Minocycline Inhibits PDGF‐BB‐induced Human Aortic Smooth Muscle Cell Proliferation and Migration via Induction of RECK

Abstract

Minocycline is an FDA‐approved tetracycline antibiotic that exerts anti‐inflammatory and anti‐apoptotic effects independent of its bactericidal activity. For example, it attenuates neointima formation after arterial injury and atherosclerotic plaque development in an animal model of diet‐induced atherosclerosis via inhibition of smooth muscle cell (SMC) proliferation and migration; however, the molecular mechanisms underlying this inhibition are not completely known. Here we investigated whether minocycline inhibits PDGF‐BB‐induced primary human aortic SMC (HASMC) proliferation and migration in vitro. Our results show that PDGF‐BB stimulates proliferation (~2.92‐fold, P<0.05) and migration (~1.8‐fold, P<0.05) of HASMC via a redox‐sensitive mechanism involving NOX2 (inhibited by gp91dstat) and NOX4 (inhibited by GKT137831). Reversion‐Inducing‐Cysteine‐Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored matrix metalloproteinase inhibitor. For the first time, our results show that PDGF‐BB downregulates RECK expression in a time‐dependent manner (by ~80% at 6 h, n=3, P<0.01), but induces miR‐221 and miR‐222 expression (~1.8‐fold and ~2.23‐fold, respectively). The reporter assays revealed that both miRs bind the 3′UTR of RECK mRNA, inhibiting its expression by ~50% (P<0.01). Supporting these observations, transduction of miR‐221 or miR‐222 mimics suppress RECK expression, but their antagomirs reverse PDGF‐BB‐induced inhibition of HASMC proliferation and migration. Further investigations revealed that PDGF‐BB activates the redox‐sensitive nuclear transcription factors NF‐kB and AP‐1 in HASMC, and shRNA directed against the AP‐1 subunit c‐Jun and the p65 subunit of NF‐kB each inhibit PDGF‐BB‐induced miR‐221 or miR‐222 expression, suggesting that PDGF‐BB suppresses RECK expression in HASMC in part via AP‐1‐ and NF‐kB‐dependent miR‐221 and miR‐222 induction. Notably, minocycline increases RECK expression dose‐dependently within the therapeutic dose of 1–100 μM, and inhibits PDGF‐BB‐induced HASMC proliferation and migration. Intriguingly, silencing RECK reverses the inhibitory effects of minocycline on PDGF‐BB‐induced migration and proliferation. Together, these results demonstrate that PDGF‐BB downregulates RECK expression in HASMC via AP‐1‐ and NF‐kB‐dependent miR‐221 and miR‐222 induction, and minocycline reverses PDGF‐BB‐induced ROS generation, miR‐221 and miR‐222 induction, and RECK suppression. These findings suggest that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.Support or Funding InformationNIH R01‐HL‐070241 (PD)VA Merit Award I01‐BX002255 (BC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.