The treatment of IPF remains challenging, and despite intensive research and advances in the field, much remains unknown about the pathogenesis of this devastating fibrosing lung disease
Posted On March 6, 2022
The treatment of IPF remains challenging, and despite intensive research and advances in the field, much remains unknown about the pathogenesis of this devastating fibrosing lung disease. to regulate phosphorylation of Src and focal adhesion kinase (FAK), control invasiveness of synoviocytes, and mediate fibrogenic signaling to TNF, IL-1, and PDGF (83). We previously showed that allele (allele were obtained from the European Conditional Mouse Mutagenesis Program. The targeted allele was generated as FMK a knockout first with an internal ribosome entry site (IRES) LacZ cassette. Targeted embryonic stem cells were microinjected into blastocysts and transferred into pseudopregnant ICR mice. The resulting chimeric mice were crossed with wild-type (WT) C57BL/6 mice, and germline transmission of the targeted allele was confirmed by PCR genotyping. The F1 generation was interbred and genotyped, and mice homozygous for the targeted allele were selected for further breeding. To remove the IRES LacZ cassette, mice were crossed with FlpO transgenic mice (provided by the University of Michigan Transgenic Animal Model Core with the permission of the Mutant Mouse Regional Resource Centers; Ref. 45), leaving loxP sites flanking exon 4 (transgene was removed by breeding. As expected, mice produced PTP protein as determined by immunoblotting of various cell types. To generate cell-type-specific deletion of mice were crossed to (mice were treated intraperitoneally with tamoxifen at a dose of 0.25 mg/g body wt every other day for three doses to induce Cre-mediated excision of exon 3 before the initiation of lung injury. Murine models of pulmonary fibrosis. All mice used were housed in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited pathogen-free facility and treated in compliance with NJH guidelines under an institutional animal care and use committee-approved protocol. values reported were adjusted for multiple testing ( 3 impartial experiments, performed on individual samples at different times. For in vivo experiments, = 8C20 biological replicates per group, performed over multiple individual experiment days. Statistical analyses were performed by one-way analysis of variance (ANOVA) to determine overall differences between groups. A Tukey (post hoc) test was used to confirm differences within groups. values 0.05 were considered to be statistically significant. RESULTS PTP is usually highly expressed in fibroblasts in IPF lungs and in lung fibroblasts in experimental animal models of pulmonary fibrosis. With the use of immunohistochemistry with anti-PTP antibody, we first decided which cells expressed PTP in healthy and IPF human lung tissue. As shown in Fig. 1, PTP was expressed at low levels in epithelial cells and mesenchymal cells in normal human lung tissue (Fig. 1, and and and and in experimental FMK pulmonary fibrosis, we used intratracheal instillation of bleomycin in mice, a widely used animal model of pulmonary fibrosis that replicates some of the features of IPF (1, 12, 57a). This model results in an initial phase of acute lung injury, followed by a fibrotic phase peaking between 14 and 21 days characterized by enhanced TGF- expression and activation and proliferation of myofibroblasts, features that are also observed in IPF (34, 91, 94), thus providing the opportunity to study the role of PTP in these pathophysiological processes. We have FMK previously shown that mice globally deficient in PTP (in lung fibroblasts or alveolar epithelial cells conferred this protection, we generated mice with cell-type-specific deletions of (or for mesenchymal or AT2 cell deletions, respectively). We used Western blotting and genotyping of isolated fibroblasts and AT2 cells to validate cell-type-specific deletion of (Supplemental ZNF35 Fig. S2, and mice created pulmonary fibrosis of an identical magnitude to littermate control mice expressing the WT allele, while dependant on histological and biochemical assays. In Fig. 2msnow, where was erased in mesenchymal cells including lung fibroblasts, are.