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β 2-agonists are now contraindicated as monotherapy for asthma, and increased PDE4D has been speculated to contribute to this phenomenon. In this study we investigated the expression of PDE4D in asthmatic and nonasthmatic ASM cells and its regulation by formoterol and budes-onide. Primary ASM cells from people with or without asthma were stimulated with transforming growth factor (TGF)- β 1, formoterol, and/or budesonide. PDE4D mRNA was assessed by real-time PCR, or PCR to assess splice variant production. PDE4D protein was assessed by Western blotting, and we investigated the effect of formoterol on cAMP production and PDE activity. Interleukin (IL)-6 was assessed using ELISA. PDE4D mRNA was dose dependently upregulated by formoterol, with a single splice variant, PDE4D5, present. Formoterol did not induce PDE4D protein at time points between 3 to 72 h, whereas it did induce and increase IL-6 secretion. We pretreated cells with actinomycin D and a proteasome inhibitor, MG132, and found no evidence of alterations in mRNA, protein expression, or degradation of PDE4D. Finally PDE activity was not altered by formoterol. This study shows, for the first time, that PDE4D5 is predominantly expressed in human ASM cells from people with and without asthma and that formoterol does not upregulate PDE4D protein production. This leads us to speculate that continual therapy with β 2AR agonists is unlikely to cause PDE4-mediated tachyphylaxis. © 2012 the American Physiological Society.

Transforming growth factor (TGF) ß1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K) is one of the signaling pathways implicated in TGFß1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFß1 induced pro-inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110d mRNA compared to NA and COPD cells; however COPD cells produced more p110d protein. TGFß1 increased 110d mRNA expression to the same extent in the three groups. Neither the p110d inhibitor IC87114 (1, 10, 30?µM), the p110ß inhibitor TGX221 (0.1, 1, 10?µM) nor the PI3K pan inhibitor LY294002 (3, 10?µM) had any effect on basal IL-6, calponin or smooth muscle a-actin (a-SMA) expression. However, TGFß1 increased calponin and a-SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFß1 induced IL-6 release in a dose related manner in all groups of ASM cells. PI3K p110d is important for TGFß1 induced production of the contractile proteins calponin and a-SMA and the proinflammatory cytokine IL-6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling.

Niimi K, Ge Q, Moir LM, Ammit AJ, Trian T, Burgess JK, Black JL, Oliver BG. beta(2)-Agonists upregulate PDE4 mRNA but not protein or activity in human airway smooth muscle cells from asthmatic and nonasthmatic volunteers. Am J Physiol Lung Cell Mol Physi

Transforming growth factor (TGF) beta 1 increases pro-inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3' kinase (PI3K)

Background and Objective: Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired β-agonist induced ASM relaxation in asthmatics, but the mechanism is not known. Objective: To characterize the potential defect in β-agonist induced cAMP in ASM derived from asthmatic in comparison to non-asthmatic subjects and to investigate its mechanism. Methods: We examined β2-adrenergic (β2AR) receptor expression and basal β-agonist and forskolin (direct activator of adenylyl cyclase) stimulated cAMP production in asthmatic cultured ASM (n = 15) and non-asthmatic ASM (n = 22). Based on these results, PDE activity, PDE4D expression and cell proliferation were determined. Results: In the presence of IBMX, a pan PDE inhibitor, asthmatic ASM had ∼50% lower cAMP production in response to isoproterenol, albuterol, formoterol, and forskolin compared to non-asthmatic ASM. However when PDE4 was specifically inhibited, cAMP production by the agonists and forskolin was normalized in asthmatic ASM. We then measured the amount and activity of PDE4, and found ∼2-fold greater expression and activity in asthmatic ASM compared to non-asthmatic ASM. Furthermore, inhibition of PDE4 reduced asthmatic ASM proliferation but not that of non-asthmatic ASM. Conclusion: Decreased β-agonist induced cAMP in ASM from asthmatics results from enhanced degradation due to increased PDE4D expression. Clinical manifestations of this dysregulation would be suboptimal β-agonist-mediated bronchodilation and possibly reduced control over increasing ASM mass. These phenotypes appear to be "hard-wired" into ASM from asthmatics, as they do not require an inflammatory environment in culture to be observed. © 2011 Trian et al.

Background and Objective: Asthma is associated with airway narrowing in response to bronchoconstricting stimuli and increased airway smooth muscle (ASM) mass. In addition, some studies have suggested impaired beta-agonist induced ASM relaxation in asthma