|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
CTRP3/cartducin is induced by transforming growth factor-β1 and promotes vascular smooth muscle cell proliferation
Takashi Maeda1 and Satoshi Wakisaka
Department of Anatomy and Cell Biology, Graduate School of Dentistry, Osaka University, 18 Yamadaoka, Suita, Osaka 5650871, Japan
CTRP3 (C1q and tumour necrosis factor-related protein 3)/cartducin, a novel serum protein, is a member of the CTRP superfamily. Although the CTRP3/cartducin gene is markedly up-regulated in rat carotid arteries after balloon injury, little is known about its biological roles in arterial remodelling and neointima formation in injured blood vessels. We have investigated the mechanisms underlying CTRP3/cartducin up-regulation and the in vitro effects of CTRP3/cartducin on vascular smooth muscle cells. CTRP3/cartducin expression in cultured p53LMAC01 vascular smooth muscle cells was induced by TGF-β1 (transforming growth factor-β1), but not by bFGF (basic fibroblast growth factor) or PDGF-BB (platelet-derived growth factor-BB). Exogenous CTRP3/cartducin promoted the proliferation of p53LMAC01 cells in a dose-dependent manner via ERK1/2 (extracellular signal-regulated kinase 1/2)- and MAPK (p38 mitogen-activated protein kinase)-signalling pathways. In contrast, CTRP3/cartducin exhibited no effect on the migration of p53LMAC01 cells. Taken together, the results of the present study demonstrate a novel biological role of CTRP3/cartducin in promoting vascular smooth muscle cell proliferation in blood vessel walls after injury.
Key words: CTRP3/cartducin, mitogen-activated protein kinase (MAPK), p53LMAC01 cell, proliferation
Abbreviations: bFGF, basic fibroblast growth factor, BrdU, bromodeoxyuridine, CTRP3, C1q and tumour necrosis factor-related protein 3, DMEM, Dulbecco's modified Eagle's medium, ERK1/2, extracellular signal-regulated kinase 1/2, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, JNK, c-Jun N-terminal kinase, MAPK, p38 mitogen-activated protein kinase, PDGF-BB, platelet-derived growth factor-BB, RT, reverse transcription, TGF-β1, transforming growth factor-β1, VSMC, vascular smooth muscle cell
1To whom correspondence should be addressed (email firstname.lastname@example.org).
Vascular restenosis subsequent to PTCA (percutaneous transluminal coronary angioplasty) is a serious limitation of this procedure. Restenosis results from the remodelling of the vessel wall and/or accumulation of cells and ECM (extracellular matrix) in the intimal layer. The major cellular component of the restenotic lesion is the VSMC (vascular smooth muscle cell), and VSMCs of the arterial wall play a critical role in the development of the lesion (Ross, 1993). In response to vessel wall injury, many cytokines and growth factors are released from platelets, endothelium, macrophages and VSMCs within injured blood vessels (Owens et al., 2004; Tedgui and Mallat, 2006). These mediators in turn alter the gene expression patterns of vascular cells, leading to the phenotypic transition of VSMCs and neointimal formation (Austin et al., 1985).
A recent study showed that the C1q and TNF (tumour necrosis factor)-related protein 3 (CTRP3)/cartducin gene is markedly up-regulated in balloon-injured rat carotid arteries but not in normal vessels (Li et al., 2007). CTRP3/cartducin was first identified as a growth plate cartilage-derived secretory protein during a search for genes underlying the induction of chondrocyte differentiation (Maeda et al., 2001). Wurm et al. (2007) showed that CTRP3/cartducin is also abundant in human plasma. We revealed that this molecule is a novel factor that plays important roles in regulating the growth and/or differentiation of several types of cells, such as chondroprogenitor (Akiyama et al., 2006; Maeda et al., 2006), endothelial (Akiyama et al., 2007) and tumour (Akiyama et al., 2009) cells. Up-regulation during embryonal development (Maeda et al., 2006) and in response to damage, and its role in cell proliferation and/or migration, suggests a function in tissue remodelling. CTRP3/cartducin shows a structural homology with the serum protein adiponectin and belongs to a new highly conserved family of adiponectin paralogues designated as CTRPs (Kishore et al., 2004). There are ten members of the CTRP family, and the proteins in this family exhibit a similar structural organization to adiponectin and consist of four distinct domains including an N-terminal signal peptide, a short variable domain, a collagen-like domain and a C-terminal C1q-like globular domain (Wong et al., 2004, 2009). While structurally related, members of this protein family are functionally diverse. CTRP1 has been reported to be expressed in the vascular wall tissues and to inhibit collagen-induced platelet aggregation by blocking the binding of von Willebrand factor to collagen (Lasser et al., 2006). Similarly to adiponectin, CTRP2 activates AMPK (AMP-activated protein kinase) in muscle cells, resulting in increased glycogen accumulation and enhanced fatty acid oxidation (Wong et al., 2004). CTRP5 is localized to the lateral and apical membranes of the retinal pigment epithelium and ciliary body, and mutations in this gene cause late-onset retinal macular degeneration (Mandal et al., 2006).
To date, mechanisms underlying CTRP3/cartducin up-regulation and its possible biological functions in the vessel wall remain unknown. In the present study, we showed that TGF-β1 (transforming growth factor-β1), a key factor present in the vessel wall in the early phases of the arterial response to injury, induces CTRP3/cartducin expression. We further demonstrated that CTRP3/cartducin had direct effects on VSMC proliferation by activating both ERK1/2 (extracellular-signal-regulated kinase 1/2) and PI3K (phosphoinositide 3-kinase)/Akt pathways.
2. Materials and methods
Recombinant human TGF-β1 was obtained from R&D Systems. Recombinant human bFGF (basic fibroblast growth factor) was obtained from Sigma–Aldrich. Recombinant human platelet-derived growth factor-BB (PDGF-BB) was obtained from PeproTech. Mouse recombinant CTRP3/cartducin was prepared as described previously (Maeda et al., 2006). Anti-mouse CTRP3/cartducin antibody was purchased from R&D Systems. Anti-ERK1/2, anti-p38 MAPK (mitogen-activated protein kinase) and anti-β-actin antibodies were purchased from Sigma–Aldrich. p-ERK1/2 (anti-phospho-ERK1/2), p-p38 MAPK (anti-phospho-p38 MAPK), p-JNK1/2 [anti-phospho-JNK1/2 (c-Jun N-terminal kinase 1/2)] and p-Akt (anti-phospho-Akt) antibodies were purchased from Cell Signaling Technology. The MEK1/2 (MAPK/ERK kinase) inhibitor U0126 and p38 MAPK inhibitor SB203580 were purchased from Calbiochem.
2.2. Cell line and cell culture
The mouse vascular smooth muscle cell line p53LMAC01, established from aortic smooth muscles of p53 knock-out mice (Ohmi et al., 1997), was obtained from the HSRRB (Health Science Research Resources Bank) and maintained on collagen type I-coated dishes. The cells were cultured in DMEM (Dulbecco's modified Eagle's medium) (Sigma-Aldrich) supplemented with 10% FCS (fetal calf serum) (JRH Biosciences), 4 mM l-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. To investigate the effects of growth factors and cytokines on CTRP3/cartducin expression, the cells were plated at 8×105 cells per 100-mm-diameter dish coated with collagen type I (Nitta Gelatin). After 48 h, the medium was replaced with fresh medium, and cells were stimulated with one of the following: TGF-β1 (10 ng/ml), bFGF (10 ng/ml) or PDGF-BB (10 ng/ml). To investigate the effects of CTRP3/cartducin on the MAPK signalling pathways, the cells were seeded at a density of 1×104 cells/well in 24-well plates coated with collagen type I and grown for 48 h. The cells were then washed and cultured for 24 h in the medium without serum. Subsequently, 5 μg/ml of CTRP3/cartducin was added to the medium for 5, 15, 30 and 60 min. For experiments with protein kinase inhibitors, cells were pretreated with specific inhibitors for 1 h prior to CTRP3/cartducin treatment. In the control experiments, 50 mM NaH2PO4 (pH 8.0) containing 1 mM EDTA and/or 0.4% DMSO was added to the culture.
2.3. RT (reverse transcription)–PCR analysis
Total RNAs were extracted from cells using the RNeasy Kit (Qiagen). A 2 µg portion of total RNA was reverse-transcribed using the Omniscript RT Kit (Qiagen). After the RT reaction, 35 cycles of PCR were carried out, as previously described (Maeda et al., 2006). Primer sequences were as follows: CTRP3/cartducin, 5′-GAAACAATGGGAACAATGGAG-3′ and 5′-TGCTGAAGGTGAAGAAATACA-3′, amplifying a 300-bp fragment; GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 5′-CCATCACCATCTTCCAGGAG-3′ and 5′-GCATGGACTGTGGTCATGAG-3′, amplifying a 322-bp fragment.
2.4. Measurement of DNA synthesis
To determine the growth-stimulatory effect of CTRP3/cartducin on p53LMAC01 cells, the BrdU (bromodeoxyuridine) assay was performed, as described previously (Maeda et al., 2006). In brief, p53LMAC01 cells were seeded at a density of 5×103 cells/well in 96-well plates coated with type I collagen and grown for 24 h. The medium was then replaced with serum-free DMEM for 24 h. Subsequently, various concentrations of CTRP3/cartducin were added to the medium, incubated for 24 h and labelled with BrdU during the last 3 h of incubation.
2.5. Measurement of cell migration
The motility response of p53LMAC01 cells to CTRP3/cartducin was assayed using a modified Boyden chamber technique. p53LMAC01 cells were trypsinized, washed and resuspended in serum-free DMEM containing 0.25% BSA. The cell suspension (100 μl, 5×104 cells/well) was added to the transwell inserts (8.0 μm pore size, Corning), and the insert was then incubated for 2 h to allow cell attachment. Then, 500 μl of serum-free DMEM containing 0.25% BSA with or without various concentrations of CTRP3/cartducin was added to the lower chamber and incubated for 5 h. Non-migrating cells on the top of the membrane were removed by scraping, and migrated cells on the lower surface of the membrane were fixed with ethanol and stained with haematoxylin. The number of cells migrating through the membrane was counted in five random fields per well under a microscope (×400 magnification). The addition of 10 ng/ml of PDGF-BB was also performed as a positive control. All assays were carried out in triplicate.
2.6. Western blot analysis
Protein immunoblotting was performed as previously described (Akiyama et al., 2006). Briefly, total cellular proteins were prepared by lysing cells in CelLytic lysis buffer (Sigma–Aldrich) containing protease inhibitor (Sigma–Aldrich) and phosphatase inhibitor (Sigma–Aldrich) cocktails, separated by SDS/PAGE and transferred to PVDF membranes. The membranes were blocked for 30 min at room temperature (20–25°C) and then incubated with primary antibodies directed against CTRP3/cartducin, ERK1/2, p-ERK1/2, p38 MAPK, p-p38 MAPK, p-JNK1/2, p-Akt and β-actin for 18 h at 4°C. The detection of bound antibodies was performed by employing the WesternBreeze Chromogenic Detection System (Invitrogen) using AP (alkaline phosphatase)-conjugated donkey anti-rabbit IgG antibody (Promega).
2.7. Statistical analysis
An unpaired Student's t test was used for statistical analysis of the experiments. Error bars represent S.D., and P<0.05 was taken as the level of significance.
3.1. TGF-β1 induces CTRP3/cartducin mRNA and protein expression in p53LMAC01 cells
In response to injury, VSMCs in the vessel wall are exposed to a number of cytokines and growth factors released from activated platelets, inflammatory cells and damaged vascular cells. For the purpose of identifying growth factors that might be involved in the regulation of CTRP3/cartducin expression, we examined the levels of CTRP3/cartducin mRNA after growth factor stimulation in the mouse vascular smooth muscle cell line p53LMAC01. Total RNA was extracted and reverse-transcribed for PCR after stimulation with growth factors for 3, 6, 12 or 24 h. Although the expression of CTRP3/cartducin mRNA was undetectable in unstimulated p53LMAC01 cells (∼90% confluence), TGF-β1 significantly induced CTRP3/cartducin mRNA in a time-dependent manner. The expression of CTRP3/cartducin mRNA was elevated and reached a maximum at 24 h after stimulation with TGF-β1 (Figure 1A). In contrast, other growth factors, such as bFGF and PDGF-BB, did not induce CTRP3/cartducin mRNA expression in p53LMAC01 cells (Figure 1A). We subsequently examined by Western blot analysis the expression of CTRP3/cartducin protein in p53LMAC01 cells at 24 h after stimulation with TGF-β1. Similar to the mRNA results, TGF-β1 induced CTRP3/cartducin protein expression in these cells (Figure 1B).
3.2. CTRP3/cartducin promotes the proliferation, but not migration, of p53LMAC01 cells
Since the proliferation and migration of VSMCs following endothelial injury are critical events in the development of vascular occlusive disease, the function of CTRP3/cartducin in VSMCs was studied. We first examined the effect of CTRP3/cartducin on p53LMAC01 cell proliferation. A dose-dependent increase in BrdU incorporation into the DNA was observed in p53LMAC01 cells. A maximal stimulation of DNA synthesis occurred in the presence of 5 μg/ml of CTRP3/cartducin (1.9-fold when compared with controls, P<0.05) (Figure 2). We subsequently examined whether CTRP3/cartducin affected VSMC migration. Cell migration was analysed using a modified Boyden chamber technique. CTRP3/cartducin, however, had no significant effects on the migration of p53LMAC01 cells (Figure 3).
3.3. CTRP3/cartducin activates ERK1/2 and p38 MAPK pathways in p53LMAC01 cells
MAPK and PI3K/Akt pathways respond to mitogenic factors (Seger and Krebs, 1995; Lawlor and Alessi, 2001). Therefore, we analysed the effects of CTRP3/cartducin on the phosphorylation of three groups of MAPKs, such as ERK, JNK and p38 MAPK, or Akt in p53LMAC01 cells. Western blot analysis detected increased ERK1/2 phosphorylation in these cells treated with 5 μg/ml of CTRP3/cartducin after 15 min, with a maximal increase occurring after 30 min of treatment and decreasing after 1 h (Figure 4). Similarly, increased p38 MAPK phosphorylation was also detected after 15 min, with it decreasing after 1 h (Figure 4). In contrast, CTRP3/cartducin had no effect on the activities of JNK1/2 and Akt, and none of their phosphorylated forms were detected (data not shown).
3.4. ERK1/2 and p38 MAPK pathways are involved in the CTRP3/cartducin-induced proliferation of p53LMAC01 cells
Because Western blot analysis confirmed CTRP3/cartducin-induced ERK1/2 and p38 MAPK pathway activation in p53LMAC01 cells, we next determined whether CTRP3/cartducin-induced VSMC proliferation is mediated through the activation of these pathways. U0126 and SB203580 alone had no effect on proliferation, and no toxicity at the concentration used was observed. U0126, as well as SB203580, blocked CTRP3/cartducin-induced p53LMAC01 cell proliferation (Figure 5). Thus both ERK1/2 and p38 MAPK pathways are required for the CTRP3/cartducin-induced proliferation of VSMCs.
CTRP3/cartducin was originally identified as a C1q-like globular domain containing chondrocyte-specific secretory protein (Maeda et al., 2001). CTRP3/cartducin and adiponectin have highly homologous structures characteristic of the CTRP family, consisting of ten members. Using microarray analysis coupled with real-time RT–PCR. Li et al. (2007) showed that CTRP3/cartducin gene expression was strongly up-regulated in the neointima in the balloon-injured rat carotid artery during a period characterized by neointima formation. So far, no studies have addressed the expression and regulation of CTRP3/cartducin in the vasculature. In the present study, we sought to identify factor(s) that could potentially up-regulate CTRP3/cartducin expression in the context of vascular injury. We demonstrated that CTRP3/cartducin mRNA and protein expression in mouse VSMCs in vitro was markedly induced by TGF-β1, but not by bFGF or PDGF-BB. TGF-β1, a multifunctional cytokine, is a key factor present in the blood vessel wall in the early phase of the arterial response to injury and plays a significant role in modulating lesion formation (Majesky et al., 1991; Nikol et al., 1992). Interestingly, Li et al. (2007) report that not only CTRP3/cartducin, but also TGF-β1, were among the most significantly induced genes in a rat carotid artery balloon-injury model, and the temporal expression patterns of both genes were similar. On the other hand, bFGF or PDGF-BB induction was not found (Li et al., 2007). Further, the CTRP3/cartducin induction by TGF-β1 in p53 LMAC01 cells is consistent with our previous finding in C3H10T1/2 cells (Maeda et al., 2001), suggesting there is a conserved site in the CTRP3/cartducin promoter that is critical for response to TGF-β1. However, the transcriptional mechanisms are not known. Further studies are needed to investigate the mechanisms. Although it is unclear whether CTRP3/cartducin expression was detected in VSMCs in injured arteries (Li et al., 2007), it is likely that CTRP3/cartducin is induced in VSMCs in the neointima after injury.
CTRP3/cartducin was previously found to be mitogenic and to induce the migration of endothelial cells (Akiyama et al., 2007). To date, the biological roles of CTRP3/cartducin up-regulation in the injured vascular wall are unknown. In response to arterial injury, VSMCs undergo a phenotypic transition whereby they migrate and proliferate from the media into the intima, contributing to vascular lesion formation and arteriosclerosis (Owens et al., 2004). Since CTRP3/cartducin mRNA is among the most strongly up-regulated transcripts in rat carotid arteries after balloon injury (Li et al., 2007), we hypothesized that VSMC-secreted CTRP3/cartducin is involved in the proliferation and/or migration of VSMCs in the injured vascular wall. In order to investigate the direct effect of CTRP3/cartducin on VSMC proliferation and migration, recombinant CTRP3/cartducin was used. The present study, using the mouse VSMC line p53LMAC01, demonstrated that CTRP3/cartducin could promote VSMC proliferation in a dose-dependent manner; however, CTRP3/cartducin had no effect on migration in this VSMC line in vitro. On balloon catheter injury, VSMCs are induced to change from a previously quiescent to a highly proliferative state. The proliferation of VSMCs induced by injury to the intima of arteries is an important aetiological factor in vascular proliferative disorders such as restenosis after angioplasty. Our results support the hypothesis that CTRP3/cartducin secreted by VSMCs in the injured vascular wall is involved in restenosis by activating VSMCs in an autocrine/paracrine manner.
In contrast with adiponectin (Yamauchi et al., 2003), no CTRP3/cartducin-specific receptor has yet been identified and cloned. Although MAPK and/or PI3K/Akt pathways are known to be activated by CTRP3/cartducin stimulation in chondroprogenitor (Akiyama et al., 2006), endothelial (Akiyama et al., 2007) and tumour (Akiyama et al., 2009) cells, little is known about their signalling pathways in VSMCs. To gain insight into the mechanisms by which CTRP3/cartducin promotes the proliferation of VSMCs, we examined both the MAPK and PI3K/Akt pathways after CTRP/cartducin stimulation. We found that ERK1/2 and p38 MAPK signalling pathways were activated in CTRP3/cartducin-treated p53LMAC01 cells. The ERK1/2 and p38 MAPK pathways have been showed to be activated by cytokines and growth factors, such as visfatin, PDGFs and IGF-1 (insulin-like growth factor-1) in cultured VSMCs (Yamaguchi et al., 2001; Duan, 2003; Wang et al., 2009), and these pathways are known to be important for their proliferation. Using specific kinase inhibitors, we were able to study the role of these signalling pathways and clarify the pathways leading to proliferation in CTRP3/cartducin-stimulated VSMCs. CTRP3/cartducin-induced DNA synthesis in p53LMAC01 cells was inhibited by both U0126 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK), suggesting that the mitogenic effect of CTRP3/cartducin is mediated through both ERK1/2 and p38 MAPK pathways. The p53LMAC01 cells used here are derived from aortic smooth muscles of p53 knock-out mice. The cells reveals extended bipolar shape and expressed h-caldesmon and calponin as well as α-smooth muscle actin as protein markers of differentiated smooth muscle (Ohmi et al., 1997). The responses to PDGF-AA, -AB and -BB were compared with those of human aortic smooth muscle cells, and p53LMAC01 cells exhibited a distinct proliferative response to PDGF similar to that of aortic smooth muscle cells (Lu et al., 2001). Further, our previous study using a chondrocytic cell line derived from p53 knock-out mice showed no influence of p53 deficiency on the effects of CTRP3/cartducin on the cells (Maeda et al., 2006). These reports suggest that the p53LMAC01 cell line is useful for vascular smooth muscle biology experiments. In conclusion, we have demonstrated that TGF-β1 induces CTRP3/cartducin mRNA and protein expression in VSMCs. CTRP3/cartducin positively regulates the proliferation of VSMCs via distinct MAPK signalling pathways. Together with the finding that a strong induction of CTRP3/cartducin occurs after vascular injury in rat models (Li et al., 2007), our results suggest that CTRP3/cartducin secreted from VSMCs in response to injury may play an important role in directing VSMC proliferation in the pathophysiology of neointimal hyperplasia and restenosis following angioplasty.
Takashi Maeda conceived the experiments. He performed the experiments and analysed the data together with Satoshi Wakisaka. Both authors discussed the results and commented on the manuscript.
This work was supported by a Grant-in-aid for Scientific Research from
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Received 9 June 2009/1 September 2009; accepted 26 October 2009
Published as Cell Biology International Immediate Publication 26 October 2009, doi:10.1042/CBI20090043
© The Authors Journal compilation © 2010 Portland Press Ltd
ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)