Urotensin II Signaling Mechanisms Result in Vascular Smooth Muscle Cell Proliferation and New Gene Induction

Urotensin II (UII) plays an important role as an activator of vascular smooth muscle (VSM) cell proliferation and hyptertrophy when sustained and unabated. UII receptor (UIIR) signaling results in the activation of phospholipase C (PLC) and second messengers IP3/ DAG, which trigger the release of Ca2+ from the sarcoplasmic reticulum (SR). We hypothesize that UIIR- coupled Gq signaling in pulmonary arterial vascular smooth muscle cells (Pac1) and primary aortic vascular smooth muscle (PAVSM) cells activates downstream calmodulin (CaM) kinase kinase (CaMKK) as a primary downstream target. We show here that UIIR signaling in Pac1 cultures results in the immediate phosphorylation and activation of CaM Kinase I (CaMKI). Activated CaMKK phosphorylates and activates CaMKI. CaMKI activation was measured in response to UII stimulation in the presence/ absence of CaMKK inhibitor. We report here that activated CaMKK/ CaMKI has at least 4 downstream targets; ERK 1/ 2, PKD, ATF2, and P38 MAPK. Activation of these kinases were identified by measurement of UII induced phosphorylation of PKD, ERK1/ 2 kinase, P38, and ATF2 via western blot analysis. Reporter assays demonstrated that UII-induces the expression of gene markers for phenotypic modulation including SM-MHC, SM-22 α, and SM-α actin. CaMKK through UIIR stimulation results in the activation of SMC specific genes associated with phenotypic switching. Gene expression is transcriptionally driven by activation of the above kinases, which recruits and induces the expression three-transcription factors MEF2, SRF, and GATA. These genes are up-regulated for differentiation and recruitment of various factors which recovers matrix structure, and expression of adhesion molecules either during vascular injury or vascular pathological states. Taken together, these results ii identify components of an important intracellular signaling pathway through which Angiotensin II induces UIIR up-regulation and leads to increased levels of intracellular Ca2+, activation of Ca2+-dependent kinases, phosphorylates stress-activated kinases, and stimulates cellular proliferation, as well as new gene expression.