Agonist-specific desensitization of PGE2-stimulated cAMP signaling due to upregulated phosphodiesterase expression in human lung fibroblasts

Abstract

AbstractPulmonary fibrosis is characterized by fibroblasts persisting in an activated form, producing excessive fibrous material that destroys alveolar structure. The second messenger molecule cyclic 3′,5′-adenosine monophosphate (cAMP) has antifibrotic properties, and prostaglandin E2(PGE2) can stimulate cAMP production through prostaglandin E (EP)2and EP4receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2have not been characterized. To determine the effects of long-term exposure of lung fibroblasts to PGE2, human fetal lung (HFL)-1 cells were treated for 24 h with 100 nM PGE2or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2shifted the concentration-response curve to PGE2rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2responses, implying that other cAMP-elevating agents do not induce desensitization. Use of EP2- and EP4-selective agonists and antagonists suggested that PGE2-stimulated cAMP responses in HFL-1 cells are mediated by EP2receptors. EP2receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2pretreatment. PGE2treatment upregulated messenger RNA for PDE3A, PDE3B, PDE4B, and PDE4D and increased overall PDE activity. The PDE4 inhibitor rolipram partially reversed PGE2-mediated desensitization and PDE4 activity was increased, but rolipram did not alter responses to isoproterenol. The PDE3 inhibitor cilostazol had minimal effect. These results show that long-term exposure to PGE2causes agonist-specific desensitization of EP2receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.