Regulation of myofibroblast differentiation and bleomycin-induced pulmonary fibrosis by adrenomedullin

Abstract

Myofibroblast differentiation induced by transforming growth factor-β (TGF-β) is characterized by the expression of smooth muscle α-actin (SMA) and extracellular matrix proteins. We and others have previously shown that these changes are regulated by protein kinase A (PKA). Adrenomedullin (ADM) is a vasodilator peptide that activates cAMP/PKA signaling through the calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying proteins (RAMP). In this study, we found that recombinant ADM had little effect on cAMP/PKA in quiescent human pulmonary fibroblasts, whereas it induced a profound activation of cAMP/PKA signaling in differentiated (by TGF-β) myofibroblasts. In contrast, the prostacyclin agonist iloprost was equally effective at activating PKA in both quiescent fibroblasts and differentiated myofibroblasts. TGF-β stimulated a profound expression of CRLR with a time course that mirrored the increased PKA responses to ADM. The TGF-β receptor kinase inhibitor SB431542 abolished expression of CRLR and attenuated the PKA responses of cells to ADM but not to iloprost. CRLR expression was also dramatically increased in lungs from bleomycin-treated mice. Functionally, ADM did not affect initial differentiation of quiescent fibroblasts in response to TGF-β but significantly attenuated the expression of SMA, collagen-1, and fibronectin in pre-differentiated myofibroblasts, which was accompanied by decreased contractility of myofibroblasts. Finally, sensitization of ADM signaling by transgenic overexpression of RAMP2 in myofibroblasts resulted in enhanced survival and reduced pulmonary fibrosis in the bleomycin model of the disease. In conclusion, differentiated pulmonary myofibroblasts gain responsiveness to ADM via increased CRLR expression, suggesting the possibility of using ADM for targeting pathological myofibroblasts without affecting normal fibroblasts.

Antifibrotic Effects of Noscapine through Activation of Prostaglandin E2 Receptors and Protein Kinase A

“…We have shown a similar protective effect of adrenomedullin in a mouse model with genetic sensitization of adrenomedullin signaling in myofibroblasts (17). We also have shown recently that myofibroblast differentiation is regulated by microtubule dynamics through a control of actin stress fiber/SRF signaling, independent of Smads (18).…”

“…Hydroxyproline Assay-The hydroxyproline assay was performed as described previously (17). Briefly, right lungs were homogenized in 6 N hydrochloric acid and hydrolyzed for 12 h at 110°C.…”

Regulation of myofibroblast differentiation by cardiac glycosides

“…This may suggest that either the antifibrotic effects of ouabain are independent of COX-2, or COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-␤ by ouabain, but may act together with other mechanisms yet to be identified. Given the previously established antifibrotic role of COX-2 and PKA (8,22,23,33,59), the latter possibility is quite plausible. Fig.…”

“…In vivo studies have shown a protective effect of cAMP-raising agonists such as prostaglandin E 2 (PGE 2 ), prostacyclin analog, iloprost (8,59), or adrenomedullin (11,23) in a bleomycin model of pulmonary fibrosis. However, identification of suitable agonists acting through cAMP/PKA signaling that could be effective in humans remains unaccomplished.…”

Divergent fibroblast growth factor signaling pathways in lung fibroblast subsets: where do we go from here?

“…There is evidence that FGF signaling may regulate Let-7d microRNA expression in the mesenchyme (11), and, considering the recent report that Let-7d regulates lung fibroblast phenotype (21), this FGF/Let-7d pathways seems worth pursuing. Along similar lines, the ability of FGF signaling to drive the action of other newly identified modulators of lung alveolarization or myofibroblast differentiation, including soluble guanylate cyclase (3) and adrenomedullin (22), highlights just some of the other candidate pathways worth exploring.…”