Objectives:
Aberrant growth-factor signalling drives pathological vascular remodelling, restenosis after angioplasty, and structural changes that predispose to cardiac arrhythmias such as atrial fibrillation (AF). MEK1 is a central node of the Ras/Raf/MEK/Erk cascade; activation is classically defined by Ser217/221 phosphorylation, but regulatory sites such as Thr292 may tune proliferative versus non-proliferative outcomes. We asked how fibroblast growth factor-2 (FGF2) regulates MEK1 phosphorylation in porcine vascular smooth muscle cells (SMC) and adult cardiomyocytes, whether the selective MEK1/2 inhibitor UO126 alters these site-specific phosphorylations, and how these changes relate to SMC proliferation and secretory activity with potential relevance to restenosis and arrhythmogenic substrate formation.
Methods & Results:
Primary porcine SMC were treated with FGF2 and analysed by RNA-seq, 1D/2D gel electrophoresis with mass spectrometry, and Western blotting using total and phospho-specific antibodies (Ser217/221, Thr286, Thr292, Thr298). mRNA/protein levels were measured at 48 h; phosphorylation was profiled at 10 min. Parallel analyses were performed in FGF2-stimulated adult cardiomyocytes and controls. Functionally, proliferation was assessed by DNA synthesis assays.
Key findings: FGF2 stimulation elicited a defined pro-proliferative programme in SMC detected by RNA-seq: >200 transcripts were upregulated >3-fold (including multiple CDKs) and ~120 transcripts were downregulated <0.3-fold (including p27Kip1) at 48 h. Secretome/2D analyses identified increased extracellular mediators including TIMP1 and KGF, factors implicated in matrix remodelling and paracrine modulation of cardiac tissue. MEK1 mRNA increased while MEK2 remained unchanged. After 10 min FGF2, 2D analysis showed MEK1 gel-shifts consistent with multi-site phosphorylation; site-specific blots revealed strong phosphorylation at Ser217/221 and at Thr292. UO126 (selective MEK1/2 inhibitor) produced an unexpected pattern: Ser217/221 became hyperphosphorylated while Thr292 phosphorylation was abolished. Despite apparent activation-loop phosphorylation, UO126 nearly eliminated DNA synthesis and SMC proliferation, indicating that loss of Thr292 phosphorylation coincides with proliferative arrest. In adult cardiomyocytes, FGF2 increased Ser217/221 phosphorylation but had negligible effect on Thr292.
Conclusions:
Thr292 phosphorylation distinguishes proliferative SMC from post-mitotic cardiomyocytes and appears to function as a molecular switch that couples MEK1 signalling to cell-cycle competence. UO126 uncouples activation-loop and Thr292 status, suppressing SMC proliferation despite Ser217/221 hyperphosphorylation. Translationally, targeting Thr292-dependent regulation may offer a strategy to limit pathologic vascular proliferation (e.g., restenosis) and modulate paracrine drivers of atrial remodelling that contribute to AF, while preserving cardiomyocyte stability. Ongoing work will define how Thr292 controls SMC differentiation and FGF2-dependent secretory programmes.
