Endothelin-1 and H2O2-induced signaling in vascular smooth muscle cells : modulation by CaMKII and Nitric oxide
Endothelin-1 has emerged as an extremely potent vasoactive peptide exhibiting potent mitogenic activity in vascular smooth muscle cells (VSMCs). A critical role of ET-1 in many cardiovascular diseases, such as atherosclerosis, hypertension, restenosis after angioplasty, heart failure and arrhythmia has been suggested. ET-1 exerts its effects through multiple signaling pathways which include Ca2+, mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI-3K)/protein kinase B (PKB)/Akt pathways. Several studies have also demonstrated that reactive oxygen species (ROS) may play an important role in mediating the signals of several growth factors and peptides hormones linked to these pathways. We have previously reported that ET-1 generates ROS which mediates ET-1-induced signaling. H2O2, an important ROS molecule, activates both MAPKs and PKB signaling in VSMCs. In addition, we have also suggested that Ca2+ and CaM are essential to trigger H2O2-induced ERK1/2, p38 and PKB phosphorylation in A-10 VSMCs. Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase which is believed to transduce the downstream effects of Ca2+/CaM, and has been shown to be involved in H2O2-induced signaling in endothelial cells. However, a role of CaMKII in mediating ET-1 and H2O2-induced ERK1/2, PKB, Pyk2 phosphorylation, as well as its effect on hypertrophic and proliferative responses of ET-1 in VSMCs remains unexplored. Interestingly, a role of CaMKII in several cardiovascular diseases has been reported and studies showing that pharmacological inhibition of CaMKII, by using KN-93, prevent arrhythmic activity improved vascular dysfunction in diabetes or in Ang II-induced hypertension.  Nitric oxide (NO) is also an important reactive species and vasoactive molecule involved in the regulation of several hormone-mediated responses. NO has been suggested to modify growth-promoting signaling events and thus may serve as a vascular protective agent. Studies have shown that NO can attenuate EGF and Ang II-induced Ras/Raf/ERK1/2 as well as increase in PKB phosphorylation signaling pathways. There is also evidence for a potential cross-talk between ET-1 and NO, however not much information on the ability of NO to modify ET-1-induced signaling in VSMCs is available. Therefore, the work presented in this thesis has investigated the role of CaMKII system in ET-1 and H2O2-induced ERK1/2, PKB and Pyk2 phosphorylation, as well as in cell growth and proliferation evoked by ET-1 in VSMCs. We also investigated the role of NO in ET-1-induced ERK1/2, PKB and Pyk2 phosphorylation as well as protein synthesis.    In the first part of our studies, by using three different approaches, i.e. use of pharmacological inhibitors, a CaMKII AIP (autoinhibitor peptide) and siRNA techniques, we have investigated the involvement of CaMKII in ET-1-induced ERK1/2 and PKB phosphorylation in A-10 VSMC. We have demonstrated that CaMKII mediates the effect of ET-1 on ERK1/2 and PKB phosphorylation in A-10 VSMC.  By using pharmacological inhibitor alone such as, KN-93, earlier studies have reported that AngII and Ca2+ elevating agents, such as ionomycin, exert their effects on ERK1/2 phosphorylation via CaM-dependent pathways in VSMC. However, by using multiple approaches, our studies, have provided the first evidence to suggest an involvement of CaMKII in mediating the effect of ET-1 on ERK1/2 and PKB phosphorylation in A-10 VSMC. We have also reported for the first time, a crucial role of CaMKII in vascular pathophysiology related to ET-1 by regulating the growth and hypertrophic events by using the technique of [3H]leucine and [3H]thymidine incorporation. In the second part, in view of earlier studies showing that ROS mediates ET-1-induced signaling events in VSMC, we have also investigated if CaMKII is also implicated in H2O2-induced activation of ERK1/2 and PKB pathways. By using both pharmacological and molecular approaches, we show that similar to ET-1, CaMKII serves as a critical upstream component in triggering H2O2-induced ERK1/2, PKB and Pyk2 phosphorylation in VSMC. Furthermore, since we have previously reported that IGF-1R transactivation is needed for H2O2-induced PKB activation, we have investigated the effect of CaMKII inhibition and knocking-down on IGF-1R phosphorylation evoked by H2O2. Taken together, these results demonstrate that CaMKII plays a critical upstream role in mediating the effect of H2O2 on ERK1/2, PKB and IGF-1R phosphorylation.  In the third part of our studies, we have investigated the molecular mechanism by which NO exerts its anti-mitogenic and anti-hypertrophic effect on ET-1-induced signaling. By testing the effect of two different NO donors (SNAP and SNP) and L-NAME, an inhibitor of NO synthase, in ET-1-induced ERK1/2, PKB and Pyk2 phosphorylation, we observed that NO has an inhibitory effect in ET-1-induced signaling in VSMC. In addition, 8-Br-cGMP, an analogue of cGMP, exerted similar effect to that of NO donors whereas, oxadiazole quinoxalin (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), reversed the inhibitory effect of NO. We conclude that NO, in a cGMP-dependent manner, attenuated ET-1-induced phosphorylation of ERK1/2, PKB and Pyk2 and also antagonized the hypertrophic effects of ET-1, since SNAP treatment decreased the protein synthesis induced by ET-1.  In summary, the studies presented in this thesis demonstrate that both ET-1 and H2O2 induce ERK1/2, PKB and Pyk2 phosphorylation in VSMC and CaMKII activation is required for these events. We have also shown that CaMKII phosphorylation is upstream of H2O2-induced IGF-1R transactivation in VSMC. We have also provided evidence that NO attenuates ET-1-induced ERK1/2, PKB and Pyk2 phosphorylation. Finally, we have established that CaMKII activation stimulates ET-1-evoked protein and DNA synthesis, yet NO attenuates protein synthesis induced by ET-1.    Keywords : Endothelin; Hydrogen peroxide; CaMKII; Nitric oxide; Vascular; Protein Kinase B; Extracellular Signal-Regulated Kinase1/2; IGF-1R; Growth.
CaMKII PKB ERK1/2 IGF-1R Endothelin Hydrogen peroxide Nitric oxide Vascular Protein Kinase B Extracellular Signal-Regulated Kinase 1/2 Growth