Rear Polarization of the Microtubule-Organizing Center in Neointimal Smooth Muscle Cells Depends on PKCα, ARPC5, and RHAMM

Abstract

Directed migration of smooth muscle cells (SMCs) from the media to the intima in arteries occurs during atherosclerotic plaque formation and during restenosis after angioplasty or stent application. The polarized orientation of the microtubule-organizing center (MTOC) is a key determinant of this process, and we therefore investigated factors that regulate MTOC polarity in vascular SMCs. SMCs migrating in vivo from the medial to the intimal layer of the rat carotid artery following balloon catheter injury were rear polarized, with the MTOC located posterior of the nucleus. In tissue culture, migrating neointimal cells maintained rear polarization, whereas medial cells were front polarized. Using phosphoproteomic screening and mass spectrometry, we identified ARPC5 and RHAMM as protein kinase C (PKC)-phosphorylated proteins associated with rear polarization of the MTOC in neointimal SMCs. RNA silencing of ARPC5 and RHAMM, PKC inhibition, and transfection with a mutated nonphosphorylatable ARPC5 showed that these proteins regulate rear polarization by organizing the actin and microtubule cytoskeletons in neointimal SMCs. Both ARPC5 and RHAMM, in addition to PKC, were required for migration of neointimal SMCs.