Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation.
OBJECTIVE
Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation.


RESULTS
We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation.


CONCLUSIONS
Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.
Group of Cardiovascular Physiopathology Lab 113, Department of Medical Physiology and Biophysic, Institute of Biomedicine of Seville, Hospital of Virgen del Rocío/CSIC/University of Sevilla, Avenida Manuel Siurot s/n, Sevilla 41013, Spain.
María
M
Rodríguez-Moyano
Ignacio
I
Díaz
Natalia
N
Dionisio
Xuexin
X
Zhang
Javier
J
Avila-Medina
Eva
E
Calderón-Sánchez
Mohamed
M
Trebak
Juan Antonio
JA
Rosado
Antonio
A
Ordóñez
Tarik
T
Smani
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D066246:ErbB Receptors / Q000235:genetics*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D008297:Male; D008562:Membrane Glycoproteins / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D032389:Myocytes, Smooth Muscle / Q000502:physiology*; D000071740:ORAI1 Protein; D010766:Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D043562:Receptors, G-Protein-Coupled / Q000502:physiology; D015398:Signal Transduction; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000502:physiology; D015533:Transcriptional Activation; D014579:Urotensins / Q000494:pharmacology*
D015220:Calcium Channels; D017362:Cyclic AMP Response Element-Binding Protein; D008562:Membrane Glycoproteins; D000071740:ORAI1 Protein; C532493:Orai1 protein, rat; D043562:Receptors, G-Protein-Coupled; C518857:Stim1 protein, rat; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; D014579:Urotensins; C473142:Uts2r protein, rat; C096351:transient receptor potential cation channel, subfamily C, member 1; C029677:urotensin II; D066246:ErbB Receptors; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium
EGFR; Ion channels; Proliferation; Smooth muscle