Combined anti-inflammatory effects of β2-adrenergic agonists and PDE4 inhibitors on astrocytes by upregulation of intracellular cAMP
2011-11, Christiansen, Søren Hofman, Selige, Jens, Dunkern, Torsten, Rassov, Andreas, Leist, Marcel
Inflammation is an important hallmark of all neurodegenerative diseases and activation of different glial populations may be involved in the progression of some of these disorders. Especially, the activation of astroglia can lead to long-term detrimental morphological changes, such as scar formation. Therefore, improved strategies to modulate inflammation in these cells are currently being investigated. We investigated the interaction of phosphodiesterase (PDE) 4 inhibitors, such as rolipram, with other agents raising cellular cAMP levels. When used alone, none of the PDE4 inhibitors increased cAMP levels. The adenylate cyclase activator forskolin, the b2-adrenergic agonist clenbuterol and the mixed b1/b2-adrenergic agonist isoproterenol increased intracellular cAMP levels of cortical murine astrocytes. This increase was synergistically elevated by rolipram or the PDE4 inhibitor RO-201724, but not by inhibition of PDE3. Inflammatory stimulation of the cells with the cytokines TNF-a, IL-1b and IFN-c strongly induced PDE4B and augmented overall PDE4 activity, while PDE3 activity was low. Clenbuterol and forskolin caused downregulation of cytokines and chemokines such as IL-6 and MCP-1. This effect was further enhanced by rolipram, but not by the PDE3 inhibitor milrinone. The cAMP-raising drug combinations attenuated the upregulation of TNF-a and IL-6 mRNA and the secretion of IL-6, but did not affect initial NF-jB signalling triggered by the stimulating cytokines. These results indicate that PDE4 may be a valuable anti-inflammatory target in brain diseases, especially under conditions associated with stimulation of cAMP-augmenting astrocyte receptors as is observed by clenbuterol treatment.
Cytokine-dependent balance of mitogenic effects in primary human lung fibroblasts related to cyclic AMP signaling and phosphodiesterase 4 inhibition
2010, Selige, Jens, Tenor, Hermann, Hatzelmann, Armin, Dunkern, Torsten
Interleukin-1β (IL-1β) and basic fibroblast growth factor (bFGF) are important regulators of proliferation, and their expression is increased in lungs of patients with asthma, idiopathic pulmonary fibrosis (IPF), or chronic obstructive pulmonary disease (COPD). We investigated the effect of IL-1β and bFGF on proliferation of human lung fibroblasts and the role of COX-2, PGE2, and cAMP in this process. Furthermore, the effect of phosphodiesterase (PDE) 3 and 4 inhibition was analyzed. In primary human lung fibroblasts low concentrations of IL-1β (<10 pg/ml) potentiated the bFGF-induced DNA synthesis, whereas higher concentrations revealed antiproliferative effects. Higher concentrations of IL-1β-induced COX-2 mRNA and protein associated with an increase in PGE2 and cAMP, and all of these parameters were potentiated by bFGF. The PDE4 inhibitor piclamilast concentration-dependently reduced proliferation by a partial G1 arrest. The PDE3 inhibitor motapizone was inactive by itself but enhanced the effect of the PDE4 inhibitor. This study demonstrates that bFGF and IL-1β act in concert to fine-tune lung fibroblast proliferation resulting in amplification or reduction. The antiproliferative effect of IL-1β is likely attributed to the induction of COX-2, which is further potentiated by bFGF, and the subsequent generation of PGE2 and cAMP. Inhibition of PDE4 inhibition (rather than PDE3) may diminish proliferation of human lung fibroblasts and therefore could be useful in the therapy of pathological remodeling in lung diseases.
The differential impact of PDE4 subtypes in human lung fibroblasts on cytokine-induced proliferation and myofibroblast conversion
2011-08, Selige, Jens, Hatzelmann, Armin, Dunkern, Torsten
Lung fibroblast proliferation and differentiation into myofibroblasts are pathological key events during development of lung fibrosis. Cyclic nucleotide signaling is described as a negative modulator of these cellular processes, and cyclic nucleotide degrading type 4 phosphodiesterases (PDE4) are important regulators of these pathways. In this study, we elucidated expression and the role of individual subtypes of PDE4 in primary normal human lung fibroblast (NHLF) in controlling cytokines-induced proliferation and conversion to myofibroblasts by short-interfering RNAs (siRNAs) induced knockdown. We verified the expression of PDE4A, B, and D, while PDE4C was only minor or even not expressed in NHLF. An efficient liposome-mediated transfection method for mRNA silencing and a knockdown of the expressed PDE4 subtypes was achieved in these cells. This knockdown was further validated by PDE4 protein expression analysis and PDE4 activity measurements. Functionally, the knockdown of PDE4A and PDE4B inhibited proliferation induced by the cytokine combination of bFGF and IL-1β, whereas knockdown of PDE4D was ineffective. In contrast, TGF-β induced differentiation into myofibroblasts was affected by knockdown of PDE4B and PDE4D, but not by PDE4A knockdown. In summary, our data allow to assign different PDE4 subtypes to distinct functions of human lung fibroblasts and highlight the predominant role of PDE4B in controlling pathophysiological processes of human lung fibroblasts. This provides a scientific rationale for focused therapeutic targeting of PDE4B to treat respiratory diseases with fibrotic lesions in the lung.
Pathophysiological relevance of PDE inhibition in lung fibroblasts for the treatment of pulmonary diseases
2010, Selige, Jens
Lung fibroblast proliferation and differentiation into myofibroblast are pathological key events during fibrotic alterations occuring in pulmonary diseases such as asthma, idiopathic pulmonary fibrosis or chronic obstructive pulmonary disease. Cyclic nucleotide signaling is described as a negative modulator of these cellular processes, and cyclic AMP degrading type 4 phosphodiesterases (PDE4) are important regulators of these pathways. Together with various cAMP-generating isoforms of adenylyl cyclases and effectors, PDE4 subtypes constitute a network that regulates the signaling via numerous mediators, e.g. growth factors or cytokines. Several studies using selective PDE4 inhibitors under clinical development have shown that the induced increase of cAMP can inhibit lung fibroblast functions and might be able to mitigate lung tissue remodeling. The role of PDE4 subtypes in these functions remains unknown. This thesis aims to analyze mechanisms of cytokine-induced functions of human lung fibroblasts, such as proliferation and differentiation, and to determine the role of PDE4 subtypes within these processes. This study demonstrates, that bFGF-induced proliferation of human lung fibroblasts is either potentiated by the presence of the inflammatory cytokine IL-1β or inhibited depending on the IL-1β concentration. The antiproliferative activity of PDE4 inhibitors (but not of PDE3 inhibitors) is due to the increase of PGE2 by IL-1β via the COX-2/PGE2-synthase pathway. Moreover, this IL-1β triggered signaling pathway is further potentiated by bFGF by increasing IL-1β induced COX-2 expression. In order to assess the role of PDE4 subtypes, their expression in human lung fibroblasts was down regulated by using an optimized liposome mediated transfection method and specific siRNA´s. With this technology, different PDE4 subtypes were assigned to distinct functions of human lung fibroblast. The knockdown of PDE4A and PDE4B inhibited cytokine-induced proliferation, whereas knockdown of PDE4D did not. In contrast, TGF-β induced differentiation of lung fibroblasts to myofibroblasts was only prevented by knockdown of PDE4B and PDE4D, but not of PDE4A. The identification of the predominant role of PDE4B in controlling pathophysiological relevant functions of human lung fibroblasts provides now a rationale for pharmaceutical development of subtype specific PDE4 inhibitors with higher efficacy and a broader therapeutic window.