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12/15-Lipoxygenase (12/15-LO) has been implicated in the pathogenesis of vascular disease. Vascular smooth muscle cell (VSMC) proliferation is a key component of the response to injury in vascular disease. The role of 12/15-LO in regulating VSMC proliferation is poorly understood. Id3 has been shown to regulate growth in various cell types and is expressed in VSMCs within atherosclerotic and restenotic lesions. This study examines the role of Id3 in 12/15-LO–mediated VSMC proliferation.
Primary aortic VSMCs from leukocyte-type 12/15-LO transgenic, leukocyte-type 12/15-LO knockout (KO), and control mice were plated in equal densities and assayed for growth, Id3 protein expression, and Id3 transcription. Results demonstrated that 12/15-LO transgenic VSMCs grew faster, whereas 12/15-LO KO VSMCs grew slower relative to control VSMCs. Further, pharmacological and molecular inhibition of 12/15-LO resulted in decreased VSMC growth. Western blots demonstrated increased Id3 protein in 12/15-LO transgenic VSMCs, whereas luciferase promoter reporter assays revealed increased Id3 transcription. In addition, overexpression of 12/15-LO increased growth in control cells but not in Id3 KO cells. 12/15-LO transgenic VSMCs demonstrated increased protein kinase C (PKC) activity. Consistent with these data, PKC inhibition decreased Id3 promoter activation.
12/15-LO is an important mediator of VSMC growth. The growth-promoting effects of 12/15-LO are at least partially mediated through induction of Id3 transcription.
The lipoxygenase enzymes have been implicated in the pathogenesis of vascular disease. Specifically, leukocyte-type 12/15-lipoxygenase (12/15-LO), highly homologous to human 15-lipoxygenase, and its products have been shown to be increased in animal models of native atherosclerosis and injury-induced restenosis. Further, pharmacological inhibition and knockout (KO) of the 12/15-LO gene in atherosclerosis and injury models result in significantly less vascular lesion formation.1–7 There are several theories as to how 12/15-LO induces lesion formation. It has been shown that lipoxygenases have the ability to oxidize low-density lipoprotein (LDL), a step important in lesion initiation and progression, and KO of the lipoxygenase gene decreases oxidant stress.6,8,9 It has also been demonstrated that 12/15-LO and its products have the ability to mediate the effects of angiotensin II (Ang II), platelet-derived growth factor, and cytokines, all important mediators of vascular injury.10–14
12/15-LO is expressed in macrophages, fibroblasts, endothelial cells, and vascular smooth muscle cells (VSMCs), all important contributors to vascular lesion formation and progression. 9,12,15–17 Recently, its role in the VSMC has begun to be defined. Indeed, the 12/15-LO products of arachidonic and linoleic acids 12S-hydroxy-eicosatetraenoic acid (12S-HETE), 15S-HETE, and 13S-hydroperoxyoctadecadienoic acid (13S-HPODE) are produced in VSMCs. Lipoxygenase activity has been shown to be increased by and to mediate the hypertrophic effects of Ang II, the mitogenic effects of cytokines, and the chemotactic effects of platelet-derived growth factor in VSMCs, whereas 12/15-LO inhibition blocks these effects.10,11,13,14 12/15-LO products have direct hypertrophic effects in VSMCs.10,18 12/15-LO appears to also affect VSMC proliferation because 12/15-LO increases dramatically with arterial injury in VSMCs, and inhibition of 12/15-LO decreases smooth muscle cell proliferation and neo-intimal formation after balloon injury.4 Recent studies show that VSMCs from 12/15-LO KO mice display decreased S-phase entry and growth-related responses relative to those from wild-type mice.19 However, although much is known about the effects of 12/15-LO and its products, less is known about the specific mechanisms by which 12/15-LO regulates key nuclear transcription factors related to VSMC growth.
Recently, Id3, a member of the helix-loop-helix (HLH) family of transcription factors, has emerged as an important regulator of cell cycle gene expression and growth in VSMCs and is expressed during vascular lesion formation in response to injury and in native atherosclerosis.20–22 Id3 functions as a dominant-negative inhibitor of specific gene expression. It functions by dimerizing with ubiquitously expressed basic HLH (bHLH) factors such as the E2A proteins (E12 and E47) and inhibits their dimerization with other ubiquitous or cell-specific bHLH factors. DNA binding and subsequent activation of transcription is inhibited. In the context of the cell cycle, this leads to decreased p21cip1 transcription and thus increased cellular proliferation. In the absence of Id3, E2A factors are free to dimerize and bind to DNA leading, to activation of p21cip1 transcription and subsequently growth arrest of the cell.23,24 Thus, it is appealing to hypothesize that the growth-promoting effects of 12/15-LO are mediated through increased expression of the HLH factor Id3.
Results of the present study demonstrate that indeed, leukocyte-type 12/15-LO is important to VSMC growth. Moreover, we provide the first evidence that the growth effects of 12/15-LO are mediated, at least in part, through upregulation of Id3 transcription and protein expression. Further, we provide evidence that protein kinase C (PKC) activity is increased in VSMCs derived from 12/15-LO transgenic mice and that the inhibition of PKC decreases Id3 transcription, providing evidence that 12/15-LO upregulation of Id3 transcription is at least in part mediated by PKC.
All animal studies were performed with an institutionally approved animal use protocol. Leukocyte-type 12/15-LO transgenic mice were generated on a C57BL/6J (B6) background using a 15.5-kb mouse genomic clone containing the full-length murine 4.5-kb leukocytetype 12/15-LO gene. Methods are described in detail previously in the literature.25 It has been demonstrated that 12/15-LO transgenic mice produce 2-fold elevations in levels of 12S-HETE and 13S-HPODE and develop aortic fatty streaks on a rodent chow diet.25 Leukocyte-type 12/15-LO KO mice, on a B6 background, were obtained from Jackson Laboratories (Bar Harbor, ME). 12/15-LO KO mice have been shown previously to produce significantly less 12/15-LO protein and 12S-HETE.19 Id3 KO mice were provided by Yuan Zhuang (Duke University, Durham, NC).
Primary aortic VSMCs were isolated from leukocyte-type 12/15-LO transgenic mice, leukocyte-type 12/15-LO KO mice, Id3 KO mice, Id3 KO litter mate controls, and C57/BL6 control mice and grown in DMEM F12 containing 10% FBS, 10 U/mL penicillin, and 10 µg/mL streptomycin. Cells were studied at passages 6 to 12.
Primary aortic VSMCs were plated in equal densities and treated with either 50 multiplicities of infection (MOI) of an adenovirally packaged leukocyte-type 12/15-LO ribozyme described previously in the literature,14 50 MOI of an adenovirus overexpressing 12/ 15-LO (Ad12/15-LO), 50 MOI of adenovirus alone, 50 MOI of an adenovirus-expressing green fluorescent protein (GFP), and 1 µmol/L cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC; a 12/ 15-LO inhibitor; Biomol) diluted in ethanol, or ethanol alone. Cell number was determined using the Beckman Coulter Counter.
After experiments, cells were lysed and protein harvested and electrophoresed on a 15% sodium dodecyl sulfate–polyacrylamide gel, transferred to a polyvinylidene fluoride membrane, and blocked for 1 hour at room temperature in 5% nonfat milk in PBS-Tween (PBST). The membrane was incubated with rabbit polyclonal Id3 (Santa Cruz Biotechnology) antibody or rabbit polyclonal antibody to phospho-PKC α/β II (Cell Signaling Technology), washed, and incubated in secondary antibody (horseradish peroxidase-conjugated), and diluted 1:5000 in PBST for 1 hour at room temperature. Immunoreactive bands were visualized by chemiluminescence after incubation with ECL reagent (Amersham Pharmacia Biotech). Blots were stripped and reprobed with antibody to tubulin or β-actin and processed in the same manner.
Total cellular RNA was obtained from equal numbers of VSMCs from 12/15-LO transgenic, 12/15-LO KO, and control mice using TRIzol (Invitrogen). Reverse transcription of 2 µg of total RNA was performed using Thermoscript RT (Invitrogen) and random hexamers. RNA was treated with DNase I (Invitrogen) and RNase Inhibitor (Invitrogen) at room temperature for 10 minutes and the reaction stopped with EDTA. cDNA was diluted 1:5 and 2 µL used for PCR. Qiagen real-time PCR kit with SYBR Green (Qiagen) was used for quantitative PCR. PCR conditions for 12/15-LO and cyclophilin were 95°C for 14 minutes; 40 cycles at 95°C for 15 seconds, 62°C for 30 seconds, and 72°C for 30 seconds; and 81°C for 15 seconds. Primers for mouse 12/15-LO were 5′-ctctcaaggcctgttcagga-3′ (forward) and 5′-gtccattgtccccagaacct-3′ (reverse). Primers for cyclophilin were 5′-tggagagcaccaagacagaca-3′ (forward) and 5′- tgccggagtcgacaatgat-3′ (reverse). Data were analyzed on the basis of the relative expression method with the formula relative expression 2−(SΔCT−CΔCT), where ΔCT is the difference in threshold cycle between the gene of interest and the housekeeping gene.
Primary VSMCs from leukocyte-type 12/15-LO transgenic mice were transfected with 0.5 µg of pK7GFP and either 0.5 µg of a promoterless-luciferase reporter vector (pGL3) or 0.5 µg of an Id3 promoter-luciferase reporter construct (pId3Luc). Cultures were treated with either 5 µmol/L GF109203X (GFX) bisindolemaleimide (Calbiochem), a PKC-α, PKC-βI, PKC-βII, PKC-γ, PKC-δ, and PKC-ε inhibitor, or vehicle. Luciferase activity was determined using Luciferase Assay System (Promega). Results were normalized for protein and transfection efficiency.
Differences between experimental values obtained were evaluated for statistical significance using a 2-tailed Student t test.
Primary VSMCs from leukocyte-type 12/15-LO KO, leukocyte-type 12/15-LO transgenic, and control mice were plated in equal densities, harvested, and analyzed for leukocyte 12/15-LO mRNA by real-time PCR and protein by Western blotting. Transgenic cells demonstrated significantly more 12/15-LO mRNA compared with KO and control cells (P=0.002 for KO versus B6 and transgenic versus B6; Figure 1A). Transgenic cells also demonstrated greater 12/15-LO protein expression compared with control (Figure 1B). Consistent with our mRNA data, KO cells have lower 12/15-LO protein expression in the VSMCs (demonstrated previously in the literature).19
Primary VSMCs from leukocyte-type 12/15-LO KO, leukocyte-type 12/15-LO transgenic, and control mice were plated in equal densities and analyzed for cell number at 0, 24, and 48 hours. Transgenic cells grew faster at 24 and 48 hours relative to control. Conversely, KO cells grew slower relative to control. Results are represented as a percentage of control growth. (Figure 2A). To determine whether transient 12/15-LO inhibition also can affect growth, control VSMCs were plated in equal densities and treated with either an adenoviral vector expressing a specific ribozyme targeting leukocyte-type 12/15-LO or a control adenoviral vector or either 1 µmol/L CDC (a pharmacological 12/15-LO inhibitor) diluted with ethanol or ethanol alone and analyzed for cell number. 12/15-LO inhibition resulted in a significant decrease in cell number at 48 and 72 hours (P=0.004 at 48 hours, P=0.005 at 72 hours for ribozyme versus control; and P=0.009 at 48 hours and P=0.004 at 72 hours for CDC versus control; Figure 2B and 2C).
Primary VSMCs from leukocyte-type 12/15-LO KO, leukocyte-type 12/15-LO transgenic, and control mice were plated in equal densities and Western blots performed at 48 hours. 12/15-LO KO cells demonstrated significantly less Id3 protein expression than control (P<0.001). Conversely, 12/ 15-LO transgenic cells demonstrated significantly more Id3 protein expression than control (P<0.001; Figure 3A and 3B).
To determine whether the effects of 12/15-LO on Id3 protein expression occur at the level of transcription, parallel plates were transfected with an Id3 promoter reporter construct (pId3Luc) and luciferase activity analyzed. 12/15-LO transgenic cells demonstrated significantly more Id3 transcriptional activity than controls (P<0.001 at 24 hours; P=0.03 at 48 hours; Figure 3C). Interestingly, 12/15-LO KO cells did not demonstrate decreased Id3 transcription even though less Id3 protein is present (Figure 3B). These data suggest that endogenous 12/15-LO may regulate Id3 protein expression by post-transcriptional mechanisms. However, it is evident that when 12/15-LO expression is increased, as is the case in response to vascular injury,4 Id3 transcription is significantly enhanced.
To determine whether Id3 is necessary for 12/15-LO–induced growth, parallel plates of control and Id3 KO cells were treated with an adenoviral vector expressing either 12/15-LO (Ad12/15-LO) or GFP (adenovirus GFP [AdGFP]) and assayed for growth. In response to 12/15-LO overexpression, control cells demonstrated increased cell number, whereas Id3 KO cells did not (24 hours Ad12/15-LO versus GFP, P=0.9 for Id3 KO and P=0.03 for control; 48 hours Ad12/15-LO versus GFP, P=0.9 for Id3 KO, P=0.004 for control; Figure 4). This provides strong evidence that Id3 is, indeed, necessary for 12/15-LO–mediated VSMC growth.
Given that 12/15-LO transgenic cells demonstrate increased Id3 transcription and protein expression, it would be interesting to determine possible pathways by which this occurs. Previous studies provide evidence that PKC inhibition attenuates 12S-HETE-induced S-phase entry in adrenocortical cells.26 To determine whether 12/15-LO–induced upregulation of Id3 transcription is mediated by PKC, Western blot analysis and Id3 promoter–reporter assays were performed. Results revealed increased activation of PKC in 12/15-LO transgenic VSMCs relative to control black 6 VSMCs (Figure 5A). In addition, inhibition of PKC decreased Id3 promoter–reporter activity in 12/15-LO transgenic VSMC (P<0.001; Figure 5B). Because PKC inhibition was not isoform specific, future studies to identify the specific PKC isoform would be of interest.
Convincing evidence exists implicating leukocyte-type 12/ 15-LO as a key player in the development of atherosclerosis and restenosis. KO of the 12/15-LO gene in apolipoprotein E– and LDL receptor–deficient mice, 2 models of robust atherosclerosis, resulted in a marked decrease in atherosclerosis development in vivo.5,7 Marked upregulation of 12/ 15-LO occurred in VSMCs after balloon carotid injury in rats, and restenosis was significantly blocked with a pharmacological 12/15-LO inhibitor as well as a specific ribozyme targeted to cleave 12/15-LO.4,27 Many events and pathways involving 12/15-LO have been proposed to contribute to atherosclerosis and restenosis. Much is understood concerning the ability of 12/15-LO within macrophages to participate in the oxidation of LDL, an early event in vascular lesion formation.2,7–9,28 However, 12/15-LO activity and products also increase in endothelial cells and VSMCs at the time of injury and in response to growth factors and cytokines. 12S-HETE, the arachidonic acid product of 12/15-LO, has been shown to increase VSMC migration, hypertrophy, and extracellular matrix production.10,13,29 13S-HPODE, the linoleic acid product of 12/15-LO and the predominant initial product of LDL oxidation, has been shown to increase VSMC proliferation, cytotoxicity, leukocyte chemotaxis, adhesion molecule expression, and inflammatory gene expression. 28,30,31 Interestingly, 12/15-LO and its products remain, to a large extent, intracellular, indicating that 12/15-LO or its products may function to some extent as a type of intracellular second messenger in addition to events possibly initiated from the cell surface by extracellular lipoxygenase products.14
It has been demonstrated that 12/15-LO initiates growth-related signal events within the VSMCs. Lipoxygenase products have been shown to directly activate mitogen-activated protein kinases (MAPKs), and lipoxygenase pathway activation has been shown to lead to the induction of oxidative stress and the formation of reactive oxygen species (ROS).18,19,30,33,34 Further, 12/15-LO products have been shown to activate PKC.26,33,35 Specifically, 12S-HETE has been shown to increase VSMC hypertrophy and fibronectin expression in VSMCs via activation of p38 MAPK and cAMP response element-binding protein (CREB).10,18,19 13S-HPODE enhances c-fos, c-jun, and c-myc expression and activation of MAPK, each of which are potent signal transduction modifiers; and 13-HPODE serves as a ligand for peroxisome proliferator-activated receptors, thus contributing to enhanced uptake of oxidized LDL via the scavenger receptor CD36.9,28,30,31,36 Further, the DNA binding activity of the key MAPK target transcription factors activator protein-1 (AP-1) and CREB is attenuated in 12/15-LO KO cells.19 As well, the expression of the immediate early genes c-fos and c-jun, components of AP-1, are reduced in 12/ 15-LO KO cells.19 Interestingly, the Id3 promoter contains a binding site for AP-1, providing a potential mechanism whereby 12/15-LO may effect the downstream transcription of Id3.37 However, although much is known about molecules that stimulate 12/15-LO and signaling molecules induced by 12/15-LO, little is known about the downstream effects of 12/15-LO on nuclear transcription factors that regulate gene expression related to VSMC proliferation in response to injury. The nuclear transcription factor Id3 has emerged as a key regulator of cell cycle gene expression, and growth in VSMCs and is expressed in response to injury and in native atherosclerosis.20–22 In the present study, we provided the first evidence that leukocyte-type 12/15-LO increases the expression and transcriptional activity of the downstream transcription factor Id3 in VSMCs.
The evidence that 12/15-LO induces Id3 transcription and protein expression is consistent with the role of Id3 as a redox-sensitive gene.21 Previous studies have shown that delivery of Id3 antisense to VSMCs inhibited Ang II– and xanthine/xanthine oxidase–induced increases in DNA synthesis, suggesting that levels of Id3 are important determinants of mitogen- and ROS-induced G1-S progression.21,38 Indeed, 12/15-LO activation increases levels of oxidant stress via generation of oxidized LDL (mainly 13S-HPODE), 12S-HETE, and formation of ROS.28,32,34 Moreover, oxidized LDL induces Id3 expression (data not shown), suggesting that Id3 may be a final common pathway in ROS-induced VSMC proliferation. Results of the present study provide evidence that increased 12/15-LO activity induces PKC expression, and PKC inhibition decreases12/15-LO–induced Id3 transcription, suggesting that 12/15-LO–induced Id3 transcription occurs at least in part through a PKC-mediated pathway. Further identification of specific PKC isoforms, other kinases, and the specific transcription regulatory mechanisms involved in 12/15-LO–induced Id3 expression may provide important insight into the molecular mechanisms that regulate the effects of 12/15-LO on VSMC growth and vascular lesion formation.
This work was supported by National Institutes of Health (NIH) grants RO1 HL-62522 (C.A.M.), P01 HL-55798 (C.C.H., C.A.M., J.L.N., R.N.), and NIH training grant T32 HL-07355 (A.M.T.). We thank Linda Lanting and Suseela Srinivasan for technical assistance and Yuan Zhuang for providing the Id3 KO mice.