Elsevier

Cellular Signalling

Volume 57, May 2019, Pages 10-20
Cellular Signalling

Minocycline inhibits PDGF-BB-induced human aortic smooth muscle cell proliferation and migration by reversing miR-221- and -222-mediated RECK suppression

https://doi.org/10.1016/j.cellsig.2019.01.014Get rights and content

Highlights

  • PDGF suppresses RECK, and induces SMC migration and proliferation

  • Minocycline induces RECK expression

  • Minocycline reverses PDGF-induced SMC migration and proliferation

  • RECK inducers have the potential to block vascular proliferative diseases

Abstract

Minocycline, a tetracycline antibiotic, is known to exert vasculoprotective effects independent of its anti-bacterial properties; however the underlying molecular mechanisms are not completely understood. Reversion Inducing Cysteine Rich Protein with Kazal Motifs (RECK) is a cell surface expressed, membrane anchored protein, and its overexpression inhibits cancer cell migration. We hypothesized that minocycline inhibits platelet-derived growth factor (PDGF)-induced human aortic smooth muscle cell (SMC) proliferation and migration via RECK upregulation. Our data show that the BB homodimer of recombinant PDGF (PDGF-BB) induced SMC migration and proliferation, effects significantly blunted by pre-treatment with minocycline. Further investigations revealed that PDGF-BB induced PI3K-dependent AKT activation, ERK activation, reactive oxygen species generation, Nuclear Factor-κB and Activator Protein-1 activation, microRNA (miR)-221 and miR-222 induction, RECK suppression, and matrix metalloproteinase (MMP2 and 9) activation, effects that were reversed by minocycline. Notably, minocycline induced RECK expression dose-dependently within the therapeutic dose of 1–100 μM, and silencing RECK partially reversed the inhibitory effects of minocycline on PDGF-BB-induced MMP activation, and SMC proliferation and migration. Further, targeting MMP2 and MMP9 blunted PDGF-BB-induced SMC migration. Together, these results demonstrate that minocycline inhibits PDGF-BB-induced SMC proliferation and migration by restoring RECK, an MMP inhibitor. These results indicate that the induction of RECK is one of the mechanisms by which minocycline exerts vasculoprotective effects.

Introduction

Dysregulated proliferation and migration of arterial smooth muscle cells lead to vascular complications, including intimal hyperplasia. Among various growth factors, the Platelet-derived growth factor (PDGF) family exerts potent mitogenic and migratory effects on vascular smooth muscle cells, ultimately resulting in intimal hyperplasia [1,2]. There are four members in PDGF family, PDGF-A, –B, –C and –D, that dimerize and signal via PDGF receptors alpha and beta. Both PDGFRα and PDGFRβ are class III receptor tyrosine kinases, and form either homo or heterodimers depending upon the ligand [3]. Among various homo- or heterodimers of members of the PDGF family, the PDGF-BB homodimer appears to be highly potent in exerting mitogenic and migratory effects [3] via activation of multiple kinase-dependent signaling cascades, including activation of PI3K/AKT and mitogen activated protein kinases [4,5]. Furthermore, a significant role for oxidative stress and oxidative stress-sensitive NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells)-dependent signaling and its downstream effectors microRNA (miR)-221 and miR-222 have also been implicated. However, the potential targets of these miRs in the context of human arterial smooth muscle cell proliferation and migration have yet to be fully identified.

Minocycline (7-dimethylamino-6-dimethyl-6-deoxytetracycline) is a US Food and Drug Administration (FDA)-approved second-generation, semi-synthetic, orally active tetracycline antibiotic. However, its antibiotic-independent effects have also been described, including vasculoprotection. In fact, minocycline has been shown to inhibit vascular endothelial growth factor (VEGF)-induced aortic smooth muscle cell (SMC) migration by suppressing ERK (Extracellular Signal-Regulated Kinase)- and PI3K (Phosphoinositide 3-Kinase)/AKT (V-Akt Murine Thymoma Viral Oncogene Homolog)-dependent signaling and matrix metallopeptidase (MMP)-9 induction [6]. In fact, in a rat model of balloon injury, minocycline blunted neointima formation by inhibiting MMP expression and SMC migration [6,7]. Furthermore, minocycline reduced atherosclerotic plaque size in apolipoprotein E-deficient mice, a murine model of atherosclerosis, via a PARP1 (Poly[ADP-Ribose]Polymerase 1) and p27Kip1-dependent suppression of SMC proliferation [8]. These reports suggest that minocycline may have therapeutic potential in vascular proliferative diseases.

RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs) is first described as a tumor suppressor gene [[9], [10], [11], [12]]. It is a glycosylphosphatidylinositol (GPI) anchored membrane protein known to inhibit secretion and activation of MMPs. It is expressed in various cell types, including adipocytes, vascular endothelial cells, smooth muscle cells, cardiomyocytes and cardiac fibroblasts [13]. We have previously reported that ectopic expression of RECK suppresses angiotensin II-mediated cardiac fibroblast migration in vitro [[14], [15], [16]].

Many types of cancer cells express low levels of RECK, possibly contributing to their malignant potential. Supporting this hypothesis, forced expression of RECK has been shown to suppress cancer cell migration and proliferation [17,18], suggesting that RECK inducers may have therapeutic potential in lowering tumor growth and metastasis. In an attempt to identify small molecular compounds that can induce Reck gene expression, Noda et al. have identified minocycline as a potent activator of RECK promoter activity using a reporter gene assay in RAS-transformed fibroblasts [19]. Here, we hypothesized that by upregulating RECK, minocycline inhibits PDGF-induced human ASMC (SMC) proliferation and migration. Our results show that PDGF-BB differentially regulates RECK and MMPs; suppresses RECK, but induces MMPs activation; resulting ultimately in increased SMC proliferation and migration. Further supporting our hypothesis, minocycline reversed PDGF-BB-induced RECK suppression and inhibited SMC proliferation and migration. To our knowledge, this is the first report demonstrating the role of RECK in minocycline-induced suppression of PDGF-BB-mediated SMC proliferation and migration. Our data suggest a possible therapeutic role for minocycline and other RECK inducers in vascular proliferative diseases.

Section snippets

Materials

Carrier-free recombinant human PDGF-BB protein was purchased from R&D Systems (#220-BB; Minneapolis, MN). The gp91 ds-tat (YGRKKRRQRRRCSTRIRRQL - NH2; #AS-63818) and its scrambled peptide control sgp91 ds-tat (YGRKKRRQRRRCLRITRQSR - NH2; #AS-63821) were purchased from AnaSpec (Fremont, CA, USA). One of the major sources of superoxide generation is NADPH oxidase (NOX). SMC express Nox2 (gp91phox) along with its cell membrane and cytosolic components [20]. Binding of gp91phox to the p47-p67-p40

Minocycline inhibits PDGF-BB-mediated PI3K/AKT- and ERK-dependent SMC migration and proliferation without affecting cell viability

Multiple signal transduction pathways are implicated in PDGF-induced SMC proliferation and migration, including AKT and ERK activation. Therefore, we investigated whether PDGF-BB induces SMC migration and proliferation via AKT and ERK activation, and whether minocycline blunts these responses. Indeed, PDGF-BB induced time dependent AKT activation, as evidenced by the increased levels of phosphor-AKT (Ser473) levels (Fig. 1A), an effect inhibited by the PI3K inhibitor Wortmannin, the AKT

Discussion

The molecular mechanisms underlying growth factor-induced smooth muscle cell migration and proliferation have yet to be fully investigated. Here we report for the first time that minocycline, a tetracycline antibiotic, inhibits PDGF-BB-mediated human aortic smooth muscle cell (SMC) migration and proliferation by reversing RECK suppression. RECK is an MMP inhibitor, and PDGF-BB suppressed its expression via AKT and ERK activation, ROS generation, NF-κB and AP-1 activation, and miR-221 and

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgments

BC is a recipient of the Department of Veterans AffairsResearch Career Scientist award (#IK6BX004016-01), and is supported by the U.S. Department of Veterans Affairs, Office of Research and Development-Biomedical Laboratory Research and Development (ORD-BLRD) Service Award VA-I01-BX002255. Work in SM's lab is supported by NIH/NIAID R01AI119131. SS is supported by NIHR21-HL113705, PD by HL080682 and 1-U54 GM104940, and TY by American Heart Association15SDG25240022.

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