C71-29 The Cyclic Amp/pka Axis Regulates Processing Body (p-body) Dynamics Within Lung Myofibroblasts

Rationale

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease with no cure. The ultimate effector cell of fibrosis is the activated fibroblast/myofibroblast which drives scarring and stiffness characteristic of IPF. Phenotypic de-differentiation of myofibroblasts into quiescent fibroblasts and subsequent clearance of these cells is thought to be necessary for fibrosis resolution. Myofibroblast de-differentiation involves rapid alteration in the expression of thousands of genes, but the mechanisms underlying these complex and timely changes are not well understood. Processing bodies (P-bodies) - cytoplasmic membraneless organelles that form through liquid-liquid phase transition - regulate gene programs by storing mRNA pools for degradation and/or translation. P-bodies therefore mediate post-transcriptional regulation, an understudied process in lung fibroblasts and pulmonary fibrosis. Although the structure of P-bodies has been characterized in yeast, their presence and influence on lung fibroblast/myofibroblast function and phenotype is entirely uncharacterized. In this study, we sought to determine the effect of increasing intracellular cyclic AMP (cAMP) - known to promote myofibroblast de-differentiation - on P-body dynamics in lung fibroblasts/myofibroblasts.

Methods

P-bodies were labeled using a GFP tag to Dcp1a - an mRNA decapping enzyme involved in P-body formation. MRC5 fibroblasts were infected with a lentivirus containing a Dcp1a-GFP plasmid construct designed to promote constitutive expression of Dcp1a-GFP in live cells. MRC5 fibroblasts and myofibroblasts (generated by treatment of fibroblasts with TGFβ - 2 ng/mL) expressing Dcp1a-GPF were treated with cAMP activators/mimics or agonists of its downstream effectors: PGE2, forskolin, dibutyryl cAMP, 6BNZ-cAMP (PKA agonist), and 8pCPT-cAMP (Epac agonist) at various time points (4 - 24 h) and imaged by immunofluorescence microscopy. P-body number, size, and intensity were subsequently measured and quantified using ImageJ.

Results

P-bodies were successfully visualized in live Dcp1a-GFP fibroblasts and myofibroblasts. In each, P-body numbers increased by nearly 10-fold 24 h following treatment with PGE2 and forskolin. The same effect was observed following treatment with 6BNZ-cAMP, but not 8pCPT-cAMP. Additionally, there was a significant reduction in the average size (but increased overall volume) of P-bodies 24 h post-treatment with PGE2, forskolin, and 6BNZ-cAMP.

Conclusions

This is the first description, to our knowledge, of P-bodies in lung fibroblasts/myofibroblasts. Our data demonstrate that the cAMP/PKA axis robustly increases P-body number and combined volume suggesting an important role for these organelles in myofibroblast de-differentiation. Additional work in our lab is ongoing to further characterize the role of P-bodies in regulating fibrotic gene programs within lung myofibroblasts using CRISPR/Cas9-specific perturbation of P-body structure.

This abstract is funded by: K08 HL163178