Abstract
Pulmonary 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)2 and EP4 receptors. Although EP receptors are attractive therapeutic targets, the effects of long-term exposure to PGE2 have 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 PGE2 or other cAMP-elevating agents. cAMP levels stimulated by acute exposure to PGE2 were measured using a fluorescent biosensor. Pretreatment for 24 h with PGE2 shifted the concentration-response curve to PGE2 rightward by approximately 22-fold but did not affect responses to the beta-adrenoceptor agonist isoproterenol. Neither isoproterenol nor forskolin pretreatment altered PGE2 responses, 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 EP2 receptors. EP2 receptors are resistant to classical mechanisms of agonist-specific receptor desensitization, so we hypothesized that increased PDE activity mediates the loss of signaling after PGE2 pretreatment. PGE2 treatment 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 PGE2 causes agonist-specific desensitization of EP2 receptor-stimulated cAMP signaling through the increased expression of PDE isozymes, most likely of the PDE4 family.
Highlights
Pulmonary fibrosis (PF) is characterized by continuous scarring of the lungs (Dackor et al 2011)
Some studies demonstrate diminished COX-2 expression in lung fibroblasts cultured from patients with PF, which would lead to reduced prostaglandin E2 (PGE2) synthesis (Wilborn et al 1995)
It is possible that PGE2 loses its antifibrotic action following long-term exposure due to attenuated cAMP signaling in lung fibroblasts (Michalski et al 2012)
Summary
Pulmonary fibrosis (PF) is characterized by continuous scarring of the lungs (Dackor et al 2011). The second messenger molecule cyclic 3′,5′-adenosine monophosphate (cAMP) will (1) slow fibroblast migration to the site of injury, (2) prevent differentiation of fibroblasts into myofibroblasts, (3) reduce the amount of collagen 1α(II) and 5α(I), and (4) increase the activity of certain matrix metalloproteinases, which are responsible for degrading ECM proteins (Yamaguchi et al 1988; Kohyama et al 2002a; Kolodsick et al 2003; Liu et al 2004). CAMP is generated in the cell when certain agonists bind to their cognate G protein–coupled receptors (GPCRs) to activate the G protein Gαs, which, in turn, stimulates adenylyl cyclase (AC) (Liu et al 2004). Gα has several different isoforms, only Gαs is responsible for stimulating cAMP production in the cell, and the GαS-AC-cAMP pathway is pertinent to the study of cAMP and its antifibrotic effects (Liu et al 2004)
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