Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli

Normal primary human fetal (IMR-90) lung fibroblasts were obtained from ATCC (Manassas, VA). Cells between passages 8 and 16 were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum (FBS) to 60% confluence and growth arrested for 16–24 h prior to treatment. Fibroblasts were cultured on tissue culture plastic or on polyacrylamide hydrogel substrates with stiffness of either 400 Pa or 6400 Pa. The polyacrylamide hydrogels were prepared in the Tschumperlin laboratory and functionalized with type 1 collagen as previously described [23].

Porcine TGF-β1, recombinant human tumor necrosis factor alpha (TNF-α) and recombinant human interferon gamma (IFN-γ) were from R&D Systems (Minneapolis, MN). Cycloheximide was from Sigma (St. Louis, MO). The activating anti-Fas antibody (clone CH11, designated as Fas-Ab) was from Millipore (Upstate, NY). PF573228, which directly blocks FAK kinase activity without significantly inhibiting PI3K or Rho-kinase (ROCK) was from Tocris Bioscience (Ellisville, MO) [24]. LY294002, (a PI3K inhibitor) was from Cell Signaling Technology (Danvers, MA). Rabbit monoclonal antibodies to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and poly-(ADP-ribose) polymerase (PARP) were from Cell Signaling (Danvers, MA; 1:1000 dilution used). The rabbit polyclonal antibody to Fas used for Western blots was from Novus Biologicals (Littleton, CO; 1:1000 dilution). The Cell Death Detection ELISA Kit detecting histone-associated DNA fragments was from Roche Applied Science (Indianapolis, IN). Horseradish peroxidase–conjugated secondary antibodies were from Pierce (Rockford, IL).

Whole cell lysates were subjected to SDS-PAGE and Western blotting as previously described [24]. All Western blots were stripped and re-probed for GAPDH. Band densities were analyzed using the public domain NIH ImageJ program version 1.50i available at http://​rsbweb.​nih.​gov/​ij. The ratio of the band density for the target protein and for the corresponding GAPDH was determined and indexed such that the average expression in untreated controls was 1.0 and differences in expression represent “fold change”.

Quantitative real-time RT-PCR was performed using StepOne Plus Real Time PCR Systems by Applied Biosystems (Foster City, CA). Relative quantitation was based on the ΔΔC_T method. We used the human Fas cell surface death receptor, FAS gene (Hs00236330_m1) primer probe set and the housekeeping gene ACTB (Hs99999903_m1) from ThermoFisher Scientific (Ann Arbor, MI).

Cells were collected by scraping and incubated with Fc Block (1:100) clone 24G2 (BD Pharmingen, San Diego, CA) for 15 min, then stained with propidium iodide, FITC Mouse Anti Human CD95 (Clone DX2) or FITC Mouse IgG1 Isotype Control, (Clone MOPC-21) antibodies (BD Pharmingen) for 30 min. Cells were run on a FACS Canto (BD Biosciences, Mountain View, CA) and further analyzed using the Flowjo single cell software analysis software.

A 96-well MaxiSorp Immuno Plate (ThermoFischer Scientific, Ann Arbor, MI) was coated with 2 μg/ml Fas capture antibody (MAB144) from R&D Systems (Minneapolis, MN) and incubated overnight at 4 degrees Celsius. The coated plate was washed and blocked with casein in PBS for 1 h. After washing, samples and recombinant standards (326-FS-050 from R&D Systems, Minneapolis, MN) were loaded and incubated at room temperature for 2. After washing, the plate was incubated with 0.1 μg/ml Fas detection/biotinylated antibody (BAF 326, R&D Systems, Minneapolis, MN) for one hour and washed. Substrate development was done with peroxidase–conjugated streptavidin solution (Jackson ImmunoReserach Labs Inc., West Grove, PA) for 30 min. The plate was washed and color developed with 3, 3′, 5, 5’-Tetramethylbenzidine in 0.1 M acetate buffer with hydrogen peroxide (all from Sigma) for 20 min. Reaction was stopped with sulfuric acid. Absorbance was read at 450 nm. Data was analyzed SpectraMax M3 SofMax Pro 5 (Molecular Devices, Sunnyvale, CA).

20,000 fibroblasts per well were seeded on polystyrene glass slides (Falcon Brand) and cultured for 24 h prior to growth arrest in serum-free media for another 24 h prior to treatment with/without TGF-β1 (10 ng/ml) for 24 h. Cells were fixed (without permeabilization) in 4% paraformaldehyde for 30 min and non-specific binding was blocked with 3% bovine serum albumin for 1 h prior to sequential incubation with rabbit polyclonal anti-Fas antibody (Novus Biologicals catalog # NBP1–89034) at 1:50 dilution for 16 h (4 °C) and then a FITC-labeled secondary antibody (Invitrogen) at a 1:200 dilution for 1 h (room temperature). Nuclei were stained with DAPI. Fluorescence intensity was measured with NIH Image J and quantified as the “corrected total cell fluorescence” as previously reported [25].

Statistical analysis was done using GraphPad Prism version 6.01. Comparisons for experiments with more than two conditions were made using one-way ANOVA with Tukey’s multiple comparisons test. Comparisons between two cohorts were done using an unpaired t-test. Data shown are the mean ± standard error of the mean. Each figure legends report the numbers of independent experimental replicates represented in each figure.

Biochemical and biomechanical properties of the extracellular matrix significantly impact fibroblast phenotypes including basal rates of apoptosis [26]. To determine whether substrate stiffness impacted fibroblast susceptibility to apoptosis triggered by activation of the extrinsic pathway, normal lung fibroblasts cultured on polyacrylamide hydrogels with the compliance of normal lung parenchyma (400 Pa) or on hydrogels that recapitulate the stiffness of fibrotic lung tissue (6400 Pa) were treated with/without a Fas-activating antibody (Fas-Ab, 250 ng/ml) [23]. Supporting a key role for increased matrix stiffness supporting an apoptosis-resistant fibroblast phenotype, cells cultured on the compliant (400 Pa) substrates were more susceptible to Fas-induced apoptosis than those cultured on the more rigid (6400 Pa) substrates (Fig. 1a). We observed no significant differences in susceptibility to Fas-mediated apoptosis with further increases in substrate stiffness, including glass substrates (Additional file 1: Figure S1).

Others have shown that fibroblast susceptibility to Fas-ligand induced apoptosis can be regulated by enhancing cell surface Fas expression and that apoptosis-resistant fibroblasts from patients with fibrotic lung disease have decreased cell-surface Fas expression [14, 19]. To determine the effect of substrate stiffness on Fas expression, normal lung fibroblasts were seeded in serum-free media on 400 or 6400 Pa substrates for 24 h. Both Fas mRNA and protein were significantly decreased in cells on the 6400 Pa substrate (Fig. 1b and c).

TGF-β1 is a pro-fibrotic cytokine that promotes fibroblast resistance to apoptosis and is activated, in part, through non-proteolytic mechanisms that are dependent on substrate stiffness [27]. Consistent with a necessary role for increased substrate stiffness, we found that TGF-β had no significant effect on Fas expression when fibroblasts were cultured on compliant (400 Pa) polyacrylamide hydrogels (Additional file 1: Figure S2). To evaluate the effect of TGF-β1 on fibroblast Fas expression on stiff substrates, we measured Fas mRNA and protein in response to different doses and durations of TGF-β1 treatment (Fig. 2). First, we found that low-dose TGF-β1 (2 ng/ml) failed to significantly impact Fas transcription over 48 h, but a higher dose (10 ng/ml) that is commonly used to study fibroblast behavior [28, 29] induced a time-dependent decrease in Fas mRNA that was significant at 24 h and persisted at 48 h (Fig. 2a and b). Using Western blot, we observed a small but significant decline in Fas over 24 h with the 2 ng/ml dose and a more pronounced decline at 24 h following treatment with 10 ng/ml (Fig. 2c and d). To improve the sensitivity of our assessment, we also measured total Fas in whole cell lysates (“cellular Fas”) using ELISA (Fig. 2e and h). With this method, we did detect a statistically significant decline in Fas with 2 ng/ml which was accentuated with the 10 ng/ml dose of TGF-β1. Finally, we confirmed the dose-response relationship by assessing Fas in fibroblasts treated with 2, 5 or 10 ng/ml of TGF-β1 for 24 h via Western blot (Fig. 2g) and ELISA (Fig. 2h). Based on the clear and consistent suppression of Fas established in these experiments, additional studies were done using TGF-β1 at a dose of 10 ng/ml.

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that is critical in mechanotransduction signaling, is activated by TGF-β1, mediates myofibroblast differentiation, promotes myofibroblast resistance to apoptosis, and is necessary for lung fibrogenesis [30‐33]. Having found that both TGF-β1 and stiff ECM substrates suppress Fas expression in fibroblasts, we next examined the role of FAK in this process. In fibroblasts treated with the FAK inhibitor PF573228 [24], TGF-β1 failed to suppress Fas mRNA expression (Fig. 3a). In contrast, inhibition of the pro-survival PI3K/AKT signaling pathway with LY294002 had no impact on TGF-β1-mediated suppression of Fas transcription (Fig. 3a). Consistently, FAK inhibition mildly increased basal Fas expression and prevented the TGF-β-mediated reduction in Fas protein (Fig. 3b). Taken together, these experiments demonstrate that FAK activation has a critical role in TGF-β1 stimulated suppression of Fas in normal fibroblasts.

Receptor expression represents the balance of protein synthesis and degradation. However, Fas is a member of the TNF-alpha superfamily, and studies have shown that these receptors, including Fas itself, can be secreted from cells as a biologically active soluble receptor [34]. Moreover, increased concentrations of soluble Fas (sFas) have been reported in the cell culture supernatants of fibrotic lung fibroblasts compared to normal lung fibroblasts [14]. In concert with proteins known to be secreted by fibroblasts, such as TIMP1 and TIMP2, we identified sFas in cell culture supernatants from normal lung fibroblasts treated with TGF-β1 for 24 h (Fig. 4a). Using ELISA, we confirmed that TGF-β1 treatment led to a significant increase in sFas in fibroblast conditioned media between 8 and 24 h, and that there was a dose-response effect to TGF-β1 (Fig. 4b). To determine if the sFas in conditioned media represented the consequence of shedding/cleavage of cell-surface receptors or if it was due to the secretion of existing cytoplasmic Fas, we pre-treated cells for one hour with Brefeldin-A, which inhibits protein transport from the endoplasmic reticulum to the Golgi and thereby blocks the protein secretory pathway. Supporting a role for the secretory pathway, TGF-β1 failed to increase the sFas in the cell culture supernatants when cells were treated with Brefeldin-A (Fig. 4c). Consistent with secretion of Fas as the primary mechanism underlying decreased fibroblast Fas, we used flow-cytometry to demonstrate that TGF-β1 did not significantly alter cell-surface Fas expression at 24 h (Fig. 4d). Similarly, there was no significant difference observed in cell-surface Fas expression when assessed by immunofluorescence staining (Fig. 4e and f).

Fibroblast susceptibility to Fas-induced apoptosis is enhanced by increased cell surface Fas expression induced by combined treatment with TNF-α and IFN-γ [15, 19]. Prior studies from our laboratory have shown that TGF-β1 reduces fibroblast susceptibility to Fas-mediated apoptosis through activation of pro-survival protein kinase signaling pathways and up-regulation of intracellular inhibitors of apoptosis [4, 21, 35]. Having now shown that TGF-β also reduces fibroblast expression of Fas we sought to determine whether the anti-apoptotic effects of TGF-β1 would counter the effects of TNF-α and IFN-γ and reverse the enhanced sensitivity to apoptosis sensitivity afforded by these inflammatory cytokines. To address this, we treated normal lung fibroblasts with the combination of TNF-α and IFN-γ for between 0 and 48 h (cells treated for less than 48 h were washed with PBS and maintained in serum-free media for the remaining time until completion of the 48-h period). After 48 h, the cells were washed and treated with or without TGF-β1 for an additional 24 h, so that all of the cells were in culture for the 72-h duration of the experiment. Consistent with prior studies, fibroblasts exposed to the inflammatory cytokines for at least 8 h showed enhanced Fas expression which peaked at 24 h and persisted in the absence of TGF-β1 for the entire 72-h duration of the experiment (Fig. 5a) [15]. Interestingly, the cells treated with the inflammatory cytokines for 24 or 48 h followed by TGF-β1 for 24 h had no significant decline in cellular Fas. Next, we treated fibroblasts with the combination of inflammatory cytokines for 24 h prior to exposure to Fas-activating antibody alone or in combination with TGF-β1 and assessed apoptosis by Western blot for identification of cleaved PARP. The combination of cytokines alone was not sufficient to induce apoptosis, but apoptosis was apparent in cells treated with the combination of cytokines and Fas-activating antibody. Consistent with cellular Fas-expression impacting susceptibility to apoptosis, TGF-β1 failed to decrease the susceptibility to apoptosis in the cells that had been pre-treated with inflammatory cytokines (Fig. 5b). Collectively, these findings indicate that TGF-β1 does not antagonize the increase in cell surface Fas induced by inflammatory cytokines. Moreover, the increased susceptibility to Fas-induced apoptosis afforded by treatment with the inflammatory cytokines is not altered by TGF-β1, suggesting that increasing cell-surface expression of Fas is sufficient to overcome the non-Fas mediated anti-apoptotic mechanisms induced by TGF-β1.