Fig 2: Pathological MAPK activation represses Nfatc1-dependent transcription of lymphatic basement membrane genes.(A) Top consensus lymphatic endothelial motif enriched in phosphorylated-MAPK CUT&Tag DNA fragments isolated from KRASG12D mutant lungs compared with controls. n = 3, P < 1 × 10–9 (known motif), P < 1 × 10–310 (de novo motif). (B and C) CUT&Tag analyses of Nfatc1 showing enrichment at transcriptional start sites (B) and basement membrane genes Col4a1, Nid1, and Lama4 regulatory loci (C) in P12 WT mice. Blue peaks (tracks) represent Nfatc1-occupied DNA sequences. Red boxes represent consensus Nfatc1 binding sites identified by ChIP-Seq (HOMER). (D) Immunofluorescent staining for Col4a (green)/Nid1 (green) or Lama4 (red) expression in intercostal lymphatic vessels marked by Pdpn (green) or Lyve1 (red) in P10 WT mice (n = 3) treated with vehicle (top row) or Nfatc1 inhibitor (bottom row). Scale bar: 10 µm. (E) Immunofluorescent staining of human lymphangiectasia tissue samples (n = 7) show decreased nuclear localization of NFATC1 (red arrows) and reduced LAMA4 expression (yellow arrows) compared with nonpathological areas (white arrows). Scale bar: 10 µm.

Fig 3: NFATc1 inhibition attenuates PIM1-promoted lung fibroblast activation.(A) Normal human lung fibroblasts transduced with control lentivirus (pLV-EGFP-CMV-mCherry) or lentivirus carrying the human PIM1 gene (pLV-EGFP-CMV-hPIM1) for 48 hours, followed by FACS to collect GFP+ cells. Shown is the mRNA expression of PIM1 evaluated by qPCR. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 2-tailed, paired Student’s t test. (B and C) mRNA expression of ACTA2 (B) and COL1A1 (C) in control and PIM1 overexpressing cells treated with 2 ng/mL of TGF-ß for 24 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) Normal human lung fibroblasts transduced with control lentivirus or PIM1 lentivirus were treated with 2 ng/mL of TGF-ß for 24 and then analyzed by Western blotting. Shown is a representative blot of gels run in parallel. (E) Representative images of collagen-I fibers from a collagen deposition assay carried out in control and PIM1-overexpressing cells. (F) Quantification of collagen-I secretion measured as mean fluorescence intensity. n = 12. Data are shown as mean ± SEM. P values were calculated using 2-tailed Student’s t test. (G) Control and PIM1-overexpressing cells were transfected with scrambled or NFATc1 siRNAs for 48 hours, followed by Western blotting analysis. Shown is a representative blot of 3 independent experiments. (H) Schematic showing mechanisms of NFATc1 inhibition by VIVIT and tacrolimus. Following phosphorylation, NFATc1 is sequestered in the cytoplasm. Upon dephosphorylation by calcineurin, NFATc1 translocates into the nucleus, where it becomes transcriptionally active. In the nucleus, PIM1 can phosphorylate NFATc1 to enhance its transcription. (I and J) Western blotting analysis of control and PIM1 overexpressing lung fibroblasts cotreated with 2 ng/mL of TGF-ß and the NFATc1 inhibitors VIVIT (5 µM) and tacrolimus (1 µM). Shown is a representative blot of 3 independent experiments.

Fig 4: Inhibition of PIM1 signaling pathway reduces prosurvival gene expression and sensitizes IPF-derived lung fibroblasts to apoptotic cues.(A) qPCR analysis of aging-associated prosurvival genes and those with NFATc1 binding sites in IPF-derived fibroblasts transfected with scrambled siRNA or siRNA for NFATc1 for 48 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 2-tailed, paired Student’s t test. (B) Western blot analysis of FOXM1 and BIRC5 in IPF-derived fibroblasts transfected with scrambled siRNA or siNFATc1 for 48 hours and then stimulated with 50 ng/mL of PDGF-BB or 10% FBS for an additional 24 hours. Shown is a representative blot of 2 independent experiments. (C) qPCR analysis of prosurvival genes in IPF-derived fibroblasts transfected with scrambled siRNA or siRNA for PIM1 for 48 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) qPCR analysis of prosurvival gene expression in IPF-derived lung fibroblasts cotreated with 10 µM of AZD1208 and 50 ng/mL of PDGF-BB for 24 hours. Data are shown as mean ± SEM of n = 3 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (E) IPF-derived lung fibroblasts were cotreated with 10 µM of AZD1208 and 50 ng/mL of PDGF-BB for 24 hours and analyzed by Western blotting using antibodies against phospho-BAD, total-BAD, BIRC5, and GAPDH. (F) IPF-derived lung fibroblasts were pretreated for 24 hours with 10 µM of AZD1208 and with 300 nM of staurosporine for an additional 2 hours, followed by Western blotting analysis using antibodies against procaspase-3, cleaved caspase-3, and GAPDH. (G) IPF-derived lung fibroblasts were preincubated for 24 hours with 10 µM of AZD1208, followed by treatment with 0.5 µg/mL of FAS-activating antibody for an additional 24 hours. Procaspase-3 and cleaved caspase-3 were detected by Western blotting. Shown is a representative blot of 3 independent experiments.

Fig 5: Pharmacological MAPK inhibition reverses lymphangiectasia in endothelial KRASG12D mutant mice.(A) Schematic of vehicle or ravoxertinib administration to neonatal mice to inhibit MAPK activity in endothelial KrasG12D mutant mice. (B) Kaplan Meier survival plot depicting improved neonatal survival of KrasG12D fl/+; Cdh5CreERT2 mice treated with ravoxertinib compared with vehicle. (C) Immunofluorescent staining of intercostal lymphatic vessels, marked by Lyve1 (red), show reduced nuclear expression of Nfatc1 (red arrows, top row) and increased nuclear expression of phosphorylated-MAPK (red arrows, bottom row) in sections of KrasG12D fl/+; Cdh5CreERT2; R26RmTmG+/– mice compared with controls (white arrows) and MAPK inhibitor–treated (yellow arrows) mice (n = 3). Green arrows show cytoplasmic Nfatc1. Hoechst nuclear counterstain (blue). Scale bar: 10 µm. (D and E) Immunofluorescent staining for Col4a (green, top row), Nid1 (green, middle row), and Lama4 (red, bottom row) expression in intercostal (D) and lung (E) lymphatic vessels, marked by Lyve1 (red, top 2 row) or Pdpn (green, bottom row). Yellow arrows show restoration of expression in ravoxertinib-treated mutant KrasG12D fl/+; Cdh5CreERT2; R26RmTmG+/– mice similar to control mice (white arrows) and in contrast to untreated mutant mice (red arrows) (n = 3). Hoechst nuclear counterstain (blue). Scale bar: 10 µm. L, Lymphatic vessel; B, Bronchus. See also Supplemental Figure 4.