PCR and Western blotting analysis were used to detect the expression of e IF6 in WT cells and the e IF6+/- cells identified by RT-PCR.Results:1. We successfully isolated and cultured the dermal cells displaying the spindle shape or polygonal shape. 2. Both Vimentin and α-SMA were presented intensively in the cytoplasma of the dermal cells with the positive rate being 100%. 3. The RT-PCR results demonstrated that the sample with two bands in the position of 650 bp and 320 bp was derived from e IF6+/- mice, while the sample with only one band in the position of 320 bp was derived from WT mice.4. Using Western blottingting and q PCR analysis, we confirmed that isolated dermal cells from the skin of e IF6+/- mice exhibited approximately a 40% reduction of e IF6 expression at m RNA levels and a 60% reduction at the protein level.Part Two The effect of e IF6 on the biological function of the dermal myofibroblastMethods:1. The flow cytometry analysis was carried out to detect the cell cycle of e IF6+/- cells and WT cells. 2. The TUNEL analysis was performed to detect the apoptosis of e IF6+/- cells and WT cells. 3. The Real-Time PCR and Western blotting analysis were used to detect the expression of α-SMA in WT cells and the e IF6+/- cells. The Real-Time PCR was carried out to test the collagen I m RNA expression in WT cells and the e IF6+/- cells. The WT cells and the e IF6+/- cells were stained with phalloidin-tetramethylrhodamine to observe the characterization of stress fiber with confocal microscopy. The collagen contraction assays in a fibroblast-populated collagen lattice(FPCL) model was used to compare the contractility of the WT cells and the e IF6+/- cells.Results:1. The results of flow cytometry analysis demonstrated that there was a reduction in the percentage of e IF6+/- cells in S phase(6.92% versus 11.16% in WT) but the percentage of e IF6+/- cells in G2/M phase was increased(42.13% vs 27.59% in WT). 2. The results of TUNEL analysis demonstrated that there was no significant difference in apoptosis observed between e IF6+/- and WT cells. 3. The expression of α-SMA was significantly increased in e IF6+/-cells compared to WT cells at both the m RNA(2.9-fold difference, P=0.020, n=5) and protein(1.8-fold difference, P=0.029, n=5) levels. Expression of collagen I(Col1a1 and Col1a2) m RNA was also increased in e IF6+/-cells(Col1a1: 4.9-fold difference, P=0.001, n=3; Col1a2: 4.6-fold difference, P=0.007, n=3). In line with these observations, increased stress fiber formation was found in e IF6+/-cells as compared with WT cells. The collagen contraction assays in a fibroblast-populated collagen lattice(FPCL) model demonstrated that e IF6+/-cells were shown to contract collagen lattice more extensively compared to WT cells(P=0.001, n=3).Part Three The effect of e IF6 on the signal transduction during myofibroblast defferetaitionMethods:1. The Real-Time PCR and ELISA analysis were used to detect the expression of TGF-β1 in WT cells and the e IF6+/- cells. 2. The Real-Time PCR was used to detect the TGF-βRI and TGF-βRII m RNA expression in WT cells and the e IF6+/- cells. And the Western blotting analysis was used to test the Smad proteins expression in WT cells and the e IF6+/- cells within the exogenous TGF-β1 stimulation. 3. The Western blotting analysis was used to test the p-Smad2 and α-SMA expression in WT cells and the e IF6+/- cells after SB431542 inhibiting the TGF-β/Smad signal pathway activation.Results:1. Both TGF-β1(Tgfb1) m RNA and TGF-β1 protein levels were found to be significantly increased in e IF6+/-cells versus WT cells(m RNA: 2.7-fold increase, P<0.001,n=5; protein: 1.8-fold increase, P<0.001, n=5), as revealed by the Real-Time PCR and ELISA analyses. 2. The levels of TGF-βRI(Tgfbr1) and TGF-βRII(Tgfbr2) m RNA expression did not differ in e IF6+/-cells vs. WT cells(P=NS, n=3). The level of phosphorylated Smad2(p-Smad2) was increased to 1.5-fold(P=0.008, n=3) and coupled with a decreased Smad7 level of 50%(P=0.002, n=3) in e IF6+/-cells as compared to WT cells. The level of phosphorylated Smad3 was slightly but not significantly elevated in e IF6+/-cells(P=NS, n=3). 3. A TGF-βR-I-specific pharmacological inhibitor SB431542 significantly inhibited p-Smad2 in e IF6+/-cells to the WT level, leading to the suppression of the upregulation of α-SMA expression in e IF6+/-cells, in which the level was alike in WT cells.Part Four The investigation of the underlying mechanism of the regulatory effect of e IF6 on TGF-β1 expressionMethods:1. The polysomal profile analysis was applied to detect the TGF-β1 m RNA translation efficiency of WT cells and the e IF6+/- cells. 2. The Real-Time PCR was performed to determine the level of TGF-β1 m RNA followed by treatment with actinomycin D for various times to inhibit de-novo RNA synthesis. 3. The bisulfite sequencing(BSP methods) was used to evaluated the DNA methylation patterns of the core region of TGF-β1 promoter in WT and e IF6+/-cells. 4. The i TRAQ-based quantitative proteomic approach to screen the differentiated expressed proteins between the WT and e IF6+/- cells followed by Real-Time PCR and Western blotting analyses confirmation. 5. The TGF-β1 promoter assay and chromatin immunoprecipitation(Ch IP)-q PCR analysis were performed to detect the presence of H2 A.Z and the binding of Sp1 to the TGF-β1 promoter.Results:1. The polysomal profile result exhibited that TGF-β1 m RNA associated with heavier polysomes did not show significant differences between e IF6 knockdown cells and control cells. 2. The Real-Time PCR analysis demonstrated that the half-life of TGF-β1 m RNA was not altered by e IF6 knockdown in e IF6+/- cells. 3. The BSP result showed that the methylation level of the TGF-β1 promoter region was similar for WT cells(61.9%) and e IF6+/- cells(73.8%). 4. The i TRAQ results demonstrated that a total of 302 differently expressed proteins induced by e IF6 deficiency was identified. Notably, proteins down-regulated by e IF6 deficiency were involved in chromosome organization, chromatin organization, chromatin as SDbly or disas SDbly, DNA packaging, protein-DNA complex as SDbly and nucleosome as SDbly. Moreover, we found that there was an association between the expression of e IF6 and Histone proteins confirmed by Real-Time PCR and Western blotting analyses. The results demonstrated that both m RNA and protein levels of H2 A.Z(H2afz) were decreased in e IF6+/-cells as compared to those present in WT cells(m RNA: 20% reduction, P=0.015, n=3; protein: 40% reduction, P=0.051, n=3). And a 45% reduction of H2 B protein was found in e IF6+/-cells as compared to those present in WT cells. 5. The Ch IP results showed that after the stimulation with exogenous TGF-β1, H2 A.Z binding to the different regions of TGF-β1 promoter was greatly reduced in e IF6+/-cells while it was enriched at the corresponding regions of TGF-β1 promoter in WT cells(region 1:-120/+73; region 2:-37/+175; region 3:-118/+246). Moreover, Sp1 recruitment to TGF-β1 promoter was remarkably increased in e IF6+/-cells compared to that in WT cells after TGF-β1 stimulation.Conclusion:In this study, we observed that: 1. e IF6 plays an important role in myofibroblast activation and differentiation. e IF6 deficit promoted myofibroblast phenotypic by heightening α-smooth muscle actin(α-SMA) expression, stimulating collagen and extracellular matrix(ECM) and cellular contractility/motility. 2. e IF6 inversely modulates the expression of TGF-β1, mainly at transcription level. 3. e IF6 evicts the H2 A.Z from the TGF-β1 promoter and facilitates the Sp1 recruitment to TGF-β1 promoter, because H2 A.Z converts chromatin from transcriptionally repressed state to an active state under e IF6 deficiency. 4. TGF-β/Smad signaling pathway was involved in e IF6-regulated myofibroblast differentiation and activation. In summary, we provide novel insights of e IF6 functional role in the regulation of TGF-β1 transcription and myofibroblast differentiation. Our data mechanically explain e IF6 modulating the transcription of TGF-β1 by regulating Sp1 recruitment to TGF-β1 promoter via H2 A.Z occupancy. We also demonstrate that e IF6 deficiency-induced myofibroblast differentiation through the TGF-β/Smad pathway. The role of e IF6 in the regulation of TGF-β1 and myofibroblast differentiation extends the roles for the multifunctional e IF6 in different and as yet undescribed cellular processes. Since myofibroblast plays a key role in extracellular matrix deposition during fibrogenesis, these findings further support the hypothesis that the e IF6 might have a relevant role during fibrogenesis. |
PDF Full Text Request