Calcium/calmodulin-dependent protein kinase II-δ isoform regulation of vascular smooth muscle cell proliferation

There is accumulating evidence that Ca2+-dependent signaling pathways regulate proliferation and migration of vascular smooth muscle ( VSM) cells, contributing to the intimal accumulation of VSM that is a hallmark of many vascular diseases. In this study we investigated the role of the multifunctional serine/ threonine kinase, calmodulin ( CaM)- dependent protein kinase II ( CaMKII), as a mediator of Ca2+ signals regulating VSM cell proliferation. Differentiated VSM cells acutely isolated from rat aortic media express primarily CaMKII gamma gene products, whereas passaged primary cultures of de-differentiated VSM cells express primarily CaMKII delta(2), a splice variant of the delta gene. Experiments examining the time course of CaMKII isoform modulation revealed the process was rapid in onset following initial dispersion and primary culture of aortic VSM with a significant increase in CaMKII delta 2 protein and a significant decrease in CaMKII gamma protein within 30 h, coinciding with the onset of DNA synthesis and cell proliferation. Attenuating the initial upregulation of CaMKII delta(2) in primary cultured cells using small-interfering RNA ( siRNA) resulted in decreased serum-stimulated DNA synthesis and cell proliferation in primary culture. In passaged VSM cells, suppression of CaMKII delta 2 activity by overexpression of a kinase-negative mutant, or suppression of endogenous CaMKII content using multiple siRNAs, significantly attenuated serum-stimulated DNA synthesis and cell proliferation. Cell cycle analysis following either inhibitory approach indicated decreased proportion of cells in G1, an increase in proportion of cells in G2/M, and an increase in polyploidy, corresponding with accumulation of multinucleated cells. These results indicate that CaMKII delta 2 is specifically induced during modulation of VSM cells to the synthetic phenotypic and is a positive regulator of serum- stimulated proliferation.