CaM kinase IIδ₂-dependent regulation of vascular smooth muscle cell polarization and migration

Abstract

Previous studies indicate involvement of the multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) in vascular smooth muscle (VSM) cell migration. In the present study, molecular loss-of-function studies were used specifically to assess the role of the predominant CaMKII delta2 isoform on VSM cell migration using a scratch wound healing assay. Targeted CaMKII delta2 knockdown using siRNA or inhibition of activity by overexpressing a kinase-negative mutant resulted in attenuation of VSM cell migration. Temporal and spatial assessments of kinase autophosphorylation indicated rapid and transient activation in response to wounding, in addition to a sustained activation in the leading edge of migrating and spreading cells. Furthermore, siRNA-mediated suppression of CaMKII delta2 resulted in the inhibition of wound-induced Rac activation and Golgi reorganization, and disruption of leading edge morphology, indicating an important function for CaMKII delta2 in regulating VSM cell polarization. Numerous previous reports link activation of CaMKII to ERK1/2 signaling in VSM. Wound-induced ERK1/2 activation was also found to be dependent on CaMKII; however, ERK activity did not account for effects of CaMKII in regulating Golgi polarization, indicating alternative mechanisms by which CaMKII affects the complex events involved in cell migration. Wounding a VSM cell monolayer results in CaMKII delta2 activation, which positively regulates VSM cell polarization and downstream signaling, including Rac and ERK1/2 activation, leading to cell migration.

Calcium/Calmodulin-dependent Protein Kinase II Delta 6 (CaMKIIδ6) and RhoA Involvement in Thrombin-induced Endothelial Barrier Dysfunction

“…6 as the Endogenous HUVEC Isoform-To design specific molecular approaches for probing functions of endogenous CaMKII in endothelial cells, we first identified the CaMKII isoform(s) expressed in several cell lines by Western blotting with CaMKII subtype-specific antibodies. Blotting with a antibody recognizing all CaMKII isoforms (11-14, 19, 22), indicated that the predominant isoform expressed in primary cultures of endothelial cells has an apparent molecular mass of 50 kDa, smaller than the predominant 52-kDa isoform in cultured rat VSM, which we previously identified as the CaMKII␦ 2 variant (11,12,14,15) (Fig. 1A, top panel).…”

Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn

“…In our hands, overexpression of Fyn had detrimental effects on VSM proliferation and viability. 4 Based on previous studies and the fact that Fyn has dual palmitoylation sites in its SH4 domain that target it directly to lipid rafts (39), Fyn association with the Golgi and peripheral membrane as shown in Fig. 4 is not surprising.…”

“…To determine whether localization of CaMKII␦ 2 and/or Fyn is dependent on activation conditions, VSM cells were stimulated with PDGF (100 ng/ml), a stimulus known to activate CaMKII and to stimulate migration in VSM cells (4). Within minutes after the addition of PDGF, Fyn and, to a lesser extent, CaMKII␦ 2 localized at cell margins.…”

“…The appearance of endogenous CaMKII␦ 2 and Fyn in a protein complex provides a potential mechanism for this pattern of CaMKII␦ 2 localization. Our previous study (4) showed that loss of CaMKII␦ 2 expression prevents both the Golgi orientation and lamellipodial formation needed for VSM cells to migrate in a directed manner (40). Based on the results presented in this study, it is reasonable to speculate that Fyn may also regulate VSM cell migration through an effect on Golgi function or organization.…”

“…Studies from our laboratory have focused on potential mechanisms and identified a role for CaMKII␦ 2 in mediating VSM cell adhesion and spreading, important early components of cell migration, through regulation of focal adhesion proteins and the ERK1/2 signaling pathway (9). We have also reported that CaMKII␦ 2 -dependent regulation of VSM cell migration involves activation of Rac1, a Rho family protein (4). Recently, CaMKII␦-dependent regulation of VSM cell migration through post-transcriptional stabilization of MMP9 mRNA levels was reported (10).…”