Molecular Medicine Cooperative Binding of KLF4, pELK-1, and HDAC2 to a G/C Repressor Element in the SM22 Promoter Mediates Transcriptional Silencing During SMC Phenotypic Switching In Vivo

Rationale: We previously identified conserved G/C Repressor elements in the promoters of most smooth muscle cell (SMC) marker genes and demonstrated that mutation of this element within the SM22␣ promoter nearly abrogated repression of this transgene after vascular wire injury or within lesions of ApoE؊/؊ mice. However, the mechanisms regulating the activity of the G/C Repressor are unknown, although we have previously shown that phenotypic switching of cultured SMC is dependent on Krupple-like factor (KLF)4. Objective: The goals of the present studies were to ascertain if (1) injury-induced repression of SM22␣ gene after vascular injury is mediated through KLF4 binding to the G/C Repressor element and (2) the transcriptional repressor activity of KLF4 on SMC marker genes is dependent on cooperative binding with pELK-1 (downstream activator of the mitogen-activated protein kinase pathway) and subsequent recruitment of histone de-acetylase 2 (HDAC2), which mediates epigenetic gene silencing. Key Words: KLF4 Ⅲ pELK-1 Ⅲ HDAC2 Ⅲ smooth muscle cells Ⅲ acetylation Ⅲ smooth muscle Ⅲ gene transcription Ⅲ transcription factors Ⅲ vascular disease S mooth muscle cells (SMC) are remarkably plastic and transition from a quiescent contractile state to a proliferative-migratory state during vascular injury and development of atherosclerosis. 1 Collectively, this process is termed " phenotypic switching " 2 and is characterized by the coordinate downregulation of markers of differentiated SMCs including SM22␣, smooth muscle myosin heavy chain (SM-MHC), and SM ␣-actin, gene products required for SMC contraction. 1 SMC phenotypic plasticity probably evolved for optimization of vascular repair after injury, 3 although it is also widely accepted that SMC phenotypic switching plays a key role in development and progression of atherosclerotic lesions 4 and regulation of plaque stability. Our laboratory 5–14 and many others 15–28 have studied molecular mechanisms and factors that repress SMC differentiation marker gene expression as a means to elucidate processes involved in mediating SMC phenotypic switching. Importantly, these studies have clearly established that SMC phenotypic switching is actively regulated (reviewed in Owens et al, 2 Physiol Rev) and is mediated through complex processes including extracellular signal-related kinases (ERK)-dependent phos-phorylation of ELK-1, loss of SRF-myocardin (MRTF) binding to CArG boxes in SMC marker gene promoters, 15,16,29 –32 HERP, and epigenetic silencing processes. 29 –31,33,34 Moreover, we have presented multiple lines of evidence that phenotypic switching of cultured SMC in response to platelet-derived growth factor (PDGF)-BB, PDGF-DD, and oxidized phospholipids is dependent on the embryonic stem cell (ESC) pluripotency …