Thy-1 attenuates TNF-alpha-activated gene expression in mouse embryonic fibroblasts via Src family kinase

Abstract

Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-alpha. Our study demonstrates distinct profiles of TNF-alpha-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1-) subsets of mouse embryonic fibroblasts (MEF). TNF-alpha induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1- MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1- MEFs significantly attenuated TNF-alpha-activated gene expression. Mechanistically, TNF-alpha activated Src family kinase (SFK) only in Thy-1- MEFs. Blockade of SFK activation abrogated TNF-alpha-activated gene expression in Thy-1- MEFs, whereas restoration of SFK activation rescued the TNF-alpha response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-alpha-activated gene expression via interfering with SFK- and NF-kappaB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

Figure 1. Differential Expression of MMP-9 and ICAM-1 in TNF-α-stimulated MEF Thy-1 subsets.

A) Serum starved Thy-1+ and Thy-1− MEFs were stimulated with TNF-α (5 ng/ml) for 24 hours. Total RNA was isolated from the stimulated MEFs. Quantitative RT-PCR was carried out to determine the mRNA levels of MMP-9. The fold change of MMP-9 mRNA in the stimulated cells over control was obtained by setting the values from the untreated MEFs to one. B) The culture conditions were similar to part A except that the mRNA levels of ICAM-1 were determined. C) The conditioned medium was collected from MEFs exposed to the indicated doses of TNF-α and TGF-β1 for 48 hrs. Gelatin zymography was carried out to determine the expression MMP-2/-9 in the conditioned medium. A representative gel image from three experiments was shown. D) Thy-1 MEF subsets were seeded into Matrigel-coated transwell chambers. The cells were stimulated by TNF-α (5 ng/ml) for 24 hours. The invading cells on the bottom surface of the membrane were stained and enumerated under light microscopy at 400× amplification. The sum of invading cells from 5 randomly selected fields was compared across the groups. The invasion assays were performed in triplicates for each treatment group. Results shown were mean and standard deviations from at least three independent experiments. *, P value<0.05.

Figure 2. Reduced TNF-α-activated gene expression by Thy-1 expression in Thy-1– fibroblasts.

A) Thy-1– MEFs were transiently transfected with either the Thy-1 expression vector (Thy-1) or the backbone vector (Vec) followed by exposure to TNF-α (5 ng/ml) for 24 hrs. Total RNA was isolated from the stimulated MEFs. Quantitative RT-PCR was carried out to determine the mRNA levels of MMP-9 and ICAM-1. The fold change of each mRNA in the stimulated cells over control was obtained by setting the values from the untreated backbone vector transfected Thy-1– MEFs to one. B) The culture conditions were similar to part A except that the transfected Thy-1– MEFs were exposed to TNF-α (5 ng/ml) for 48 hrs. The conditioned medium was collected and analyzed for the expression of MMP-2/-9 by gelatin zymography. A representative gel image from three experiments was shown. C) The culture conditions were similar to part A except that the IL-8-Luc and RL-TK reporters were co-transfected with Thy-1 or the backbone vector. A fold increase in IL-8-Luc by TNF-α (5 ng/ml) was determined by setting the values from the unstimulated Thy-1– MEFs to one. D) The serum-starved RLE6 variants were stimulated with the indicated doses of TNF-α for 24 hrs. The mRNA levels of MMP-9 were determined as described in part A. The results were average from two independent experiments. E) Similar to Part D except that MMP-9 expression in the conditioned medium was determined using gelatin zymogram in RLE6 variants stimulated with TNF-α for 48 hrs. The gel image is representative of two independent experiments. Vec and V refer to Thy-1– MEFs transfected with the backbone vector. Thy-1 and T refer to Thy-1– MEFs transfected with the Thy-1 expression vector. In parts A & C, the results shown are the mean and standard deviation from at least three independent transfections. *, P value<0.05.

Figure 3. TNF-α-mediated activation of SFK in MEF Thy-1 subsets.

A) MEF Thy-1 subsets were exposed to TNF-α (5 ng/ml) for 5 and 15minutes. The treated MEFs were lysed and immunoblotted for tyrosine 416 phosphorylated-SFK, total SFK, I-κBα, and β-actin. B) Similar to part A except that MEF Thy-1 subsets were pretreated with PP2 (10 µM) for 30 minutes, and then exposed to TNF-α (5 ng/ml) for 5 minutes. The cell lysates were immunoblotted for tyrosine 416 phosphorylated-SFK and total SFK. C) Serum-starved MEFs were exposed to TNF-α (5 ng/ml) ± PP2 (10 µM) for 5 minutes and cell lysates were immunoprecipitated with an SFK-specific antibody. The level of phosphorylated FAK in SFK-specific antibody immunoprecipitates was measured by immunoblots with a phosphotyrosine-specific antibody. The blot was then stripped and reprobed with a FAK-specific antibody to determine the amount of SFK-associated FAK. D) The MEFs were treated and immunoprecipitated with an SFK-specific antibody as in part C. SFK activity was determined using in vitro SFK assay as described in Methods. Results shown were mean and standard deviations from three independent experiments. *, P value<0.05. The results represent the average and standard error from three separate assays. The immunoblots in parts A to C are representative of two independent experiments. “SFK-P” refers to SFK with tyrosine 416 phosphorylated; “FAK-P” refers to FAK with tyrosine residues phosphorylated.

Figure 4. Rescue of TNF-α-activated gene expression by PP2 in Thy-1+ MEFs.

A) Serum-starved MEFs were pre-treated with either DMSO (solvent control) or PP2 (10 µM) for 30 minutes followed by exposure to TNF-α (5 ng/ml) for 24 hours. Total cellular RNA was isolated and quantitative RT-PCR was performed to determine the mRNA levels of MMP-9. The fold change of each mRNA was obtained by correction with the corresponding values of the house keeping gene 36B4 and by setting the values from the control group to one. B) Similar to part A except that the mRNA levels of ICAM-1 was determined by quantitative RT-PCR. C) MEF Thy-1 subsets were treated as in part A for 48 hrs. The conditioned medium was collected from each treatment group and processed for gelatin zymography to determine the expression of MMP-9 in the conditioned medium. “S” refers to the solvent DMSO. The results represent the mean and standard deviation from three independent experiments. *, P value<0.05.

Figure 5. Abrogation of TNF-α-activated gene expression by SFK blockade.

A & B) Serum-starved MEFs were pre-treated with either DMSO (solvent control) or SU6656 (20 µM) for 30 minutes followed by exposure to TNF-α (5 ng/ml) for 24 hours. Total cellular RNA was isolated and subject to quantitative RT-PCR for MMP-9 and ICAM-1 as described in Methods. The fold change of each mRNA was obtained by correction for the corresponding values of the house keeping gene 36B4 and by setting the values from the control group to one. The results were presented in means and standard deviations from three independent experiments. *, P value<0.05. C) The conditioned medium was collected from each treatment group of MEFs as in parts A & B and processed for gelatin zymography as described in Methods. D) Serum-starved MEF were pretreated with SU6656 (20 µM) for 30 minutes followed by exposure to TNF-α for 5 minutes. The exposed cells were then lysed and immunoblotted for tyrosine 416 phosphorylated-SFK, total SFK. The level of phosphorylated FAK in SFK-specific antibody immunoprecipitates was measured by immunoblots using a phosphotyrosine-specific antibody. The blot was then stripped and reprobed with a FAK-specific antibody to determine the amount of SFK-associated FAK. “SFK-P” refers to SFK with tyrosine 416 phosphorylated; “FAK-P” refers to FAK with tyrosine residues phosphorylated. A representative image from at least two independent experiments is presented in parts C and D.

Figure 6. Attenuated TNF-α-activated gene expression by dnFyn in Thy-1– MEF.

The dnFyn expression vector or the backbone vector was transiently transfected into Thy-1– MEF prior exposure to TNF-α. A & B) Serum-starved MEFs were exposed to TNF-α (5 ng/ml) for 24 hours. Total cellular RNA was isolated and subject to quantitative RT-PCR for MMP-9 and ICAM-1, respectively. The fold change of each mRNA was obtained by correction for the corresponding values of the house keeping gene 36B4 and by setting the values from the control group to one. The results are presented as the mean and standard deviation from three independent transfections. *, P value<0.05. C) The conditioned medium was collected from each treatment group and processed for gelatin zymography to determine the expression of MMP-9 in the conditioned medium. A representative image is shown from two independent transfections.

Figure 7. Activation of NF-κB-mediated gene expression by PP2 in Thy-1+ MEFs.

A) MEF Thy-1 subsets were transfected with IL-8-LUC reporter (IL-8-LUCwt) or its variants with either the NF-κB or the AP-1 binding sites mutated (IL-8-LUCmtNF-κB or IL-8-LUCmtAP-1). The reporter activity was examined upon exposure to TNF-α. The ratios of IL-8-LUC variants vs. the cotransfected RL-TK were compared across the groups. B) Similar to part A except that the activity of IL-8-LUC variants was examined in Thy-1+ MEFs exposed to TNF-α ± PP2. C) Similar to part B except that activity of NF-κB-LUC and AP-1-LUC was examined in Thy-1+ MEFs exposed to TNF-α ± PP2. D) Similar to part C except that the experiments were carried out in Thy-1− MEFs. The results are presented in mean and standard deviations obtained from at least three independent transfections preformed in duplicates. * indicates a P value<0.05.