ABSTRACT
Title
cAMP system inhibits EMT in MDCK cells and purine receptor-mediated activity may represent a novel insight in the modulation of this phenotypic conversion.
Authors
S. Romano1, P. Giuliani1, P. Ballerini2, R. Ciccarelli2, F. Caciagli2, P. Di Iorio1.
1Dept. of Human Movement Sciences, “G. d’Annunzio” University of Chieti-Pescara;2Dept. of Biomedical Sciences “G. d’Annunzio” University of Chieti-Pescara.
1Dept. of Human Movement Sciences, “G. d’Annunzio” University of Chieti-Pescara;2Dept. of Biomedical Sciences “G. d’Annunzio” University of Chieti-Pescara.
Abstract
Epithelial to mesenchymal transition (EMT) is an extreme form of cellular plasticity that leads epithelial cells to lose their polarization and specialized junctional structures to undergo cytoskeleton reorganization and to acquire morphological and functional features of mesenchymal-like cells. Originally described in embryonic development, EMT is involved in several pathophysiological conditions, including tissue remodeling, wound healing, organ fibrosis and cancer progression. It can be triggered by different conditions such as immunological and/or ischemic injuries.
One of the most commonly used agent able to induce an experimental EMT is TGF-b1 (5–10ng/ml of culture medium for 3–7days). It causes characteristic morphological changes with loss of cohesion, a decline in the expression of E-cadherin and induction of several mesenchymal markers including a-SMA, vimentin, fibronectin, N-cadherin and of transcription factors such as ZEB1, ZEB2 and Slug. Although a number of inflammation mediators as well as an alteration of the redox balance have been recognized to be involved in the EMT induction, the mechanisms underlying the inhibition/reversion of EMT are still poorly known. It has been recently reported that the overexpression of TGF-b1, observed after a myocardial infarction, was associated with a reduced expression of Epac1 (the exchange protein activated by cAMP) which in turn, when it was overexpressed in cardiac fibroblasts, inhibited the collagen synthesis (Yokoyama U. et al., PNAS 2008). Thus the first aim of the study was to evaluate whether the cAMP system inhibits TGF-b1-induced EMT. We used epithelial Madin Darby canine kidney (MDCK) cells and we observed that the intracellular cAMP increase obtained by treating the cells with cAMP permeable analogues such as 8-CPT-cAMP, N6-Phenyl-cAMP, 8-pCPT-2’-O-Me-cAMP or with forskolin inhibited EMT both modifying the mesenchymal phenotype assessed by phalloidin staining and reducing the expression of mesenchymal markers and transcription factors cited above (Western Blot and real time RT-PCR).
Besides the direct activation of the cAMP system we studied the possible modulation of EMT through receptor-mediated mechanisms. To this aim, we tested the effect of extracellular purines which are recognized as a super-family of signaling molecules playing key roles in a variety of physiological and pathological responses and deeply involved in the control of cell homeostastis. ATP and adenosine as well as GTP and guanosine are known to be widely released during hypoxia/hypoglycemia (Ciccarelli R. et al., Glia 1999) and post-ischemic reperfusion. They act as modulators of inflammatory/immune response and their metabolites such as hypoxanthine (HYPO) or guanine (GUA) generate ROS (Tarozzi A. et al., 2009) while uric acid behaves as ROS scavenger. Interestingly, oxidative stress has been recently proposed to be involved, together with NF-kB, in the initial cell-cell dissociation and, more in general, in the TGF-b1-induced EMT.
In this scenario, we found that the pretreatment with ADPbS, a non-hydrolyzable agonist of P2Y receptors or with CGS-21680, a selective agonist of A2 adenosine receptors notoriously able to stimulate cAMP formation, inhibited the TGF-b1-induced mesenchymal phenotype of MDCK cells and this effect resulted to be associated with a reduction of ROS generation.
On the other hand, HYPO and GUA, per se unable to influence MDCK cells phenotype, slightly enhanced the effect of TGF-b1 on EMT whereas the simultaneous cell pre-treatment with HYPO or GUA and aminotriazole, an inhibitor of catalase used to produce a disbalance between H2O2 and superoxide, partially reduced the TGF-b1-induced EMT.
Thus, receptor-mediated signaling able to stimulate cAMP formation as well as anti-oxidant drugs or pharmacological tools able to modulate ROS formation and catabolism inhibit EMT and purinergic signaling plays a key homeostatic role in the modulation of this phenotypic conversion.
One of the most commonly used agent able to induce an experimental EMT is TGF-b1 (5–10ng/ml of culture medium for 3–7days). It causes characteristic morphological changes with loss of cohesion, a decline in the expression of E-cadherin and induction of several mesenchymal markers including a-SMA, vimentin, fibronectin, N-cadherin and of transcription factors such as ZEB1, ZEB2 and Slug. Although a number of inflammation mediators as well as an alteration of the redox balance have been recognized to be involved in the EMT induction, the mechanisms underlying the inhibition/reversion of EMT are still poorly known. It has been recently reported that the overexpression of TGF-b1, observed after a myocardial infarction, was associated with a reduced expression of Epac1 (the exchange protein activated by cAMP) which in turn, when it was overexpressed in cardiac fibroblasts, inhibited the collagen synthesis (Yokoyama U. et al., PNAS 2008). Thus the first aim of the study was to evaluate whether the cAMP system inhibits TGF-b1-induced EMT. We used epithelial Madin Darby canine kidney (MDCK) cells and we observed that the intracellular cAMP increase obtained by treating the cells with cAMP permeable analogues such as 8-CPT-cAMP, N6-Phenyl-cAMP, 8-pCPT-2’-O-Me-cAMP or with forskolin inhibited EMT both modifying the mesenchymal phenotype assessed by phalloidin staining and reducing the expression of mesenchymal markers and transcription factors cited above (Western Blot and real time RT-PCR).
Besides the direct activation of the cAMP system we studied the possible modulation of EMT through receptor-mediated mechanisms. To this aim, we tested the effect of extracellular purines which are recognized as a super-family of signaling molecules playing key roles in a variety of physiological and pathological responses and deeply involved in the control of cell homeostastis. ATP and adenosine as well as GTP and guanosine are known to be widely released during hypoxia/hypoglycemia (Ciccarelli R. et al., Glia 1999) and post-ischemic reperfusion. They act as modulators of inflammatory/immune response and their metabolites such as hypoxanthine (HYPO) or guanine (GUA) generate ROS (Tarozzi A. et al., 2009) while uric acid behaves as ROS scavenger. Interestingly, oxidative stress has been recently proposed to be involved, together with NF-kB, in the initial cell-cell dissociation and, more in general, in the TGF-b1-induced EMT.
In this scenario, we found that the pretreatment with ADPbS, a non-hydrolyzable agonist of P2Y receptors or with CGS-21680, a selective agonist of A2 adenosine receptors notoriously able to stimulate cAMP formation, inhibited the TGF-b1-induced mesenchymal phenotype of MDCK cells and this effect resulted to be associated with a reduction of ROS generation.
On the other hand, HYPO and GUA, per se unable to influence MDCK cells phenotype, slightly enhanced the effect of TGF-b1 on EMT whereas the simultaneous cell pre-treatment with HYPO or GUA and aminotriazole, an inhibitor of catalase used to produce a disbalance between H2O2 and superoxide, partially reduced the TGF-b1-induced EMT.
Thus, receptor-mediated signaling able to stimulate cAMP formation as well as anti-oxidant drugs or pharmacological tools able to modulate ROS formation and catabolism inhibit EMT and purinergic signaling plays a key homeostatic role in the modulation of this phenotypic conversion.