MAPK phosphatase 1 inhibition of p38α within lung myofibroblasts is essential for spontaneous fibrosis resolution

Fibrosis following tissue injury is distinguished from normal repair by the accumulation of pathogenic and apoptosis-resistant myofibroblasts (MFs), which arise primarily by differentiation from resident fibroblasts. Endogenous molecular brakes that promote MF dedifferentiation and clearance during spontaneous resolution of experimental lung fibrosis may provide insights that could inform and improve the treatment of progressive pulmonary fibrosis in patients. MAPK phosphatase 1 (MKP1) influences the cellular phenotype and fate through precise and timely regulation of MAPK activity within various cell types and tissues, yet its role in lung fibroblasts and pulmonary fibrosis has not been explored. Using gain- and loss-of-function studies, we found that MKP1 promoted lung MF dedifferentiation and restored the sensitivity of these cells to apoptosis — effects determined to be mainly dependent on MKP1’s dephosphorylation of p38α MAPK (p38α). Fibroblast-specific deletion of MKP1 following peak bleomycin-induced lung fibrosis largely abrogated its subsequent spontaneous resolution. Such resolution was restored by treating these transgenic mice with the p38α inhibitor VX-702. We conclude that MKP1 is a critical antifibrotic brake whose inhibition of pathogenic p38α in lung fibroblasts is necessary for fibrosis resolution following lung injury.

Cell culture.CCL210 normal adult and MRC5 normal fetal HLFs were obtained from the American Type Culture Collection.MRC5 HLFs were utilized for all gain-and loss-of-function studies while CCL210s were used for all other in vitro studies involving normal HLFs unless otherwise specified.
Primary IPF fibroblast lines and normal patient-derived HLFs were obtained from our institutional biorepository as described previously (1).All cells were initially cultured in low glucose DMEM (Invitrogen) supplemented with 10% FBS (Hyclone), 100 units/mL penicillin, and 100 μg/mL streptomycin (both from Invitrogen).Cells were then serum starved in FBS-free DMEM overnight, and differentiation to MFs was induced by treatment with TGFβ for 48 h.TGFβ-elicited MFs were then treated for specified time points as described followed by harvesting.Fibroblasts from lung tissue of saline-or bleomycin-treated mouse lungs were obtained during lung harvest at the indicated times as previously described (2).For all in vitro experiments, a minimum of three independent experiments were performed, and the results are presented as mean ± SEM.
qPCR.Analysis of transcript expression was performed by extracting total cellular RNA using an RNeasy kit (Qiagen).cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), amplified with Fast SYBR Green Master Mix, and analyzed on a StepOne real time PCR system (Applied Biosystems).Fold changes were normalized to the expression levels of the housekeeping gene GAPDH.Primer pair sequences used for qPCR are listed in Supplemental Table 1.
Apoptosis assays.Apoptosis was determined either by measuring caspase 3/7 activity, extracellular Annexin V expression, or by TUNEL staining of fixed lung sections (Roche, 11684795910).For caspase 3/7 and Annexin V readouts, fibroblasts or MFs were treated with 100 ng/mL of anti-Fas activating antibody (EMD Biosciences, CH11 05-201) overnight to induce apoptosis.Caspase 3/7 activity was measured using the luciferase Caspase-Glo 3/7 kit (Promega, G8091) per the manufacturer's protocol (3).Annexin V+ cells were quantified using flow cytometry (BD Fortessa flow cytometer).For MKP1 overexpression in which Annexin V was quantified by flow cytometry, a vehicle control was used in lieu of GFP overexpression to avoid fluorescence interference.Briefly, fibroblasts or MFs were treated with the indicated therapies followed by exposure to anti-Fas (100 ng/mL) for 24 h.Cells were then lifted with 0.25% trypsin, pelleted in FACS tubes, and stained with AlexaFluor 488 conjugated Annexin V antibody (Invitrogen, V13241) per the manufacturer's protocol (4).TUNEL staining of mouse lung tissue was achieved using 10 μg/mL proteinase K digestion at 37 o C for 30 minutes followed by signal development per the manufacturer's protocol.
Immunofluorescence microscopy and immunohistochemistry. CCL210 or MRC5 fibroblasts were plated and cultured in single-chamber slides and serum starved overnight.Fibroblast differentiation into MFs was achieved by addition of TGFβ at 2 ng/mL for 48 h and treatment with either vehicle or the indicated therapies to elicit MF dedifferentiation.Chamber slides were then washed twice with chilled PBS, fixed with freshly prepared 4% formaldehyde for 20 min, washed with PBS, and quenched with 100 mM glycine for 15 min.Blocking and permeabilization were achieved by incubating the slides for 1 h in PBS containing 10% FBS and 0.1% Triton X-100 (Sigma-Aldrich).
Mouse lungs were fixed with 10% formalin, embedded in paraffin, sectioned, and mounted on glass slides.Subsequent deparaffinization, antigen retrieval blocking and staining was performed as previously described (5).Staining of fixed fibroblasts and murine lung tissue was performed with the following primary antibodies: anti-αSMA-FITC (1:500; F3777, Sigma-Aldrich) or anti-αSMA-Cy3