Regulation of Vascular Smooth Muscle Cell Proliferation

    The Medizinische Klinik mit Schwerpunkt Kardiologie (F.B.M., R.D.), Universittsklinikum Charitee, Campus Virchow Klinikum, Berlin
    Max-Delbre筩k-Centrum for Molecular Medicine (F.B.M., R.S.-U., R.D., C.S.), Berlin, Germany

    Abstract

    The transcription factor NF-B regulates cell cycle progression and proliferation in a number of cell types. An important unresolved issue is the potential role of NF-B in the proliferation of vascular smooth muscle cells (VSMCs) as a basis for the development of vascular disease. To investigate the contribution of NF-B to mitogen-induced proliferation of VSMCs, a knock-in mouse model expressing the NF-B superrepressor IBN (cIBN) was used. Comparing wild-type and IBN-expressing VSMCs, we found that proliferation rates did not differ after mitogenic stimulation by platelet-derived growth-factor-BB (PDGF-BB) or serum. In line with this, NF-B activation was not observed in VSMCs derived from transgenic mice expressing an NF-BeCdependent lacZ reporter (c(Igk)3conalacZ). We further show, that classical mitogenic signaling pathways (namely mitogen-activated protein kinase and the phosphatidyl-inositol-3-OH-kinase [PI3K] pathways) control VSMC proliferation, but independently of NF-B activation. In contrast to VSMCs, mouse embryonic fibroblasts (MEFs) derived from IBN-expressing mice showed significantly impaired proliferation rates after mitogenic stimulation. This was reflected by strongly impaired cyclin D1 expression in serum-stimulated MEFs derived from (cIBN) mice. These results implicate that essential pathogenetic functions of NF-B in the development of atherosclerosis involve apoptotic and inflammatory signaling of VSMCs rather than proliferation. They further provide genetic evidence for a cell-type restricted requirement of NF-B in the control of cellular proliferation.

    Key Words: VSMC proliferation  atherosclerosis  NF-B  PI3K  MAPK

    Introduction

    Vascular smooth muscle cell (VSMC) proliferation and neo-intima formation are important events in the pathophysiological course of atherosclerosis and restenosis after balloon angioplasty. After endothelial cell activation, locally produced growth factors and cytokines mediate an inflammatory response within the vessel wall, which involves monocyte recruitment, stimulation of macrophage proliferation, migration of VSMCs from the medial layer of the vessel and finally deposition of collagen and other extracellular matrix proteins leading to the formation of a fibrous cap.1,2 Diverse signal transduction systems have been proposed to translate the mitogenic stimulus within VSMCs, among them NF-B,3eC6 the MAPK 7eC9 or the PI3K pathways.10,11

    A role for MAPKs in induction of cellular proliferation has been described not only in smooth muscle cells, but also in a variety of cell types and tissues after interaction of growth factors with their receptors.12 The main feature of the extracellular signal regulated kinase (ERK) cascade (to distinguish it from other MAPKs) involves activation of Raf, which then initiates a series of phosphorylation steps, resulting in the activation of ERK1 and ERK2, which in turn act on several substrates including transcription factors, protein kinase C or p90 ribosomal S6 kinase.13 As blockade of the ERK cascade prevents proliferation, it is commonly believed that activation of the ERK pathway is critical for cell replication.14,15

    A variety of growth factors trigger activation of PI3K followed by generation of phosphatidylinositol diphosphate and triphosphate in the cell membrane.16 Protein kinase B (PKB, also referred to as AKT) is recruited to the cell membrane, where it is phosphorylated and activated by phosphoinositide-dependent kinases (PDKs).17,18 One important target of activated PKB is ribosomal p70-S6 kinase,19 which is associated with a mitogenic signal-transduction cascade that is distinct from the ERK pathway.20,21 p70-S6 kinase has been reported to be activated in VSMCs after balloon catheter injury.7 Furthermore, wortmannin (a PI3K-inhibitor) administered to rats at the time of balloon injury, reduced early VSMC replication.11

    NF-B belongs to the Rel family of transcription factors, which regulate gene expression in immune and inflammatory responses. The 5 members of the NF-B family (p50, p52, p65, RelB, and c-Rel) can form various homo- and heterodimeric complexes with diverse DNA-binding and transcriptional activating properties. Cellular stimulation by proinflammatory cytokines and other agents activates an IB-kinase (IKK) complex to phosphorylate the IB proteins. Subsequent polyubiquitination and proteasomal degradation of IB leads to the release of NF-B into the nucleus.22 Involvement of NF-B in the process of atherosclerosis has become evident in a variety of studies. Activated nuclear NF-B has been detected in smooth muscle cells after balloon injury of rat carotid arteries and in the smooth muscle cells of human atherosclerotic lesions. It further was identified in situ in macrophages, endothelial cells and VSMCs in the intima and media of atherosclerotic vessel sections. On the other hand, only little activated NF-B is detected in healthy vessels, where the key NF-B/IB components like p50 or p65 are expressed diffusely in the cytoplasm.23eC26 These data strongly suggest a causative role for NF-B in development and maintenance of atherosclerosis.

    In earlier studies, we could demonstrate an important role for NF-B in control of proliferation in various cell types. In Hodgkin’s lymphoma cells constitutive NF-B activity is required for cell cycle progression and accounts for apoptosis resistance of these cells.27 In mouse embryonic fibroblasts (MEFs), mitogenic stimuli activate NF-B, which in turn stimulates transcription of cyclin D1. Inhibition of NF-B activation in these cells strongly delayed G1- to S-phase cell cycle progression.28 In an in vivo model it was demonstrated recently, that in mammary epithelial cells impaired NF-B activation via RANK (receptor-activator of NF-B) and IKK leads to defective proliferation of mammary epithelial cells, a process that could also be rescued by cyclin D1 overexpression.29 In this study we investigated the role of NF-B in mitogenic signal transduction in VSMCs. We used the classical VSMC growth factor PDGF-BB as well as serum stimulation to induce mitogenic responses in VSMCs and analyzed primary cells derived from mice engineered either to ubiquitously express the NF-B superrepressor IBN or transgenic mice carrying an NF-BeCregulated reporter gene.

    Methods

    Reagents

    PDGF-BB was purchased from R&D Systems; TNF- was from Biomol; AKT, ERK1/2 phospho-AKT and phospho-ERK1/2 antibodies were from Cell Signaling Technology; cyclin D1 antibody was from Pharmingen; IB antibody from Santa Cruz, -actin and -smooth muscle actin antibody from Sigma. Pharmacological inhibitors U0126 (20 eol/L) and wortmannin (100 nmol/L) were purchased from Calbiochem. PDGF-BB was used at 10 ng/mL and TNF- at 30 ng/mL unless stated otherwise.

    Cell Culture

    Knock-in mice (C57B1/6) engineered to express the NF-B superrepressor IBN (cIBN) as well as transgenic mice (C57B1/6 x SJL) carrying a Gal reporter under control of a B-dependent promoter (c(Igk)3conalacZ) have been described before.30,31 VSMCs from these animals were obtained by the explant method as previously described.32 In brief, arterial explants from carotid arteries of adult mice were washed in PBS and subsequently cultured in DMEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and 100U of penicillin/streptomycin/mL. After 2 weeks, cells that had migrated onto the tissue culture dish were collected by trypsinization and subcultured successively; cells were used up to passage 6. Purity of cultures was confirmed by staining for -smooth muscle actin and was higher than 95%. MEFs from the same transgenic model were isolated and cultured as described before.30 For each experiment, cells from at least 3 different animals were used.

    Proliferation Assay

    The proliferation rate of stimulated cells was determined using a nonradioactive cell proliferation assay (Promega) as described.33 Briefly, after serum deprivation for 36 to 48 hours, cells were stimulated with the indicated mitogen in 96-well plates. 20 e蘈 of a methyltetrazolium salt/phenazine ethosulfate were added and the absorbance was recorded after 90 minutes incubation at 37°C at 490 nm with a microtiter plate reader (Bio-Rad). Results are reported as relative optical densities from at least 3 independent experiments using VSMCs from different animals; microtiter plate readings were performed in triplicate.

    EMSA and Western Blotting

    Whole cell lysates were prepared and analyzed by electrophoretic mobility shift assay (EMSA) and Western Blotting essentially as described previously.34 For densitometric analysis of scanned blots, the NIH Imager software was used.

    Immunofluorescence and -Galactosidase Staining

    Primary cultures of mouse vascular smooth muscle cells were fixed in the culture dish either with ice-cold methanol for 10 minutes (immunofluorescence) or using 1% formaldehyde and 0.2% glutaraldehyde in PBS at 4°C for 20 minutes (X-Gal staining). For immunofluorescence cells were stained with an -smooth muscle actin antibody followed by a Cy3-labeled anti-mouse secondary antibody. X-Gal staining was performed as described earlier.31

    Statistical Analysis

    The results are expressed as mean±SEM of at least 3 independent experiments, unless stated otherwise. Statistical significance was determined using an unpaired t test. A value of P<0.05 was considered significant.

    Results

    Mitogen-Induced VSMC Proliferation Does not Differ Between Wild-Type and cIBN-Derived VSMCs

    To investigate a NF-B requirement for VSMC proliferation in response to mitogenic stimuli, VSMCs derived from wild-type or cIBN transgenic mice were growth arrested for 36 to 48 hours and subsequently stimulated with serum, TNF- or increasing concentrations of PDGF-BB for 36 hours. Proliferation rates were measured using a nonradioactive proliferation assay. As shown in Figure 1, PDGF-BB induced concentration-dependent growth of VSMCs. Serum stimulation also resulted in a pronounced proliferative response. Importantly, VSMCs from wild-type or cIBN transgenic mice presented no differences in mitogen induced proliferation rates. Because higher doses of PDGF-BB did not significantly increase VSMC proliferation (data not shown), we used a dose of 10 ng/mL for subsequent experiments, which was within the ranges of other studies using PDGF to stimulate VSMC proliferation. Treatment with TNF-, a classical NF-B activator, resulted in no significant proliferation in wild-type VSMCs whereas it significantly reduced numbers of viable cIBN-derived VSMCs, because of apoptotic cell death (data not shown).

    PI3K/AKT and MAPK/ERK1/2 Signaling Pathways Mediate Mitogenic Signal Transduction in VSMCs Independent of NF-B Activity

    We next explored the contribution of diverse signaling pathways to mitogen-induced proliferation of VSMCs. Utilization of phospho-specific antibodies against AKT and ERK1/2 for immunoblotting revealed activation of the PI3K and the ERK1/2 pathway in wild-type and cIBN-derived VSMC after 15 minutes of PDGF-BB stimulation (Figure 2A). Activation of PI3K- or MEK1-dependent signaling was blocked by the use of specific pharmacological inhibitors (wortmannin and U0126 respectively). Induction of both pathways was observed as early as 10 minutes after stimulation with PDGF-BB or serum (data not shown). TNF- did not act on any of the 2 pathways significantly.

    We also investigated the influence of the pharmacological inhibitors on proliferation rates of wild-type or cIBN-derived VSMCs. As demonstrated in Figure 2B, parallel inhibition of PI3K and MEK1 significantly interfered with mitogen-induced proliferation of wild-type as well as cIBN-derived VSMCs, and reduced the proliferation rates of PDGF-BBeCstimulated VSMCs to baseline levels in both. Incubation of nonstimulated VSMCs with the inhibitors had no effect on cell viability.

    In VSMCs Mitogenic Stimuli Do not Activate NF-B

    To monitor NF-B activation in VSMCs after different stimuli, we studied degradation of the cytoplasmatic inhibitor IB and regulation of NF-B DNAeCbinding activity in these cells. Figure 3A displays IBN expression in cIBN-derived VSMCs compared with control cells. Whereas stimulation with TNF-, but not mitogenic stimuli led to rapid degradation of endogenous IB, IBN is not regulated by TNF-. Figure 3B demonstrates impaired NF-B DNAeCbinding activity in cIBN-derived VSMCs (EMSA) after TNF- stimulation, as expected. Neither serum nor PDGF-BB stimulation induced NF-B DNAeCbinding activity in VSMCs. In a control EMSA experiment an octamer probe did not show significantly regulated Oct-1 binding activity of the same cellular extracts.

    We furthermore examined NF-BeCdependent transcriptional activity after mitogenic stimulation in VSMCs. Taking advantage of a mouse model carrying a NF-BeCdependent lacZ-reporter gene (c(Igk)3conalacZ), we isolated VSMCs from these mice and stimulated with serum, PDGF-BB or TNF-, as described before. As shown in Figure 4, both serum and PDGF-BB stimulation failed to induce reporter gene activity, whereas TNF- stimulation resulted in a strong nuclear X-Gal signal, indicating NF-B transcriptional activity in these cells.

    Cyclin D1 Expression and Cell Cycle Regulation Are Differentially Regulated in VSMCs and MEFs From cIBN Mice

    We have reported before that cyclin D1, a key regulator of G1 check-point control is positively regulated by NF-B after mitogenic stimulation in various cell types, including MEFs.28 To demonstrate NF-B activation induced by mitogenic stimuli in MEFs we monitored DNA-binding activity. Figure 5A shows PDGF-BB, serum and TNF-eCinduced NF-B DNAeCbinding activity above the basal levels in wild-typeeCderived MEFs, whereas basal and induced NF-B activation were sharply suppressed in cIBN-derived MEFs. Fifteen minutes of PDGF-BB, serum, or TNF- treatment resulted in a rapid induction of NF-B activation, which declined after longer periods of stimulation (data not shown). We also studied cell growth of MEFs from cIBN mice in response to different mitogens. In contrast to VSMCs, treatment of MEFs with PDGF-BB resulted in a less pronounced proliferative response compared to stimulation with fetal calf serum. We therefore chose serum stimulation to induce proliferation of MEFs. Figure 5B demonstrates significantly impaired growth of serum-stimulated cIBN-MEFs in comparison to wild-typeeCderived MEFs (). Furthermore, it became clear that inhibition of PI3K as well as MEK1 significantly reduced proliferation rates of wild-typeeCderived MEFs ().

    We also investigated mitogen-induced cyclin D1 expression in MEFs and VSMCs. A dramatically reduced cyclin D1 expression level was observed after serum stimulation of cIBN-derived MEFs compared with wild-type cells (Figure 6A). In VSMCs, however, time course and levels of cyclin D1 expression after PDGF-BB stimulation did not differ significantly between wild-typeeC and cIBN-derived cells (Figure 6B). Pharmacological inhibition of PI3K and MEK1 reduced PDGF-induced cyclin D1 expression both in wild-type and transgenic VSMCs (data not shown). These results clearly demonstrate a cell-type specific requirement of NF-B for mitogen-induced proliferation.

    Discussion

    In addition to its well-established role in regulation of the immune system, various reports suggest a role for the ubiquitous transcription factor NF-B in mitogenic growth control of a variety of cell types. Enhanced NF-B activity is apparent during G0/G1 transition in fibroblasts35 and is induced by mitogenic stimuli, including serum, in G0-arrested 3T3 fibroblasts.36 Deregulated NF-B activity is also associated with oncogenesis and cellular transformation, as has been shown for constitutive NF-B activation in Hodgkin’s lymphoma cells.27 Lymphocytes from mice lacking p50, p65 or c-rel are defective in mitogenic responses.37eC39 The best explored link between NF-B activation and cell cycle progression involves cyclin D1 and the RB checkpoint. After mitogenic stimulation, NF-B activates transcription of the cyclin D1 promoter through several binding sites, promoting G1 to S-phase transition in fibroblasts and skeletal muscle cell precursors. Inhibition of NF-B activity in these cells results in impaired proliferation rates, which can be rescued by ectopic cyclin D1 expression.28,40 The significance of these data are supported by Cao et al,29 who describe a defect of mammary epithelial cell proliferation in IKK-kinaseeCdead animals. Defective signaling via the IKK complex and NF-B could be overcome by crossing these mice with mammary-specific cyclin D1 transgenics.

    However, a general growth-promoting effect of NF-B can be ruled out. Systematic inhibition of NF-B by ubiquitous IBN expression in mice resulted in a number of distinct developmental defects, including impaired hair follicle and tooth formation, and development of secondary lymphoid organs.30 These animals and diverse NF-B and IKK knock-outs did not reveal generalized defects in diverse cell types or tissues that can be attributed to inhibited proliferation. In the present study, we further demonstrate a differential role for NF-B in regulation of proliferative responses in different cell types. Our data provide definitive genetic evidence, that NF-B activation is not required for serum- or PDGF-induced VSMC proliferation. In contrast to VSMCs derived from cIBN mice, MEFs from these animals presented a dramatic growth impairment, after serum stimulation. This phenomenon was accompanied by a significant retardation and down regulation of cyclin D1-expression. In VSMCs cyclin D1/Rb is regulated by distinct pathways, most likely including the PI3K and MAPK cascades.

    The data presented here supply a possible explanation for previous studies, using the chemokine monocyte chemotactic protein-1 (MCP-1), which is important for recruitment of circulating monocytes to areas of vascular injury. MCP-1, like PDGF-BB, induced VSMC proliferation via PI3K/AKT activation, independently of NF-B activation.41 Further downstream in the PI3K/AKT signaling cascade, p70-S6-kinase (a serine/threonine kinase) is involved in cell cycle progression and protein synthesis. p70-S6-kinase significantly contributes to VSMC replication in vitro and has been shown to be activated in rat arteries after balloon injury.11 Rapamycin, a potent inhibitor of p70-S6-kinase, inhibits VSMC DNA synthesis in vitro,42 and antagonizes intimal thickening after balloon angioplasty in an animal model.43 These findings have already been transferred to a clinical application with the introduction of rapamycin-coated stents, leading to local and prolonged release of rapamycin in balloon-injured vessel sections and diminishing the amount of restenosis after balloon angioplasty dramatically.44,45

    The NF-B system has been shown to be regulated by hemodynamic factors within the vascular organ system, predisposing regions exposed to high levels of flow disturbances for the development of atherosclerotic lesions.46 These data, as well as the immunohistochemical findings of activated NF-B family members in atherosclerotic lesions,25,26 strongly suggest a causative role for NF-B in development and maintenance of atherosclerosis. It is noteworthy that in the process of atherosclerosis VSMC migration and proliferation is only part of the pathophysiological course. Thus, activation of NF-B in atherosclerotic lesions could be entirely unrelated to mitogen induced VSMC proliferation. This topic has been discussed controversially. In earlier studies, growth factor induced NF-B activation has been reported in diverse cell types, including cultured rat aortic SMC.5,47 These findings were challenged by 2 recent publications.48,49 In VSMCs, the growth factor PDGF-BB did not activate NF-B directly, but enhanced the IL1-eCinduced persistent activation of NF-B and augmented iNOS expression.49 In contrast to our work, none of these studies directly investigated NF-BeCdependent VSMC proliferation.

    Apart from proliferation of VSMCs, which is independent of NFB, two other important pathophysiological events in the development of atherosclerosis, apoptosis and inflammation, are regulated by NF-B. Apoptosis of VSMCs contributes to the instability of advanced atherosclerotic plaques50 and NF-B regulates apoptosis of VSMCs through expression of cIAP-1 in these cells.51 Furthermore, the immunoinflammatory balance within the plaque is critically dependent on NF-B activity. Two previous models of NF-B inhibition demonstrated different phenotypes of atherosclerotic plaques, depending on how NF-B activity is inhibited. In LDL receptoreCdeficient mice a macrophage-restricted lesion of IB kinase- resulted in a significant reduction in the antiinflammatory and antiatherogenic cytokine interleukin-10 (IL-10), accompanied by the unexpected finding of increased atherosclerotic lesion formation and inflammation.52 In another study using the same LDL receptoreCdeficient mice, hematopoietic deficiency in the NF-B component p50 resulted in a significant decrease in lesion size despite enhanced accumulation of T- and B-lymphocytes within the lesions.53 The findings of the present study therefore support the concept of NF-B as a regional regulator of SMC survival rather than a direct promoter of proliferation of these cells. Possibly, the most important role of NF-B in atherosclerosis is the fine-tuning of the inflammatory response in the injured vessel wall.

    Acknowledgments

    This study was supported in part by a Max Delbre筩k Center fellowship to F.B.M. and by a grant from BMBF to C.S. We thank Yoshiaki Sunami for his help with the octamer shift.

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