cAMP-elevating agents and adenylyl cyclase overexpression promote an antifibrotic phenotype in pulmonary fibroblasts

Abstract

Pulmonary fibroblasts are recruited to sites of lung injury, where they are activated to produce extracellular matrix proteins and to facilitate repair. However, these cells become dysregulated in pulmonary fibrosis, producing excess collagen at sites of injury and forming fibrotic loci that impair lung function. In this study, we used WI-38 human lung fibroblasts and evaluated the ability of G protein-coupled receptor agonists to increase cAMP production and regulate cell proliferation and collagen synthesis. WI-38 cells increase cAMP in response to the beta-adrenergic agonist isoproterenol (Iso), prostaglandin E(2) (PGE(2)), certain prostanoid receptor-selective agonists (beraprost, butaprost), an adenosine receptor agonist, and the direct adenylyl cyclase (AC) activator forskolin (Fsk). Responses to Iso, PGE(2), and Fsk were studied in more detail. Each induced a dose-dependent inhibition of serum-stimulated cell proliferation (as measured by [(3)H]thymidine incorporation) and collagen synthesis (as measured by [(3)H]proline incorporation, collagenase-sensitive [(3)H]proline incorporation, or levels of procollagen type 1 C-peptide). Quantitative RT-PCR analyses indicated that elevation in cellular cAMP levels decreases expression of collagen types 1alpha(II) and 5alpha(I) and increases expression and activity of matrix metalloproteinase 2 (MMP-2). Overexpression of AC type 6 or inhibition of cyclic nucleotide phosphodiesterases also increased cellular cAMP levels and decreased cell proliferation and collagen synthesis. Thus multiple approaches that increase cAMP signaling reduce proliferation and differentiated function in human pulmonary fibroblasts. These results suggest that therapies that raise cAMP levels may prove useful in the treatment of pulmonary fibrosis.

Hyaluronan Fragments Promote Inflammation by Down-Regulating the Anti-Inflammatory A2a Receptor.

“…In addition, IL-13 transgenic mice, which have increased A1, A2b, and A3, but not the anti-inflammatory A2aR, have recently been shown to have high levels of lung adenosine and increased lung inflammation (39). These data are consistent with the previously described proinflammatory role of A2bR in adenosinedependent lung inflammation via stimulation of fibroblast proliferation, matrixmetallo proteinase (MMP)-2 activity, and collagen deposition (40,41). Interestingly, whereas A2b has been shown to have proinflammatory activity in airway epithelial cells, A2aR stimulation in lung epithelial cells has been shown to promote wound healing (42,43).…”

Inhibition of cardiac myofibroblast formation and collagen synthesis by activation and overexpression of adenylyl cyclase

“…CFs were separated from cardiac myocytes by gravity separation and grown to confluency on 10-cm cell culture dishes at 37°C with 90% air͞10% CO 2 in growth media (DMEM͞10% FBS͞1% penicillin͞1% streptomycin). For overexpression studies, CFs were incubated with Ϸ100 viral particles per cell of an adenovirus containing either lacZ (control) or the murine AC6 gene as described (17,19). Cells were incubated with adenovirus for 18-24 h before stimulation with serum or agonists of interest.…”

“…Assays were terminated by aspiration of media and addition of 250 l of ice-cold trichloroacetic acid (7.5%) to each well. RIA was used to quantitate cAMP (14,17); results were normalized to the amount of protein per sample, as determined by a colorimetric protein assay (Bio-Rad).…”

“…G protein-coupled receptor (GPCR) agonists that signal through G s to activate AC and stimulate cAMP production can inhibit collagen synthesis (13)(14)(15). Forskolin and GPCR agonists that stimulate AC were recently shown to inhibit serum or TGF-␤-stimulated ␣-SMA expression and collagen formation by human lung fibroblasts (16,17). Although such data suggest that increased cAMP production inhibits collagen synthesis via an inhibition of fibroblast-to-myofibroblast transformation, no direct evidence has linked the two events and no studies have examined the effects of increased cAMP on myofibroblast formation in the heart.…”

The β2-adrenergic receptor activates pro-migratory and pro-proliferative pathways in dermal fibroblasts via divergent mechanisms

“…conversely, decrease (Liu et al, 2004) cell proliferation, depending on the cell type studied. Thus it was important to determine what effect ␤2-AR activation would have on human dermal fibroblast proliferation.…”