Desensitization of PGE2‐stimulated cAMP Signaling Due to Upregulated Phosphodiesterase Activity in Human Lung Fibroblasts

Abstract

Pulmonary fibroblasts play an essential role in maintaining the structure and function of the lung. In idiopathic pulmonary fibrosis, fibroblast cells persist in an activated form and produce excessive fibrous material, leading to loss of alveolar structure. PGE2, which specifically activates EP2 and EP4 receptor subtypes, is a cAMP elevating agent and thus has anti‐fibrotic potential, since cAMP has been shown to have anti‐fibrotic properties. Although PGE2 levels are increased in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis (and in animal models of pulmonary fibrosis), it does not prevent pulmonary fibrosis. One hypothesis is that PGE2 effects are self‐limiting because of desensitization caused by prolonged exposure. To test this hypothesis, we pretreated HFL‐1 fibroblasts with PGE2 and other cAMP‐elevating agents then measured PGE2‐stimulated cAMP levels using the downward cAMP difference detector in situ (cADDis) cAMP sensor (Montana Molecular, Bozeman MT). After 24‐hour pretreatment with 100 nM PGE2, we changed the media and measured cAMP kinetics in response to various concentrations of PGE2. Pretreatment with PGE2 shifted the concentration‐response curve of PGE2 rightward approximately 50‐fold. Neither 100 nM isoproterenol or 1μM forskolin pretreatment for 24 hours altered PGE2 response, implying that other cAMP elevating agents do not induce desensitization. We sought to uncover the underlying mechanism responsible for PGE2 desensitization. Desensitization could result from either receptor internalization through the GRK/β‐arrestin mediated pathway or via increase PDE activity. To determine if PGE2 pretreatment increases PDE activity, we used the PDE3 inhibitor, cilostazol, and the PDE4 inhibitor, rolipram after PGE2 pretreatment. Rolipram, increased both the EC50 and Emax of PGE2 in vehicle pretreated HFL‐1 fibroblasts. In cells pretreated with PGE2, rolipram partially reversed the desensitization, indicating that PDE4 may be induced by long term PGE2 exposure. Cilostazol had minimal effects on either control or desensitized PGE2 responses, implying that PDE3 is not involved. Examination of PDE isoform mRNA levels via quantitative RT‐PCR, show that PGE2 treatment upregulates PDE3A, PDE4C and PDE4D, while pretreatment with isoproterenol or forskolin only upregulated PDE4D. Taken together, these results show that long‐term exposure to PGE2 causes desensitization of EP receptor‐stimulated cAMP signaling, partly through the increased expression of a PDE4 isoform. Defining the precise PDE isoform involved in the desensitization of PGE2 responses may lead to new therapeutic strategies for treating pulmonary fibrosis.Support or Funding InformationThis work was supported by NIH grant GM107094 (RO)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.