Proprotein Convertase PC5 Regulation by PDGF-BB Involves PI3-Kinase/p70s6-Kinase Activation in Vascular Smooth Muscle Cells
We have recently demonstrated that furin, PC5, and PC7, members of the subtilisin/kexin-like mammalian proprotein convertases (PCs), are found in rodent aorta. These PCs have been identified to activate several growth factors, adhesion molecules and extracellular matrix compounds by endoproteolytic cleavage. In the present study, we investigated the regulation of PC5 in vascular smooth muscle cells (VSMCs) in vitro and in vivo. Stimulation of rat aortic VSMCs with platelet-derived growth factor (PDGF)-BB (20 ng/mL), angiotensin II (Ang II, 1 μmol/L), or 10% fetal calf serum (FCS) for 48 hours increased DNA synthesis, as assessed by proliferating cell nuclear antigen (PCNA) immunoblotting. PC5 was strongly upregulated by PDGF-BB and 10% FCS (both 8-fold, P<0.05), whereas Ang II had no effect on PC5 protein levels compared with controls. The PCs furin and PC7, which display a comparable subcellular localization and cleavage activity, were found in VSMCs, but their levels did not increase following PDGF-BB, Ang II, or FCS stimulation. Time-course analysis revealed a rapid increase in PC5 levels after 30 minutes of PDGF-stimulation of VSMCs. PDGF-stimulated PC5 induction was inhibited by the PI3-kinase inhibitor wortmannin, and by rapamycin, an inhibitor of mTOR/p70s6-kinase (both P<0.05). In contrast, the mitogen-activated protein kinase (MAPK)-pathway inhibitor PD98059 did not inhibit PDGF-stimulated PC5 induction. Immunocytochemistry and in situ hybridization revealed low PC5 protein and mRNA levels in intact rat aorta in vivo. After balloon injury, PC5 protein and mRNA levels were strongly increased in proliferating PCNA-positive VSMCs. The present data demonstrate that PC5 is upregulated during proliferation of VSMCs in vivo and in vitro. We show that PDGF-induced PC5 expression is PI3-kinase/p70s6-kinase dependent. Thus, growth factors regulate the proprotein convertase PC5, which may play an important role during VSMC growth.
Several growth factors and adhesion molecules undergo limited endoproteolytic cleavage downstream from the endoplasmic reticulum to become biologically active. This type of cleavage takes place at the carboxyl side of basic residues with the general cleavage motif (K/R)-(X)n-(K/R)↓. Generally, this requires the presence of suitable proprotein convertases (PCs), which are processing enzymes that belong to the kexin/subtilisin-like family. So far, 7 mammalian proprotein convertases (PCs) have been identified, including furin, PC1 (also named PC3), PC2, PC4, PACE4, the two PC5 isozymes A and B (also known as PC6), and PC7.1
PC1 and PC2 are mostly found in neuroendocrine tissue, where they are sorted into the secretory granules of the regulated pathway of protein secretion.1 PC4 is unique in that it is exclusively confined to testicular germ cells.2 In contrast, furin, PC5, and PC7 display a more widespread and overlapping tissue distribution.1 These PCs are sorted to the constitutive pathway of protein secretion,1 where they activate a number of diverse substrates, including growth factors such as endothelin-13 and transforming growth factor (TGF)-β1,4 extracellular matrix proteins,5 matrix metalloproteinases,6 viral envelope glycoproteins,7 as well as adhesion molecules.8
So far, little is known about the regulation of PCs in vascular smooth muscle cells (VSMCs). Using a serum-free organ culture system, we have recently reported the expression of furin, PC5, and PC7 in extracts of the rat aorta, whereas PC1 and PC2 were undetectable in vascular tissue.9 Based on this observation, PCs might be important intermediates during VSMC growth, thereby participating in proliferation and migration of VSMCs.
Angiotensin II (Ang II) is a key player in vascular remodeling involved in hypertension, arteriosclerosis, and restenosis. Beside its role as a vasoconstrictor, Ang II is a potent growth factor that leads to hypertrophy and protein synthesis in VSMCs.10 The platelet-derived growth factors (PDGFs) are the most potent known mitogens and are thought to play a major role in arterial remodeling.11 Both growth factors exert their effects via activation of numerous intracellular signaling cascades. The ERK1/2 MAP-kinase and the ribosomal S6 kinase (p70s6-kinase) are key regulators, which have been implicated in mitogen-induced cell growth.12,13⇓ ERK1/2 is activated by the Ras-Raf-mitogen–activated protein kinase-kinase (MEK-signaling) cascade and regulates the expression of a variety of transcription factors and early response genes.14 The ribosomal p70s6-kinase, a downstream target of the phosphatidylinositol 3-kinase (PI3K)→ Akt-pathway, is the major in vivo regulator of the phosphorylation of the 40S ribosomal protein S6, thereby controlling protein synthesis and cell cycle progression.15
We therefore investigated the regulation of PCs by the growth factors Ang II and PDGF-BB in VSMCs and the involvement of the ERK-MAPK pathway and PI3K/p70s6-kinase pathway in these signaling events.
Materials and Methods
Ang II and PDGF-BB were purchased from Bachem. Wortmannin, PD98059, and rapamycin were from Biomol. Cell culture media and materials were from GIBCO. All other chemicals were purchased from Sigma. The following antibodies were used: anti α-smooth muscle actin (α-SMA; Dako); anti proliferating cell nuclear antigen (PCNA; Novocastra Laboratories), anti–trans-Golgi-network 38 (TGN38; ABR Affinity Bioreagents), rabbit-anti PC5 NT antibody,16 rabbit-anti PC717, and rabbit-anti furin.17 Levels of signaling pathway induction were assessed by the use of phospho-specific antibodies, recognizing ERK1/2 MAP-kinase when phosphorylated at threonine 202 and tyrosine 204 (Promega), Akt when phosphorylated at serin 473 (Cell Signaling), and p70s6-kinase when phosphorylated at threonine 421 and serine 424 (Cell Signaling).
Rat VSMCs were derived from normal Sprague-Dawley rat thoracic aortae through the explant technique as described.18 Cells were kept at 95% relative humidity and 5% CO2 at 37° in 10% FCS (fetal calf serum)-DMEM, supplemented with penicillin 100 U/mL, 100 μg/mL streptomycin, and 200 mmol/L glutamine. By staining with a monoclonal antibody against α-SMA, cells were identified as VSMCs. Cells were passaged by trypsination, and passage 4 to 6 subconfluent cells were used throughout this study. For experiments, VSMCs were rendered quiescent by serum-starvation in serum-free DMEM for 48 hours. At the time of experiments, medium was changed and cells were stimulated as indicated. In inhibition experiments, inhibitors were added 1 hour before stimulation. All experiments were done in triplicates, with different preparation of VSMCs.
Western Blot Analysis
Immunoblotting was done as described9 with modifications. Proteins were extracted in RIPA-buffer (×1 PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) containing freshly dissolved protease inhibitors (Complete EDTA-free, Boehringer). Up to 50 μg of proteins were mixed with sample buffer and applied on 8% SDS-polyacrylamide gel electrophoresis. After migration, proteins were transferred onto nitrocellulose membranes (BioRad). The membranes were first incubated overnight at 4°C with the primary antibody, followed by incubation with donkey anti-rabbit or donkey anti-mouse IgG-peroxidase (Dianova) diluted 1:20 000. Peroxidase activity product was revealed with the enhanced chemiluminescence method (ECL) (Amersham). Semiquantitative densitometry was performed with the National Institutes of Health (NIH) software program, Image 1.62, and is expressed in arbitrary units (AU). Analysis of variance and paired and unpaired t test were used for statistical analysis as appropriate. Statistical significance was designated as P<0.05.
VSMCs were platted on 0.2% gelantine-coated plastic camber slides (Nunc). Cells were fixed with 4% buffered formaline (10 minutes) at room temperature. Nonspecific binding was blocked with 10% normal-goat serum. Cells were washed with PBS/0.01% Triton-X100 and incubated with the primary antibody overnight at 4°C. After washing with PBS/0.01% Triton-X100 several times, FITC- and Texas Red (TXR)-conjugated secondary antibodies were used as secondary antibodies (Vector). In double-label experiments, species-specific antibodies were used to distinguish primary antibodies.
Immunocytochemical stainings were performed with the ABC Zymed Histostain-Plus kit (Zymed Laboratories). Double immunocytochemical stainings were done by combining alkaline phosphatase and immunostaining with horseradish peroxidase as described previously.9
In Situ Hybridization
In situ hybridization (ISH) was performed as described17 with paraffin tissue sections using a [35S]-UTP cDNA probe (rPC51089–1925 equivalent). The sections were hybridized overnight at 55°C. Hybridization was examined on x-ray films (exposure time 2 to 3 days), followed by autoradiography with NTB-2 emulsion (Kodak) for 15 days at 4°C and developed in D19 solution (Kodak). The sections were then stained with hematoxylin-eosin and viewed under bright-field as well as dark-field illumination.
Balloon Injury Model of the Rat Aorta
Adult male Spargue-Dawley rats weighing 300 to 350 g were used in this study. All procedures were in accordance with the guidelines of the Canadian Council on Animal Care. Animals were anesthetized with ketamine (60 mg/kg, IM) and xylazine (6.6 mg/kg, IM) and received 100 IU heparin, IV, before surgery. The left common carotid was exposed by blunt dissection. The proximal artery was ligated and a 2F Fogarty catheter (Baxter Healthcare Corp) was introduced, placed in the distal aorta approximately above the bifurcation, inflated to create a distending resistance, and pulled back to the carotid (repeated 5 times). Sham operated animals were subject to the same procedure, except balloon inflation. After removal of the catheter the distal carotid was ligated and wounds were closed with surgical clips. Four to six animals were killed at 2, 7, 14, and 28 days after the operation. They received a dose of 100 IU heparin, IV, and were sacrificed by lethal intraperitoneal injection of phentobarbital (50 mg/kg). Sections of the vessels from various regions along the injured segment were fixed in 4% buffered formaldehyde for 24 hours, then washed for 24 hours in 70% ethanol and embedded in paraffin.
PC5 is Induced by PDGF-BB, but Not by Ang II in VSMCs
VSMCs were made quiescent by serum-starvation for 48 hours and then stimulated with Ang II (1 μmol/L), PDGF-BB (20 ng/mL), or 10% FCS for 48 hours. Immunoblotting revealed the presence of furin, PC5, and PC7 in unstimulated VSMCs (Figure 1A). Low amounts of PC5 were found in 48-hour serum-starved VSMCs. The level of PC5 was significantly increased by stimulation with PDGF-BB (20 ng/mL) and 10% FCS (both 8-fold; P<0.05 versus control) (Figures 1A and 1B). This increase of PC5 by PDGF-BB and FCS was accompanied by increased PCNA levels (Figures 1A and 1C). Ang II (1 μmol/L) stimulation moderately increased PCNA by 10-fold (P<0.05 versus control), but had no effect on PC5. Compared with PC5, relatively high levels of furin and PC7 were found in serum-starved VSMCs, which did not change on stimulation with Ang II, PDGF-BB, or FCS (Figure 1A). Time-course analysis revealed that PDGF-BB induces a rapid upregulation of PC5 expression with a significant increases by 3-fold after 30 minutes stimulation (P<0.05 versus control) (Figures 2A and 2B).
Induction of PC5 by PDGF-BB Requires Activity of PI3-Kinase and p70S6-Kinase Phosphorylation, but Not ERK1/2 MAP-Kinase Phosphorylation
To investigate which signaling pathways are involved in PDGF-induced PC5 upregulation, inhibition experiments were performed using the pharmacological ERK-MAPK inhibitor PD98059,19 the PI3K inhibitor wortmannin20, and the mTOR (mammalian target of rapamycin)/p70S6-kinase inhibitor rapamycin.20
Inhibitors were added 1 hour before stimulation with PDGF-BB (20 ng/mL) and proteins extracted following 1-hour PDGF stimulation in the presence of inhibitors. PD98059 (30 μmol/L), significantly inhibited PDGF-induced ERK1/2 MAP-kinase phosphorylation by ≈70% (P<0.05 versus PDGF alone), but had no effect on PC5 induction (Figure 3A). PDGF-BB stimulated phosphorylation of Akt, and p70s6-kinase was not affected by PD98059. Wortmannin (200 nmol/L), an inhibitor of the PI3K pathway, completely inhibited phosphorylation of its downstream target Akt and did not change levels of pMAPK. In contrast to PD98059, wortmannin significantly inhibited PDGF-stimulated PC5 induction (P<0.05 versus PDGF alone) (Figures 3A and 3B) and also significantly lowered the levels of phosphorylation of p70s6-kinase (P<0.05 versus PDGF alone) (Figure 3C). Rapamycin (10 ng/mL), an inhibitor of mTOR, completely abrogated p70s6-kinase phosphorylation and was the most potent inhibitor of PDGF-induced PC5 upregulation (P<0.05 versus PDGF alone) (Figures 3A and 3C).
Interestingly, Ang II also induced the rapid and potent activation of ERK1/2 MAPK, pAkt, and p70s6-kinase (data not shown), although it did not induce PC5 expression.
PC5 Is Localized in the Trans-Golgi Network of VSMCs
Immunofluorescence double-labeling with PC5 and α-SMA antibodies (Figures 4A and 4B) confirmed PC5 expression in VSMCs. To identify the subcellular localization of PC5, double-staining with the monoclonal marker-antibody TGN38 was performed following brefeldin A (BFA) (10 μg/mL; 30 minutes) treatment before fixation.21 After treatment with BFA, PC5 concentrated in the condensed paranuclear compartment, which is characteristic for the trans-Golgi network (TGN). Double-labeling with TGN38 confirmed that PC5 staining is predominantly localized to the TGN (Figure 4C and 4D). Staining of PC5 without BFA showed a wider distribution (Figure 4A). Using epitope-tagged PC5B constructs, Xiang et al21 recently demonstrated that PC5B staining dispersed with BFA treatment, because it localizes to the earlier lamellae of the TGN, which collapse by BFA. However, in constitutive cells, PC5A is localized in the noncollapsible part of the TGN8 and, hence, colocalizes with TGN38 following BFA. The antibody used in this study recognizes both PC5 isoforms.16 Similar to PC5, and as demonstrated in other cell lines,21 furin also localizes to the TGN (Figures 4E and 4F).
PC5 mRNA and Protein are Upregulated During Arterial Remodeling in the Rat Aorta
In intact vessels, in situ hybridization revealed low steady-state PC5 mRNA signals distributed throughout intima, media, and adventitia (Figures 5A and 5A′). Following balloon injury, a strong increase of PC5 mRNA levels was evident within the established neointima on day 28 (Figures 5B and 5B′). Immunocytochemical analysis revealed a similar distribution of PC5 protein. Little PC5 protein was found in VSMCs in the media in intact vessels, whereas strong upregulation was found in VSMCs on day 2 in the media and on days 7, 14, and 28 in VSMCs in the established neointima (data not shown). Colocalization studies demonstrated that PC5 localized mostly to PCNA-positive VSMCs on day 2 in the media and on day 14 in the neointima (Figures 5D and 5E).
In the present study, we demonstrate that PC5 is upregulated by PDGF-BB, but not Ang II. Neither of the growth factors had any effect on the levels of furin and PC7. The increase in PC5 levels by PDGF-BB stimulation were PI3K/p70s6-kinase–dependent and associated with increased DNA synthesis assessed by PCNA analysis. Ang II, which moderately increased PCNA, did not change the levels of furin, PC5, or PC7 even though it stimulates PI3K/p70s6-kinase.13,22⇓ We also demonstrate upregulation of PC5 mRNA levels in the neointima of balloon-injured rat aorta. Double-labeling immunocytochemistry reveals colocalization of PC5 protein and PCNA in VSMCs in the media and, later on, in the neointima during arterial remodeling in vivo.
A common response of cells to both mitogenic and hypertrophic factors is the initiation of protein synthesis.13 One of the rate-limiting steps in protein synthesis is the initiation of translation, which is accompanied by increased activity of the eukaryotic transcription factor 4E (eIF-4E)23 and ribosomal S6 protein phosphorylation by p70s6-kinase.15 In the absence of growth factor stimulation, eIF-4E is bound to 4E-BP1/PHAS-1, which dissociates on phosphorylation.23 Both PI3K and mTOR signaling are required for 4E-BP and p70s6-kinase phosphorylation.24 The ERK1/2 MAP-kinase pathway is also involved in growth factor–induced protein synthesis. The MAPK-pathway inhibitor PD98059 inhibits growth factor–induced protein synthesis,12 even though it lies on a pathway distinct from PI3K and p70s6-kinase.15
Here, we demonstrate that the PI3K inhibitor wortmannin and the p70s6-kinase inhibitor rapamycin significantly inhibit PDGF-stimulated PC5 induction in VSMCs. Rapamycin was the most potent inhibitor of PDGF-stimulated PC5 induction. In contrast, the MAPK-pathway inhibitor PD98059 had no effect on PDGF-stimulated PC5 induction.
Our subcellular colocalization studies with TGN38 demonstrate that PC5 and furin are localized to the TGN in VSMCs. The TGN, being distinct from the Golgi stack, plays an important role in the sorting and translocation of proteins. Posttranslational processing of proproteins by PCs takes place in the TGN and/or secretory granules.1 The changes in staining patterns following BFA indicate that both PC5 isoforms are present. PC5B, a splice variant of PC5A, which differs from it by a C-terminal extension and a transmembrane domain which affects its trafficking,16 has been demonstrated to localize to a BFA-sensitive compartment distinct from furin.21 Because it has been reported that PC5A also localizes in secretory vesicles in some cell lines16, this PC seems to function in both the constitutive and regulated pathway of protein secretion.
So far, most research on PCs has focused on furin, which has served as a “model” convertase. Furin has been identified as the converting enzyme of TGF-β1 and is regulated by it.4 Even though PC5 and PC7 have several structural, biochemical, and cell biological similarities to furin, TGF-β1 did not increase the levels of PC5 or PC7 mRNAs.4 In vascular endothelial cells, furin increases with increased fluid shear stress, whereas PC5 remains unaffected.25 Furthermore, studies in other cell lines report ERK-MAPK–dependent regulation of furin.26 Although we did not observe that PDGF-BB or Ang II mediated regulation of furin and PC7 in VSMCs, further research is needed to clarify the role of these PCs in the vasculature.
In conclusion, we demonstrate that even though several PCs are redundantly expressed in VSMCs, only PC5 is upregulated by PDGF-BB via PI3K/p70s6-kinase–dependent signal transduction. The finding that PC5 is expressed in proliferating VSMCs following balloon injury in vivo and that its signaling involves pathways important for the initiation of protein synthesis, indicates that this PC might be important during arterial remodeling.
M.M. was supported by grants from the Canadian Health Medical Research Institute (MT14466) and an award from the Canadian Stroke Network (D1). The authors thank Nabil G. Seidah (Montreal) for provision of PCs antibodies and Edwige Marcinkiewicz (Montreal) for expert technical assistance.
- Received September 23, 2001.
- Revision received October 29, 2001.
- Accepted November 8, 2001.
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