Role of MAP kinase activation in bovine tracheal smooth muscle mitogenesis

Abstract

Abnormal growth of airway smooth muscle may play an important role in the pathogenesis of human airway diseases. Little is known about the proliferative responses of cultured airway smooth muscle cells, nor of the precise pathways responsible for mitogenesis in these cells. We assessed DNA synthesis, cell proliferation, and mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes after exposure to four potential mitogens: platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and 5-hydroxytryptamine (5-HT). Stimulation with either PDGF or IGF-1 induced substantial increases in DNA synthesis and cell number, as reflected by [3H]thymidine incorporation, flow cytometry, and methylene blue staining. Treatment with EGF or 5-HT, on the other hand, induced only modest DNA synthesis and no increase in cell number. Immunoblots and kinase renaturation assays of cell extracts demonstrated activation of both the 42- and 44-kDa MAP kinases within minutes of either PDGF, IGF-1, EGF, or 5-HT exposure. However, relative to EGF and 5-HT stimulation, late-phase MAP kinase activation was significantly greater after treatment with the mitogens PDGF and IGF-1. We conclude that in cultured bovine tracheal myocytes 1) PDGF and IGF-1 are potent mitogens; 2) MAP kinase may be activated subsequent to stimulation of either receptor tyrosine kinases (PDGF, EGF, IGF-1) or G protein-linked receptors lacking in known tyrosine kinase activity (5-HT); and 3) unsustained MAP kinase activation is insufficient for mitogenesis. Finally, the finding that mitogenicity correlates with the late phase of MAP kinase activation is consistent with the notion that sustained MAP kinase activation is important for bovine tracheal myocyte proliferation.

Phenotypic diversity and molecular mechanisms of airway smooth muscle proliferation in asthma

“…However, no effect of salbutamol was found on cyclin D 1 mRNA, suggesting a post-transcriptional effect on cyclin D 1 , perhaps involving accelerated degradation of the cyclin D 1 protein [159]. In contrast to the lack of effect of glucocorticoids on ERK activity [155], salbutamol prevented ERK activation between 5 min and 8 h following stimulation with a-thrombin, consistent with earlier reports that sustained ERK activation throughout G 1 is necessary for cell cycle traversal [74,75,78]. An additional mechanism that was not investigated in this study, but has been reported previously by these authors [160], was the salbutamol-induced expression of p27 kip1 , a cdk protein inhibitor which prevents phosphorylation of pRb and cell cycle progression by inhibiting cdk activity in the mitogen-activated cyclin D 1 /cdk4 complex.…”

“…Interestingly, S-phase entry and proliferation, as determined by BrdU labelling or 3 H-thymidine incorporation, could not be induced in these cells by hydrogen peroxide [79]. This may reflect a requirement for sustained ERK activation in proliferation [74,75,78], or that activation of the p38MAPK or SAPK/JNK pathway by hydrogen peroxide in these cells [79] may in some way counter the effect of ERK activation.…”

“…Although not easily explained at first, it was known that the modest proliferation of airway smooth muscle in response to ET-1 was mediated by G protein-coupled ET A [55] and ET B receptors [56,82] (depending on the species investigated), and that this was dependent upon activation of the ERK pathway. Likewise, proliferation induced by 5-HT was also shown to be dependent on ERK activation [50,74]. [76] showed that inhibiting the kinase activity of Raf-1 with forskolin prevented ERK activation by ET-1, but not by PDGF, suggesting that unlike RTK-linked receptors, G protein-coupled receptors have a requirement for Raf-1 in ERK activation.…”

“…MEK-1 activates both p44 ERK1 and p42 ERK2 by direct phosphorylation of tyrosine and threonine residues. In bovine airway smooth muscle, ERK activation is induced by a wide variety of stimuli, including PDGF, EGF, IGF-1, 5-HT [50,74], bradykinin [75], a-thrombin [75,76], ET-1 [56,76] and hydrogen peroxide [77]. Several independent groups have reported that the duration of ERK activation is critical for transduction of the mitogenic stimulus.…”

“…Several independent groups have reported that the duration of ERK activation is critical for transduction of the mitogenic stimulus. KELLEHER et al [74] and MALARKEY et al [75] observed that, in bovine airway smooth muscle cells, ERK activation was of significantly longer duration following stimulation with mitogens (PDGF and ET-1) than following treatment with nonmitogens (bradykinin). A similar finding of sustained ERK activation by PDGF was reported in guinea-pig airway smooth muscle cells by PYNE et al [78]; whereas ERK activation by bradykinin was again more transient.…”