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Cell-permeable mitochondrial ubiquinol–cytochrome c reductase binding protein induces angiogenesis in vitro and in vivo
Cancer Letters 366 (2015) 52–60
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Cell-permeable mitochondrial ubiquinol–cytochrome c reductase binding protein induces angiogenesis in vitro and in vivo Junghwa Chang a, Hye Jin Jung b, Hyun-Ji Park a, Seung-Woo Cho a, Sang-Kyou Lee a, Ho Jeong Kwon a,c,* a Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea b Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70, Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of Korea c Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea
A R T I C L E
I N F O
Article history: Received 19 April 2015 Received in revised form 1 June 2015 Accepted 2 June 2015 Keywords: Angiogenesis HIF-1α Mitochondria Pro-angiogenic agent PTD UQCRB
A B S T R A C T
Ubiquinol–cytochrome c reductase binding protein (UQCRB), a component of the mitochondrial complex III, has been recently implicated in angiogenesis. Targeting mitochondria to balance vascular homeostasis has been widely recognized. However, the effect of UQCRB replenishment by direct delivery remains unknown. To explore the biological function of UQCRB in angiogenesis, a novel protein transduction domain (PTD)-conjugated UQCRB fusion protein was generated. PTD-UQCRB localized to mitochondria as does endogenous UQCRB. Treatment with PTD-UQCRB generated mitochondrial reactive oxygen species (mROS) without cytotoxicity, following hypoxia inducible factor-1α (HIF-1α) stabilization and downstream vascular endothelial growth factor (VEGF) expression. Accordingly, PTD-UQCRB induced angiogenesis in vitro and PTD-UQCRB pro-angiogenic activity was further validated in matrigel plug assay and in cutaneous wound-healing mouse models in vivo. Together, these results demonstrate that UQCRB plays a role in angiogenesis and the developed cell-permeable PTD-UQCRB can be utilized as a pro-angiogenic agent. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Regarding angiogenesis, mitochondria play an important role in vascular homeostasis by regulating numerous biological processes such as mitochondrial ROS production [1,2], calcium loading [3], or mitochondrial enzyme activation [4]. However, the regulation of specific mitochondrial proteins to uncover their function in angiogenesis has not been fully understood. Ubiquinol–cytochrome c reductase binding protein (UQCRB), the 13.4-kDa subunit of the mitochondrial complex III, has recently been implicated in angiogenesis by virtue of its ability to modulate hypoxia-induced mROSand hypoxia inducible factor (HIF)-mediated signaling in cancer cells [2] as well as mROS-mediated vascular endothelial growth factor receptor 2 (VEGFR2) signaling in endothelial cells (ECs) [5]. Moreover, UQCRB has been identified as a target protein of the natural anti-angiogenic compound, terpestacin. In a previous study, UQCRB overexpression was shown to significantly increase tumor cell invasion under normoxic conditions by elevating mROS generation and subsequently increasing HIF-1α stability and VEGF expression [2]. In addition, UQCRB positively regulates VEGFR2 signaling
* Corresponding author. Tel.: +82 2 2123 5883; fax: +82 2 362 7265. E-mail address: [email protected] (H.J. Kwon). http://dx.doi.org/10.1016/j.canlet.2015.06.013 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
by promoting mROS generation and results in increased EC migration in vitro [5]. Therefore, overexpression experiments demonstrated that UQCRB presents a pro-angiogenic activity. Protein transduction domains (PTDs) are small cationic amphipathic peptides comprising 30 amino acids or less that can assist in the uptake of large and biologically active proteins into mammalian cells [6]. A number of PTDs have been identified and utilized for receptor- and transporter-independent protein delivery across biological membranes [7,8]. Hph-1-PTD, a novel human transcription factor-derived PTD, can also facilitate efficient uptake of fusion proteins [9]. In this study, we employed a similar PTD strategy to deliver UQCRB directly into the cells in order to explore the potential role of UQCRB in angiogenesis. Herein, we show that the PTD-UQCRB fusion protein is a potent pro-angiogenic agent in vitro and in vivo. Materials and methods Molecular cloning, expression, and purification of PTD-UQCRB To isolate the PTD-UQCRB fusion protein from bacteria, a PCR-amplified fulllength UQCRB cDNA (accession no. NM_006294) was inserted into the EcoRI/XhoI sites of a pRSET-B expression vector containing the cell permeable protein transduction domain (Hph-1 domain). Escherichia coli BL21 (DE3) StarpLysS cells that were transformed with plasmids encoding PTD-UQCRB were grown in Luria–Bertani medium containing ampicillin (50 μg/mL) and chloramphenicol (34 μg/mL) at 37 °C
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up to an absorbance of 0.6 at 600 nm. Protein expression was induced using 0.1 M isopropyl-β-d-thiogalactopyranoside and cells were grown for 16 h. The cells were collected by centrifugation, suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, protease inhibitor cocktail tablets, pH 8.0), and then disrupted by sonication. After centrifugation at 13,000 rpm for 15 min at 4 °C, Ni-NTA agarose beads (Qiagen, Hilden, Germany) were added to the supernatant and incubated at 4 °C for 2 h. After incubation with the beads, the supernatant was loaded on Poly-Prep chromatography columns (Bio-Rad, Hercules, CA). Bound proteins were washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluted proteins were desalted using PD-10 Sephadex G-25 (GE Healthcare, Buckinghamshire, UK), supplemented with 10% glycerol, separated into aliquots and flashfrozen at −20 °C. Cell culture HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GibcoBRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, GibcoBRL). HUVECs were seeded in gelatin-coated plates containing endothelial cell basal medium (Cambrex Walkersville, MD, USA) supplemented with 10% FBS. HepG2 cells were cultured in Roswell Park Memorial Institute medium 1640 (Gibco-BRL) supplemented with 10% FBS. HT1080 cells were cultured with minimum essential medium (Gibco-BRL) supplemented with 10% FBS. The cells were maintained in a humidified incubator with 5% CO2. Cell proliferation assay Cells were seeded onto 96-well plates and incubated for 24 h for stabilization. Various concentrations of PTD-UQCRB were added to each well and incubated for 2 days. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) colorimetric assay.
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Metropolitan Institute of Medical Science, Japan) and has been described previously [10]. HepG2 cells were transfected with 0.5 μg of the reporter construct and, after a 24 h incubation, cells were collected and lysed using lysis buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, pH 8.0) and centrifuged at 13,000 rpm. Cell lysates were obtained and assayed for luciferase activity by using the SteadyGlo® Luciferase Assay System (Promega, Madison, WI, USA). Luminescence was measured using a FL600 Microplate Fluorescence Reader (Bio-Tek Instrument, Inc., Winooski, VT, USA). Chemoinvasion assay HUVEC invasiveness was evaluated in vitro by using the Transwell chamber system and 8.0-μm polycarbonate filter inserts (Corning Costar, Cambridge, MA, USA). The lower and upper sides of the filter were coated with 10 μL of gelatin (1 mg/mL, SigmaAldrich) and 10 μL of matrigel (3 mg/mL, BD Biosciences), respectively. HUVECs (1 × 105) were placed in the upper chamber and conditioned medium (CM) was placed in the lower chamber. The chamber was then incubated at 37 °C for 16 h, fixed with methanol, and then stained with hematoxylin and eosin (H&E; Sigma-Aldrich). Cells that had invaded the lower part of the filter were detected using optical microscopy at 100× magnification. Wound treatment Rectangular wounds (1.5 × 1.5 cm2) were created on the dorsal skin of each 5-week-old hairless mouse (BALB/c nude female mice, Orient Co., Seongnam, Korea). Mice were anesthetized with an intraperitoneal injection of tribromoethanol (Avertin®, 250 mg/kg) during the surgery. Analgesic (ketoprofen, 0.1 mg/kg) was administered subcutaneously for 4 d post-surgery. The following 3 treatment groups were established: PTD-UQCRB, PTD-EGFP, and PBS (n = 5–7). PTD-tagged fusion protein (200 μg) or PBS mixed with fibrin matrix (Greenplast, Greencross PD Co., Yongin, Korea) was applied to the wound. After treatment, the wounds were dressed with Tegaderm (3M Health Care, St. Paul, MN, USA). The animal study was approved by the Yonsei Laboratory Animal Research Center (2011-0062).
Fluorescence imaging Histological and immunohistochemical analyses To monitor the localization of PTD-UQCRB, HeLa cells were incubated in 4 μM of PTD-UQCRB for 1 h and were then fixed with 4% formaldehyde (Sigma-Aldrich). After washing with phosphate buffered saline (PBS), cells were co-stained with 1 μg/mL Hoechst and 0.5 μg/mL MitoTracker for 30 min. Finally, cells were washed with distilled water, mounted, and analyzed by confocal microscopy (Carl Zeiss LSM 510 META, Germany). Isolation of mitochondria Mitochondria were isolated by differential centrifugation using a mitochondrial isolation kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Measurement of mitochondrial ROS Mitochondrial ROS level was measured using the red mitochondrial superoxide indicator, MitoSOX™ (Gibco-BRL). HeLa cells were grown on cover glass (Decklaser, Lauda-Konlgshofen, Germany) and treated with 1 μM of MitoSOX™. Following 20-min incubation at 37 °C, cells were washed with PBS and then treated with PTDUQCRB and incubated again for 2 h at 37 °C. After washing with PBS, cells were stained with 1 μg/mL Hoechst to visualize the nuclei. Cells were washed with PBS and then fixed with 4% formaldehyde for 15 min at 37 °C. After 3 PBS washes, samples were finally washed with distilled water, mounted, and analyzed by fluorescence microscopy (Olympus America, Inc., Melville, NY, USA). SDS-PAGE and western blot analysis HeLa cells grown in 6-well plates were lysed and the lysates were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using standard methods. Blots were blocked and immunolabeled overnight at 4 °C with the following primary antibodies: anti-UQCRB (Sigma-Aldrich, St. Louis, MO, USA), anti-penta-His (Qiagen, Hilden, Germany), anti-VDAC (Cell Signaling, Danvers, MA, USA), anti-HIF-1α (BD Bioscience, Bedford, MA, USA), anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-tubulin (Millipore, Billerica, MA, USA). Immunolabeling was detected by an enhanced chemiluminescence (ECL) kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions.
Mice were euthanized 2 weeks post-wound treatment. The repaired tissues were bisected and used for histological analysis and immunohistochemistry. For the histological analysis, specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated with a graded ethanol series, and embedded in paraffin. Specimens were sliced into 4-μm thick sections and stained using H&E and Masson’s trichrome (M-T). The tissue sections were also immunohistochemically stained with anti-involucrin (Abcam, Cambridge, UK) and anti-smooth muscle α-actin (Abcam) antibodies. Measurement of epidermal thickness Epidermal thickness was measured for each treatment in sections stained using anti-involucrin and M-T at 3 random points across the wound. Matrigel plug assay A matrigel plug assay was performed to assess in vivo angiogenesis as previously described [5]. Briefly, 7-week-old C57BL/6 mice (Orient Co.) were injected subcutaneously with 0.6 mL of matrigel supplemented with 20 μg of PTD-UQCRB or VEGF (200 ng/mL). After 5 d, the mice were euthanized and the matrigel plugs were retrieved and photographed. The hemoglobin levels were measured with the Drabkin Reagent kit 525 (Sigma-Aldrich) to quantitate the matrigel angiogenesis. Statistical analysis All quantitative data were expressed as the mean ± standard error of the mean (±s.e.m.) and all statistical analyses were calculated with GraphPad Prism (ver. 5.00 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com). Student’s t-tests were used to determine statistical significance between control and test groups. A p-value less than 0.05 was considered statistically significant (* indicates p < 0.05, ** indicates p < 0.005, *** indicates p < 0.0001).
Results Cell permeable PTD-UQCRB fusion protein was generated to replenish UQCRB protein
Hypoxia responsive element (HRE) reporter gene assay HepG2 cells were grown at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS. The cells were seeded in 6-well plates 24 h before plasmids were transfected using Lipofectamine LTX (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The reporter plasmid construct used for the HREluciferase reporter assay was kindly provided by Dr. Futoshi Shibasaki (Tokyo
To verify the potential role of UQCRB as an angiogenesis enhancer, we generated a cell-permeable Hph-1-PTD peptideconjugated UQCRB protein (PTD-UQCRB) (Fig. 1A). The full length UQCRB gene was subcloned into the pRSET-B protein expression vector, which contained the protein transduction domain (PTD),
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Fig. 1. Generation of PTD-UQCRB fusion protein. (A) PTD-UQCRB DNA construct. DNA sequence of Hph-1 PTD and individual restriction enzyme sites are represented. (B) DNA gel electrophoresis of subcloned pRSET-B-PTD-UQCRB after EcoRI and XhoI digestion. (C) Selection of a host strain for protein expression. BL21 Star pLysS was selected. (D) Purification of PTD-UQCRB using Ni-NTA agarose beads. The beads bind with expressed PTD-UQCRB which are tagged with EGFP after IPTG induction. (E) Purified PTD-UQCRB fusion protein (43 kDa).
mitochondria (Fig. 2C). As shown in Fig. 2D, western blot analysis of mitochondrial fractions confirmed that PTD-UQCRB localized to the mitochondrial inner-membrane, where endogenous UQCRB protein exists. The mitochondrial targeting signal of UQCRB (amino acids 1–35) could be responsible for delivery and functional localization of PTD-UQCRB to mitochondria [11].
Hph-1 (Supplementary Fig. S1 and Fig. 1B). The Hph-1 PTD enabled the delivery of its fusion partner, UQCRB, into the cells. A 6× His tag was inserted to allow protein purification using Ni-NTA beads. Enhanced green fluorescence protein (EGFP) was also included to detect the intracellular delivery of PTD-UQCRB fusion protein by immunofluorescence. In order to confirm that the PTD-UQCRB DNA construct was correct, PTD-UQCRB DNA construct was sequenced and the sequencing results also showed that PTD and UQCRB were perfectly inserted. To obtain the PTD-UQCRB fusion protein, three bacterial strains for protein expression were tested. Out of three strains, BL21 StarpLysS strain was chosen because of its excellent capability to express PTD-UQCRB fusion protein than the other strains (Fig. 1C). PTD-UQCRB fusion proteins were purified with Ni-NTA beads (Fig. 1D), following a series of standard purification steps using imidazole containing buffers. Imidazole was subsequently removed via desalting and purified PTD-UQCRB fusion proteins were identified by SDS-PAGE (Fig. 1E) and immunoblotting with anti-UQCRB antibody (data not shown).
Since UQCRB is a complex III subunit of the mitochondrial respiratory chain, PTD-UQCRB transduction may affect mROS production. Indeed, PTD-UQCRB treatment increased mROS levels, as indicated by the increase in MitoSOX™ fluorescence (red), an mROS indicator, compared to control (Fig. 3A). In addition, mROS levels augmented with increasing PTD-UQCRB concentrations, an effect that was observed with UQCRB treatment (Fig. 3B and C). Notably, generated mROS, resulting from PTD-UQCRB treatment, did not affect cell proliferation (Fig. 3D).
PTD-UQCRB specifically localizes to mitochondria
PTD-UQCRB induces angiogenesis through HIF-1α activation in vitro
To investigate the intracellular localization of PTD-UQCRB, the ability of PTD-UQCRB to penetrate cells was investigated. PTDUQCRB (green fluorescence) was detected mainly in the cytoplasm, rather than in nuclei (Fig. 2A), demonstrating that Hph-1 PTD helps the delivery of UQCRB across cellular membranes. PTD-UQCRB transduction was achieved in an efficient and dose-dependent manner (Fig. 2B). By co-staining the cells with the mitochondrial marker MitoTracker, we observed that PTD-UQCRB localized to the
Because increased intracellular mROS stabilizes HIF-1α protein [12] and mediates transcription of downstream angiogenic factors, including VEGF [13,14], we next investigated the effect of PTDUQCRB on HIF-1α stability in cells. Under normoxia, HIF-1α is normally hydroxylated by prolyl hydroxylases [15], resulting in oxygen-dependent degradation. However, HIF-1α protein accumulated dose dependently in PTD-UQCRB-treated cells, a phenomenon not observed in cells treated with PTD-EGFP as a negative control
Generation of mitochondrial ROS by PTD-UQCRB treatment
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Fig. 2. Subcellular localization of PTD-UQCRB. (A) Subcellular localization of PTD-UQCRB (2 μM) in HT1080 cells detected by confocal microscopy (NT: not-treated, PC: phase contrast). Scale bar, 20 μm. (B) Total fluorescence intensity of EGFP was measured in cells treated with 1, 2, and 4 μM of PTD-UQCRB. (C) Mitochondrial localization of PTDUQCRB. After a 1 h treatment with 2 μM of PTD-UQCRB, HT1080 cells were stained with Hoechst and MitoTracker to observe nuclei and mitochondria, respectively. PTDUQCRB was localized in mitochondria. Scale bars, 20 μm. (D) Cytosolic and mitochondrial cell fractions were isolated using differential centrifugation. Proteins were analyzed using antibodies against UQCRB, penta-His (PTD-UQCRB), and VDAC (mitochondrial membrane protein). C: cytosol, X: mitochondrial matrix fraction, M: mitochondrial membrane fraction.
(Fig. 4A and B). The results showed that mROS generation, resulting from PTD-UQCRB treatment, was sufficient to stabilize HIF-1α protein. In the following hypoxia-responsive element (HRE) reporter gene assay, PTD-UQCRB transduction led to a higher luminescence intensity of HRE-luciferase than that of the control (Fig. 4C). This confirmed that HIF-1α was stabilized under normoxic conditions, likely because increased mROS can contribute to the dimerization with HIF-1β [16], resulting in the formation of active transcriptional complexes that induce the expression of the downstream gene, VEGF. Indeed, intracellular delivery of PTD-UQCRB significantly increased VEGF levels in a dose-dependent manner (Fig. 4D, E and Supplementary Fig. S2). Interestingly, the increased VEGF expression by PTD-UQCRB treatment was significantly suppressed by terpestacin (Fig. 4F). Terpestacin directly targets UQCRB leading to suppression of mROS production, HIF-1α stability and VEGF expression to inhibit tumor angiogenesis [2]. Therefore, the result confirmed that elevation of mROS generation through PTD-UQCRB treatment is sufficient for VEGF expression. To determine whether PTD-UQCRB has pro-angiogenic activity in vitro, conditioned medium
(CM) from PTD-UQCRB-treated HeLa cells was collected and used to treat human umbilical vascular endothelial cells (HUVECs). The PTD-UQCRB CM treatment strongly promoted the invasive behavior of HUVECs compared to control CM (Fig. 4G). These results clearly demonstrate that PTD-UQCRB potentiates angiogenesis in vitro.
PTD-UQCRB enhances angiogenesis and wound healing in vivo To demonstrate that PTD-UQCRB can induce angiogenesis in vivo, a matrigel plug assay was performed in mice. PTD-UQCRB significantly increased angiogenesis within matrigel plugs, to a level comparable to that induced by VEGF (Fig. 5A). In addition, the level of hemoglobin consistently supported the pro-angiogenic effect of PTD-UQCRB. The pro-angiogenic activity of PTD-UQCRB was further assessed in vivo by using a cutaneous wound healing mouse model. Wounds (1.5 × 1.5 cm) were created on the dorsal skin of 5-weekold BALB/c nude mice that were subsequently treated with PTDUQCRB or PBS mixed with fibrin matrix. Notably, PTD-UQCRB
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Fig. 3. Effect of PTD-UQCRB on mROS production. (A) mROS levels were determined by measuring the MitoSOX™ fluorescence in HeLa cells. Images representing mROS production after PTD-UQCRB treatment (NT: not-treated, left) and bar graphs showing the average intensity of one cell (right). Scale bar, 20 μm. (B) Elevated mitochondrial mROS levels were measured in HeLa cells treated with 1, 2, and 4 μM of PTD-UQCRB by using MitoSOX™. (C) Measurement of MitoSOX™ fluorescence intensity in cells treated with 4 μM of PTD-UQCRB than those not-treated (NT) and 4 μM PTD-EGFP-treated control cells. (D) Effect of PTD-UQCRB on cell growth. 1, 2 and 4 μM of PTDUQCRB were added to HUVEC, HeLa, and HepG2 cells and incubated for 2 days. Cell proliferation was measured using MTT colorimetric assay. Data represent mean ± standard error of the mean (±s.e.m.) compared to control (*** indicates p < 0.0001).
resulted in remarkable wound closure activity 9 d post-treatment (Fig. 5B). The percentage of wound closure was 64.6% in the PTDEGFP control group, which increased up to 85.4% in the PTDUQCRB treatment group. Histology of the repaired tissues was examined using H&E and M-T staining methods. It showed that considerable epidermal regeneration occurred in the PTD-UQCRB
treatment group compared to the control groups (Fig. 5C). In addition, immunohistochemical staining performed using an antiinvolucrin antibody showed epidermal regeneration. Overall, PTDUQCRB treatment showed a 2-fold increase over the control groups in epidermal thickness (Fig. 5D). Furthermore, a 3-fold increase in the number of arterioles was detected in wound healing regions of
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Fig. 4. PTD-UQCRB induces angiogenesis by activating HIF-1α in vitro. (A) Effect of PTD-UQCRB treatment on HIF-1α stability. Western blot analysis was used to assess HIF1α protein levels in HeLa cells treated with PTD-UQCRB (2 and 4 μM) and PTD-EGFP (2 and 4 μM) as a control for 4 h. Tubulin served as an internal control. (B) Quantitative results of the HIF-1α western blot analysis. (C) Effect of PTD-UQCRB treatment on HRE reporter gene expression. HepG2 cells were transiently transfected with HREluciferase vectors. Cells were treated with 1 and 2.5 μM PTD-UQCRB for 4 h. Luciferase luminescence was measured to assess the effect of PTD-UQCRB on HRE reporter gene. (D) Effect of PTD-UQCRB treatment on VEGF expression. VEGF protein levels were evaluated in HeLa cells treated with PTD-UQCRB (2 and 4 μM) and PTD-EGFP (2 and 4 μM) as control for 24 h by performing western blot analysis. Tubulin served as an internal control. (E) Quantitative results of the VEGF western blot analysis. (F) Regulation of VEGF induction with UQCRB inhibitor, terpestacin. 50 μM terpestacin was used to treat HeLa cells for 12 or 24 h following pre-treatment with 2 or 4 μM PTD-UQCRB for 2 h. Tubulin was used as an internal control. (G) Pro-angiogenic effect of PTD-UQCRB in HUVECs. HeLa cells were treated with PTD-UQCRB (2 and 4 μM) for 24 h in serum free medium before collecting CM (NT: not-treated). HUVECs were incubated for 16 h in the CM. The collected CM stimulates HUVEC invasion. Quantified data are presented as mean (±s.e.m.) compared to control (* indicates p < 0.05, ** indicates p < 0.005 and *** indicates p < 0.0001).
PTD-UQCRB-treated mice assessed by smooth muscle alpha actin (SMA) staining (Fig. 5E). Taken together, these results show that PTDUQCRB treatment promotes angiogenesis and enhances wound healing in vivo.
Discussion In the present study, a novel PTD-UQCRB fusion protein was generated to allow the intracellular delivery of UQCRB. Previously, to
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Fig. 5. PTD-UQCRB transduction enhances cutaneous wound healing in vivo. (A) Effect of PTD-UQCRB on in vivo angiogenesis in matrigel plugs. C57BL/6 mice were injected subcutaneously with matrigel supplemented with PBS, PTD-UQCRB (20 μg), or VEGF (200 ng/mL). After 5 d, matrigel plugs were retrieved and analyzed (n = 6 per group). The hemoglobin levels were measured to quantify blood vessel formation in the matrigel plugs. (B) Wound closure was evaluated in BALB/c nude mice (n = 5–7) under different treatment conditions. Macroscopic analysis of wounds and wound closure analysis under the following treatment conditions: PTD-UQCRB (200 μg), PTD-EGFP (200 μg), and PBS. (C) Histological and immunohistochemical staining of the mid-wound healing region for each treatment group (H&E: hematoxylin and eosin staining and M-T: Masson’s trichrome staining). The arrows indicate the thickness of epidermis (H&E (white), M-T (white), involucrin (black)). Scale bars indicate 100 μm in the H&E and M-T stained specimens, 200 μm in those stained using anti-involucrin. (D) Quantitative analysis of the thickness of the regenerated epidermis. (E) Immunohistochemical staining of smooth muscle α-actin (SMA) in the mid-wound healing region for each treatment group and quantitative data for arterioles in healed regions. The arrows point to arterioles (SMA, red). Scale bars indicate 50 μm. (F) Schematic summary of PTD-UQCRB function. When PTD-UQCRB permeates cells, the mitochondrial complex III-derived ROS production is induced. Increased mROS stabilize HIF-1α. The stable heterodimer complex, containing HIF-1, translocates to the nucleus where it initiates the transcription of the target gene, VEGF, which leads to angiogenesis and cutaneous wound healing. Quantified data are presented as mean (±s.e.m.) compared to control (** indicates p < 0.005, *** indicates p < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. (continued)
investigate its biological role, UQCRB was overexpressed by DNA transfection. However, direct delivery of UQCRB protein into cells by using PTD enabled efficient transduction of UQCRB in vitro and in vivo. In addition, PTD-UQCRB treatment had a strong proangiogenic effect as well as effects on wound healing, suggesting that PTD-UQCRB has great potential as a potent pro-angiogenic agent. Unlike a similar mitochondrial inner membrane protein, prohibitin-1, which was also identified as an angiogenic factor [17], PTD-UQCRB enhanced angiogenesis by inducing mROS production. Although high mROS levels are known to have harmful effects on cell growth, resulting in apoptosis [18], mROS, at low levels, are thought to act as signaling molecules to regulate various cellular processes, including differentiation, metabolic adaptation, and immune cell activation [19]. Consistent with this report, PTDUQCRB treatment did not show cytotoxicity (Fig. 3D). However, the mechanism by which PTD-UQCRB transduction increases mROS and results in angiogenesis will be investigated in a future study. HIF-1α induction is commonly implemented as a strategy to enhance wound healing [14,20–22]. Consistent with previous studies, PTD-UQCRB significantly increased HIF-1α stability, resulting in an increase in VEGF expression, followed by induction of angiogenesis. Notably, however, PTD-UQCRB is different from previous agents in that it can be applied topically. Recently, our group reported the biological effect of UQCRB mutation, originated from a patient with lactic acidosis, in angiogenesis
[1]. The mutant has 7 modified amino acids as well as 14 additional amino acids on the C-terminal end. We constructed the UQCRB mutant stable cell lines, which exhibited glycolytic, proliferative, and pro-angiogenic activities through the mROS-induced HIF-1 signaling pathway. It is surprising that the biological traits of the UQCRB mutant and UQCRB supplementation present highly relevant similarities in angiogenesis. Presumably, similarities resulted from changes in complex III conformation. However, the unsolved mechanism should be examined in a future study. In this study, we showed that PTD-UQCRB presents a proangiogenic activity in vitro and in vivo. To our best knowledge, this is the first report on the application of UQCRB in vivo. UQCRB, a component of the mitochondrial complex III, plays a critical role in angiogenesis. These results provide new insights into the biological role of UQCRB in angiogenesis by demonstrating that it plays a role in mROS production and leads to HIF-1α activation (Fig. 5F). In addition, these data provide the basis for the medical application of PTD-UQCRB as a pro-angiogenic agent. Acknowledgements This work was partly supported by grants from the National Research Foundation of Korea grant funded by the Korean government (MSIP; 2010-0017984, 2012M3A9D1054520), Translational Research Center for Protein Function Control, NRF (2009-0083522),
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Ministry of Health and Welfare (0620360-1), and Brain Korea 21 Plus Project, Republic of Korea.
Conflict of interest The authors declare that they have no conflict of interest.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.06.013.
References [1] J. Chang, H.J. Jung, S.H. Jeong, H.K. Kim, J. Han, H.J. Kwon, A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species, Biochem. Biophys. Res. Commun. 455 (2014) 290–297. [2] H.J. Jung, J.S. Shim, J. Lee, Y.M. Song, K.C. Park, S.H. Choi, et al., Terpestacin inhibits tumor angiogenesis by targeting UQCRB of mitochondrial complex III and suppressing hypoxia-induced reactive oxygen species production and cellular oxygen sensing, J. Biol. Chem. 285 (2010) 11584–11595. [3] K.H. Kim, D. Kim, J.Y. Park, H.J. Jung, Y.H. Cho, H.K. Kim, et al., NNC 55-0396, a T-type Ca channel inhibitor, inhibits angiogenesis via suppression of hypoxiainducible factor-1alpha signal transduction, J. Mol. Med. (Berl) 93 (2015) 499–509. [4] R. Solito, F. Corti, C.H. Chen, D. Mochly-Rosen, A. Giachetti, M. Ziche, et al., Mitochondrial aldehyde dehydrogenase-2 activation prevents beta-amyloidinduced endothelial cell dysfunction and restores angiogenesis, J. Cell Sci. 126 (2013) 1952–1961. [5] H.J. Jung, Y. Kim, J. Chang, S.W. Kang, J.H. Kim, H.J. Kwon, Mitochondrial UQCRB regulates VEGFR2 signaling in endothelial cells, J. Mol. Med. 91 (2013) 1117– 1128. [6] L.N. Patel, J.L. Zaro, W.C. Shen, Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives, Pharm. Res. 24 (2007) 1977–1992. [7] F. Heitz, M.C. Morris, G. Divita, Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics, Br. J. Pharmacol. 157 (2009) 195– 206.
[8] M.C. Morris, J. Depollier, J. Mery, F. Heitz, G. Divita, A peptide carrier for the delivery of biologically active proteins into mammalian cells, Nat. Biotechnol. 19 (2001) 1173–1176. [9] J.M. Choi, M.H. Ahn, W.J. Chae, Y.G. Jung, J.C. Park, H.M. Song, et al., Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation, Nat. Med. 12 (2006) 574–579. [10] H. Kato, S. Tamamizu-Kato, F. Shibasaki, Histone deacetylase 7 associates with hypoxia-inducible factor 1alpha and increases transcriptional activity, J. Biol. Chem. 279 (2004) 41966–41974. [11] H. Suzuki, Y. Hosokawa, H. Toda, M. Nishikimi, T. Ozawa, Cloning and sequencing of a cDNA for human mitochondrial ubiquinone-binding protein of complex III, Biochem. Biophys. Res. Commun. 156 (1988) 987–994. [12] T. Klimova, N.S. Chandel, Mitochondrial complex III regulates hypoxic activation of HIF, Cell Death Differ. 15 (2008) 660–666. [13] J.A. Forsythe, B.H. Jiang, N.V. Iyer, F. Agani, S.W. Leung, R.D. Koos, et al., Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1, Mol. Cell. Biol. 16 (1996) 4604–4613. [14] A. Ahluwalia, A.S. Tarnawski, Critical role of hypoxia sensor – HIF-1alpha in VEGF gene activation. Implications for angiogenesis and tissue injury healing, Curr. Med. Chem. 19 (2012) 90–97. [15] M. Gruber, M.C. Simon, Hypoxia-inducible factors, hypoxia, and tumor angiogenesis, Curr. Opin. Hematol. 13 (2006) 169–174. [16] G.L. Wang, B.H. Jiang, E.A. Rue, G.L. Semenza, Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 5510–5514. [17] M. Schleicher, B.R. Shepherd, Y. Suarez, C. Fernandez-Hernando, J. Yu, Y. Pan, et al., Prohibitin-1 maintains the angiogenic capacity of endothelial cells by regulating mitochondrial function and senescence, J. Cell Biol. 180 (2008) 101–112. [18] N. Zamzami, P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch, et al., Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death, J. Exp. Med. 182 (1995) 367–377. [19] L.A. Sena, N.S. Chandel, Physiological roles of mitochondrial reactive oxygen species, Mol. Cell 48 (2012) 158–167. [20] B.D. Kelly, S.F. Hackett, K. Hirota, Y. Oshima, Z. Cai, S. Berg-Dixon, et al., Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1, Circ. Res. 93 (2003) 1074–1081. [21] L. Liu, G.P. Marti, X. Wei, X. Zhang, H. Zhang, Y.V. Liu, et al., Age-dependent impairment of HIF-1alpha expression in diabetic mice: correction with electroporation-facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells, J. Cell. Physiol. 217 (2008) 319–327. [22] E. Andrikopoulou, X. Zhang, R. Sebastian, G. Marti, L. Liu, S.M. Milner, et al., Current Insights into the role of HIF-1 in cutaneous wound healing, Curr. Mol. Med. 11 (2011) 218–235.
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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t
Original Articles
Cell-permeable mitochondrial ubiquinol–cytochrome c reductase binding protein induces angiogenesis in vitro and in vivo Junghwa Chang a, Hye Jin Jung b, Hyun-Ji Park a, Seung-Woo Cho a, Sang-Kyou Lee a, Ho Jeong Kwon a,c,* a Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science & Biotechnology, Yonsei University, Seoul 120-749, Republic of Korea b Department of BT-Convergent Pharmaceutical Engineering, Sun Moon University, 70, Sunmoon-ro 221, Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of Korea c Department of Internal Medicine, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea
A R T I C L E
I N F O
Article history: Received 19 April 2015 Received in revised form 1 June 2015 Accepted 2 June 2015 Keywords: Angiogenesis HIF-1α Mitochondria Pro-angiogenic agent PTD UQCRB
A B S T R A C T
Ubiquinol–cytochrome c reductase binding protein (UQCRB), a component of the mitochondrial complex III, has been recently implicated in angiogenesis. Targeting mitochondria to balance vascular homeostasis has been widely recognized. However, the effect of UQCRB replenishment by direct delivery remains unknown. To explore the biological function of UQCRB in angiogenesis, a novel protein transduction domain (PTD)-conjugated UQCRB fusion protein was generated. PTD-UQCRB localized to mitochondria as does endogenous UQCRB. Treatment with PTD-UQCRB generated mitochondrial reactive oxygen species (mROS) without cytotoxicity, following hypoxia inducible factor-1α (HIF-1α) stabilization and downstream vascular endothelial growth factor (VEGF) expression. Accordingly, PTD-UQCRB induced angiogenesis in vitro and PTD-UQCRB pro-angiogenic activity was further validated in matrigel plug assay and in cutaneous wound-healing mouse models in vivo. Together, these results demonstrate that UQCRB plays a role in angiogenesis and the developed cell-permeable PTD-UQCRB can be utilized as a pro-angiogenic agent. © 2015 Elsevier Ireland Ltd. All rights reserved.
Introduction Regarding angiogenesis, mitochondria play an important role in vascular homeostasis by regulating numerous biological processes such as mitochondrial ROS production [1,2], calcium loading [3], or mitochondrial enzyme activation [4]. However, the regulation of specific mitochondrial proteins to uncover their function in angiogenesis has not been fully understood. Ubiquinol–cytochrome c reductase binding protein (UQCRB), the 13.4-kDa subunit of the mitochondrial complex III, has recently been implicated in angiogenesis by virtue of its ability to modulate hypoxia-induced mROSand hypoxia inducible factor (HIF)-mediated signaling in cancer cells [2] as well as mROS-mediated vascular endothelial growth factor receptor 2 (VEGFR2) signaling in endothelial cells (ECs) [5]. Moreover, UQCRB has been identified as a target protein of the natural anti-angiogenic compound, terpestacin. In a previous study, UQCRB overexpression was shown to significantly increase tumor cell invasion under normoxic conditions by elevating mROS generation and subsequently increasing HIF-1α stability and VEGF expression [2]. In addition, UQCRB positively regulates VEGFR2 signaling
* Corresponding author. Tel.: +82 2 2123 5883; fax: +82 2 362 7265. E-mail address: [email protected] (H.J. Kwon). http://dx.doi.org/10.1016/j.canlet.2015.06.013 0304-3835/© 2015 Elsevier Ireland Ltd. All rights reserved.
by promoting mROS generation and results in increased EC migration in vitro [5]. Therefore, overexpression experiments demonstrated that UQCRB presents a pro-angiogenic activity. Protein transduction domains (PTDs) are small cationic amphipathic peptides comprising 30 amino acids or less that can assist in the uptake of large and biologically active proteins into mammalian cells [6]. A number of PTDs have been identified and utilized for receptor- and transporter-independent protein delivery across biological membranes [7,8]. Hph-1-PTD, a novel human transcription factor-derived PTD, can also facilitate efficient uptake of fusion proteins [9]. In this study, we employed a similar PTD strategy to deliver UQCRB directly into the cells in order to explore the potential role of UQCRB in angiogenesis. Herein, we show that the PTD-UQCRB fusion protein is a potent pro-angiogenic agent in vitro and in vivo. Materials and methods Molecular cloning, expression, and purification of PTD-UQCRB To isolate the PTD-UQCRB fusion protein from bacteria, a PCR-amplified fulllength UQCRB cDNA (accession no. NM_006294) was inserted into the EcoRI/XhoI sites of a pRSET-B expression vector containing the cell permeable protein transduction domain (Hph-1 domain). Escherichia coli BL21 (DE3) StarpLysS cells that were transformed with plasmids encoding PTD-UQCRB were grown in Luria–Bertani medium containing ampicillin (50 μg/mL) and chloramphenicol (34 μg/mL) at 37 °C
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up to an absorbance of 0.6 at 600 nm. Protein expression was induced using 0.1 M isopropyl-β-d-thiogalactopyranoside and cells were grown for 16 h. The cells were collected by centrifugation, suspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, protease inhibitor cocktail tablets, pH 8.0), and then disrupted by sonication. After centrifugation at 13,000 rpm for 15 min at 4 °C, Ni-NTA agarose beads (Qiagen, Hilden, Germany) were added to the supernatant and incubated at 4 °C for 2 h. After incubation with the beads, the supernatant was loaded on Poly-Prep chromatography columns (Bio-Rad, Hercules, CA). Bound proteins were washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) and eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0). The eluted proteins were desalted using PD-10 Sephadex G-25 (GE Healthcare, Buckinghamshire, UK), supplemented with 10% glycerol, separated into aliquots and flashfrozen at −20 °C. Cell culture HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, GibcoBRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, GibcoBRL). HUVECs were seeded in gelatin-coated plates containing endothelial cell basal medium (Cambrex Walkersville, MD, USA) supplemented with 10% FBS. HepG2 cells were cultured in Roswell Park Memorial Institute medium 1640 (Gibco-BRL) supplemented with 10% FBS. HT1080 cells were cultured with minimum essential medium (Gibco-BRL) supplemented with 10% FBS. The cells were maintained in a humidified incubator with 5% CO2. Cell proliferation assay Cells were seeded onto 96-well plates and incubated for 24 h for stabilization. Various concentrations of PTD-UQCRB were added to each well and incubated for 2 days. Cell proliferation was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT; Sigma-Aldrich, St. Louis, MO, USA) colorimetric assay.
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Metropolitan Institute of Medical Science, Japan) and has been described previously [10]. HepG2 cells were transfected with 0.5 μg of the reporter construct and, after a 24 h incubation, cells were collected and lysed using lysis buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, pH 8.0) and centrifuged at 13,000 rpm. Cell lysates were obtained and assayed for luciferase activity by using the SteadyGlo® Luciferase Assay System (Promega, Madison, WI, USA). Luminescence was measured using a FL600 Microplate Fluorescence Reader (Bio-Tek Instrument, Inc., Winooski, VT, USA). Chemoinvasion assay HUVEC invasiveness was evaluated in vitro by using the Transwell chamber system and 8.0-μm polycarbonate filter inserts (Corning Costar, Cambridge, MA, USA). The lower and upper sides of the filter were coated with 10 μL of gelatin (1 mg/mL, SigmaAldrich) and 10 μL of matrigel (3 mg/mL, BD Biosciences), respectively. HUVECs (1 × 105) were placed in the upper chamber and conditioned medium (CM) was placed in the lower chamber. The chamber was then incubated at 37 °C for 16 h, fixed with methanol, and then stained with hematoxylin and eosin (H&E; Sigma-Aldrich). Cells that had invaded the lower part of the filter were detected using optical microscopy at 100× magnification. Wound treatment Rectangular wounds (1.5 × 1.5 cm2) were created on the dorsal skin of each 5-week-old hairless mouse (BALB/c nude female mice, Orient Co., Seongnam, Korea). Mice were anesthetized with an intraperitoneal injection of tribromoethanol (Avertin®, 250 mg/kg) during the surgery. Analgesic (ketoprofen, 0.1 mg/kg) was administered subcutaneously for 4 d post-surgery. The following 3 treatment groups were established: PTD-UQCRB, PTD-EGFP, and PBS (n = 5–7). PTD-tagged fusion protein (200 μg) or PBS mixed with fibrin matrix (Greenplast, Greencross PD Co., Yongin, Korea) was applied to the wound. After treatment, the wounds were dressed with Tegaderm (3M Health Care, St. Paul, MN, USA). The animal study was approved by the Yonsei Laboratory Animal Research Center (2011-0062).
Fluorescence imaging Histological and immunohistochemical analyses To monitor the localization of PTD-UQCRB, HeLa cells were incubated in 4 μM of PTD-UQCRB for 1 h and were then fixed with 4% formaldehyde (Sigma-Aldrich). After washing with phosphate buffered saline (PBS), cells were co-stained with 1 μg/mL Hoechst and 0.5 μg/mL MitoTracker for 30 min. Finally, cells were washed with distilled water, mounted, and analyzed by confocal microscopy (Carl Zeiss LSM 510 META, Germany). Isolation of mitochondria Mitochondria were isolated by differential centrifugation using a mitochondrial isolation kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions. Measurement of mitochondrial ROS Mitochondrial ROS level was measured using the red mitochondrial superoxide indicator, MitoSOX™ (Gibco-BRL). HeLa cells were grown on cover glass (Decklaser, Lauda-Konlgshofen, Germany) and treated with 1 μM of MitoSOX™. Following 20-min incubation at 37 °C, cells were washed with PBS and then treated with PTDUQCRB and incubated again for 2 h at 37 °C. After washing with PBS, cells were stained with 1 μg/mL Hoechst to visualize the nuclei. Cells were washed with PBS and then fixed with 4% formaldehyde for 15 min at 37 °C. After 3 PBS washes, samples were finally washed with distilled water, mounted, and analyzed by fluorescence microscopy (Olympus America, Inc., Melville, NY, USA). SDS-PAGE and western blot analysis HeLa cells grown in 6-well plates were lysed and the lysates were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using standard methods. Blots were blocked and immunolabeled overnight at 4 °C with the following primary antibodies: anti-UQCRB (Sigma-Aldrich, St. Louis, MO, USA), anti-penta-His (Qiagen, Hilden, Germany), anti-VDAC (Cell Signaling, Danvers, MA, USA), anti-HIF-1α (BD Bioscience, Bedford, MA, USA), anti-VEGF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-tubulin (Millipore, Billerica, MA, USA). Immunolabeling was detected by an enhanced chemiluminescence (ECL) kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions.
Mice were euthanized 2 weeks post-wound treatment. The repaired tissues were bisected and used for histological analysis and immunohistochemistry. For the histological analysis, specimens were fixed in 10% (v/v) buffered formaldehyde, dehydrated with a graded ethanol series, and embedded in paraffin. Specimens were sliced into 4-μm thick sections and stained using H&E and Masson’s trichrome (M-T). The tissue sections were also immunohistochemically stained with anti-involucrin (Abcam, Cambridge, UK) and anti-smooth muscle α-actin (Abcam) antibodies. Measurement of epidermal thickness Epidermal thickness was measured for each treatment in sections stained using anti-involucrin and M-T at 3 random points across the wound. Matrigel plug assay A matrigel plug assay was performed to assess in vivo angiogenesis as previously described [5]. Briefly, 7-week-old C57BL/6 mice (Orient Co.) were injected subcutaneously with 0.6 mL of matrigel supplemented with 20 μg of PTD-UQCRB or VEGF (200 ng/mL). After 5 d, the mice were euthanized and the matrigel plugs were retrieved and photographed. The hemoglobin levels were measured with the Drabkin Reagent kit 525 (Sigma-Aldrich) to quantitate the matrigel angiogenesis. Statistical analysis All quantitative data were expressed as the mean ± standard error of the mean (±s.e.m.) and all statistical analyses were calculated with GraphPad Prism (ver. 5.00 for Windows, GraphPad Software, San Diego, CA, USA, www.graphpad.com). Student’s t-tests were used to determine statistical significance between control and test groups. A p-value less than 0.05 was considered statistically significant (* indicates p < 0.05, ** indicates p < 0.005, *** indicates p < 0.0001).
Results Cell permeable PTD-UQCRB fusion protein was generated to replenish UQCRB protein
Hypoxia responsive element (HRE) reporter gene assay HepG2 cells were grown at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS. The cells were seeded in 6-well plates 24 h before plasmids were transfected using Lipofectamine LTX (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The reporter plasmid construct used for the HREluciferase reporter assay was kindly provided by Dr. Futoshi Shibasaki (Tokyo
To verify the potential role of UQCRB as an angiogenesis enhancer, we generated a cell-permeable Hph-1-PTD peptideconjugated UQCRB protein (PTD-UQCRB) (Fig. 1A). The full length UQCRB gene was subcloned into the pRSET-B protein expression vector, which contained the protein transduction domain (PTD),
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Fig. 1. Generation of PTD-UQCRB fusion protein. (A) PTD-UQCRB DNA construct. DNA sequence of Hph-1 PTD and individual restriction enzyme sites are represented. (B) DNA gel electrophoresis of subcloned pRSET-B-PTD-UQCRB after EcoRI and XhoI digestion. (C) Selection of a host strain for protein expression. BL21 Star pLysS was selected. (D) Purification of PTD-UQCRB using Ni-NTA agarose beads. The beads bind with expressed PTD-UQCRB which are tagged with EGFP after IPTG induction. (E) Purified PTD-UQCRB fusion protein (43 kDa).
mitochondria (Fig. 2C). As shown in Fig. 2D, western blot analysis of mitochondrial fractions confirmed that PTD-UQCRB localized to the mitochondrial inner-membrane, where endogenous UQCRB protein exists. The mitochondrial targeting signal of UQCRB (amino acids 1–35) could be responsible for delivery and functional localization of PTD-UQCRB to mitochondria [11].
Hph-1 (Supplementary Fig. S1 and Fig. 1B). The Hph-1 PTD enabled the delivery of its fusion partner, UQCRB, into the cells. A 6× His tag was inserted to allow protein purification using Ni-NTA beads. Enhanced green fluorescence protein (EGFP) was also included to detect the intracellular delivery of PTD-UQCRB fusion protein by immunofluorescence. In order to confirm that the PTD-UQCRB DNA construct was correct, PTD-UQCRB DNA construct was sequenced and the sequencing results also showed that PTD and UQCRB were perfectly inserted. To obtain the PTD-UQCRB fusion protein, three bacterial strains for protein expression were tested. Out of three strains, BL21 StarpLysS strain was chosen because of its excellent capability to express PTD-UQCRB fusion protein than the other strains (Fig. 1C). PTD-UQCRB fusion proteins were purified with Ni-NTA beads (Fig. 1D), following a series of standard purification steps using imidazole containing buffers. Imidazole was subsequently removed via desalting and purified PTD-UQCRB fusion proteins were identified by SDS-PAGE (Fig. 1E) and immunoblotting with anti-UQCRB antibody (data not shown).
Since UQCRB is a complex III subunit of the mitochondrial respiratory chain, PTD-UQCRB transduction may affect mROS production. Indeed, PTD-UQCRB treatment increased mROS levels, as indicated by the increase in MitoSOX™ fluorescence (red), an mROS indicator, compared to control (Fig. 3A). In addition, mROS levels augmented with increasing PTD-UQCRB concentrations, an effect that was observed with UQCRB treatment (Fig. 3B and C). Notably, generated mROS, resulting from PTD-UQCRB treatment, did not affect cell proliferation (Fig. 3D).
PTD-UQCRB specifically localizes to mitochondria
PTD-UQCRB induces angiogenesis through HIF-1α activation in vitro
To investigate the intracellular localization of PTD-UQCRB, the ability of PTD-UQCRB to penetrate cells was investigated. PTDUQCRB (green fluorescence) was detected mainly in the cytoplasm, rather than in nuclei (Fig. 2A), demonstrating that Hph-1 PTD helps the delivery of UQCRB across cellular membranes. PTD-UQCRB transduction was achieved in an efficient and dose-dependent manner (Fig. 2B). By co-staining the cells with the mitochondrial marker MitoTracker, we observed that PTD-UQCRB localized to the
Because increased intracellular mROS stabilizes HIF-1α protein [12] and mediates transcription of downstream angiogenic factors, including VEGF [13,14], we next investigated the effect of PTDUQCRB on HIF-1α stability in cells. Under normoxia, HIF-1α is normally hydroxylated by prolyl hydroxylases [15], resulting in oxygen-dependent degradation. However, HIF-1α protein accumulated dose dependently in PTD-UQCRB-treated cells, a phenomenon not observed in cells treated with PTD-EGFP as a negative control
Generation of mitochondrial ROS by PTD-UQCRB treatment
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Fig. 2. Subcellular localization of PTD-UQCRB. (A) Subcellular localization of PTD-UQCRB (2 μM) in HT1080 cells detected by confocal microscopy (NT: not-treated, PC: phase contrast). Scale bar, 20 μm. (B) Total fluorescence intensity of EGFP was measured in cells treated with 1, 2, and 4 μM of PTD-UQCRB. (C) Mitochondrial localization of PTDUQCRB. After a 1 h treatment with 2 μM of PTD-UQCRB, HT1080 cells were stained with Hoechst and MitoTracker to observe nuclei and mitochondria, respectively. PTDUQCRB was localized in mitochondria. Scale bars, 20 μm. (D) Cytosolic and mitochondrial cell fractions were isolated using differential centrifugation. Proteins were analyzed using antibodies against UQCRB, penta-His (PTD-UQCRB), and VDAC (mitochondrial membrane protein). C: cytosol, X: mitochondrial matrix fraction, M: mitochondrial membrane fraction.
(Fig. 4A and B). The results showed that mROS generation, resulting from PTD-UQCRB treatment, was sufficient to stabilize HIF-1α protein. In the following hypoxia-responsive element (HRE) reporter gene assay, PTD-UQCRB transduction led to a higher luminescence intensity of HRE-luciferase than that of the control (Fig. 4C). This confirmed that HIF-1α was stabilized under normoxic conditions, likely because increased mROS can contribute to the dimerization with HIF-1β [16], resulting in the formation of active transcriptional complexes that induce the expression of the downstream gene, VEGF. Indeed, intracellular delivery of PTD-UQCRB significantly increased VEGF levels in a dose-dependent manner (Fig. 4D, E and Supplementary Fig. S2). Interestingly, the increased VEGF expression by PTD-UQCRB treatment was significantly suppressed by terpestacin (Fig. 4F). Terpestacin directly targets UQCRB leading to suppression of mROS production, HIF-1α stability and VEGF expression to inhibit tumor angiogenesis [2]. Therefore, the result confirmed that elevation of mROS generation through PTD-UQCRB treatment is sufficient for VEGF expression. To determine whether PTD-UQCRB has pro-angiogenic activity in vitro, conditioned medium
(CM) from PTD-UQCRB-treated HeLa cells was collected and used to treat human umbilical vascular endothelial cells (HUVECs). The PTD-UQCRB CM treatment strongly promoted the invasive behavior of HUVECs compared to control CM (Fig. 4G). These results clearly demonstrate that PTD-UQCRB potentiates angiogenesis in vitro.
PTD-UQCRB enhances angiogenesis and wound healing in vivo To demonstrate that PTD-UQCRB can induce angiogenesis in vivo, a matrigel plug assay was performed in mice. PTD-UQCRB significantly increased angiogenesis within matrigel plugs, to a level comparable to that induced by VEGF (Fig. 5A). In addition, the level of hemoglobin consistently supported the pro-angiogenic effect of PTD-UQCRB. The pro-angiogenic activity of PTD-UQCRB was further assessed in vivo by using a cutaneous wound healing mouse model. Wounds (1.5 × 1.5 cm) were created on the dorsal skin of 5-weekold BALB/c nude mice that were subsequently treated with PTDUQCRB or PBS mixed with fibrin matrix. Notably, PTD-UQCRB
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Fig. 3. Effect of PTD-UQCRB on mROS production. (A) mROS levels were determined by measuring the MitoSOX™ fluorescence in HeLa cells. Images representing mROS production after PTD-UQCRB treatment (NT: not-treated, left) and bar graphs showing the average intensity of one cell (right). Scale bar, 20 μm. (B) Elevated mitochondrial mROS levels were measured in HeLa cells treated with 1, 2, and 4 μM of PTD-UQCRB by using MitoSOX™. (C) Measurement of MitoSOX™ fluorescence intensity in cells treated with 4 μM of PTD-UQCRB than those not-treated (NT) and 4 μM PTD-EGFP-treated control cells. (D) Effect of PTD-UQCRB on cell growth. 1, 2 and 4 μM of PTDUQCRB were added to HUVEC, HeLa, and HepG2 cells and incubated for 2 days. Cell proliferation was measured using MTT colorimetric assay. Data represent mean ± standard error of the mean (±s.e.m.) compared to control (*** indicates p < 0.0001).
resulted in remarkable wound closure activity 9 d post-treatment (Fig. 5B). The percentage of wound closure was 64.6% in the PTDEGFP control group, which increased up to 85.4% in the PTDUQCRB treatment group. Histology of the repaired tissues was examined using H&E and M-T staining methods. It showed that considerable epidermal regeneration occurred in the PTD-UQCRB
treatment group compared to the control groups (Fig. 5C). In addition, immunohistochemical staining performed using an antiinvolucrin antibody showed epidermal regeneration. Overall, PTDUQCRB treatment showed a 2-fold increase over the control groups in epidermal thickness (Fig. 5D). Furthermore, a 3-fold increase in the number of arterioles was detected in wound healing regions of
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Fig. 4. PTD-UQCRB induces angiogenesis by activating HIF-1α in vitro. (A) Effect of PTD-UQCRB treatment on HIF-1α stability. Western blot analysis was used to assess HIF1α protein levels in HeLa cells treated with PTD-UQCRB (2 and 4 μM) and PTD-EGFP (2 and 4 μM) as a control for 4 h. Tubulin served as an internal control. (B) Quantitative results of the HIF-1α western blot analysis. (C) Effect of PTD-UQCRB treatment on HRE reporter gene expression. HepG2 cells were transiently transfected with HREluciferase vectors. Cells were treated with 1 and 2.5 μM PTD-UQCRB for 4 h. Luciferase luminescence was measured to assess the effect of PTD-UQCRB on HRE reporter gene. (D) Effect of PTD-UQCRB treatment on VEGF expression. VEGF protein levels were evaluated in HeLa cells treated with PTD-UQCRB (2 and 4 μM) and PTD-EGFP (2 and 4 μM) as control for 24 h by performing western blot analysis. Tubulin served as an internal control. (E) Quantitative results of the VEGF western blot analysis. (F) Regulation of VEGF induction with UQCRB inhibitor, terpestacin. 50 μM terpestacin was used to treat HeLa cells for 12 or 24 h following pre-treatment with 2 or 4 μM PTD-UQCRB for 2 h. Tubulin was used as an internal control. (G) Pro-angiogenic effect of PTD-UQCRB in HUVECs. HeLa cells were treated with PTD-UQCRB (2 and 4 μM) for 24 h in serum free medium before collecting CM (NT: not-treated). HUVECs were incubated for 16 h in the CM. The collected CM stimulates HUVEC invasion. Quantified data are presented as mean (±s.e.m.) compared to control (* indicates p < 0.05, ** indicates p < 0.005 and *** indicates p < 0.0001).
PTD-UQCRB-treated mice assessed by smooth muscle alpha actin (SMA) staining (Fig. 5E). Taken together, these results show that PTDUQCRB treatment promotes angiogenesis and enhances wound healing in vivo.
Discussion In the present study, a novel PTD-UQCRB fusion protein was generated to allow the intracellular delivery of UQCRB. Previously, to
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Fig. 5. PTD-UQCRB transduction enhances cutaneous wound healing in vivo. (A) Effect of PTD-UQCRB on in vivo angiogenesis in matrigel plugs. C57BL/6 mice were injected subcutaneously with matrigel supplemented with PBS, PTD-UQCRB (20 μg), or VEGF (200 ng/mL). After 5 d, matrigel plugs were retrieved and analyzed (n = 6 per group). The hemoglobin levels were measured to quantify blood vessel formation in the matrigel plugs. (B) Wound closure was evaluated in BALB/c nude mice (n = 5–7) under different treatment conditions. Macroscopic analysis of wounds and wound closure analysis under the following treatment conditions: PTD-UQCRB (200 μg), PTD-EGFP (200 μg), and PBS. (C) Histological and immunohistochemical staining of the mid-wound healing region for each treatment group (H&E: hematoxylin and eosin staining and M-T: Masson’s trichrome staining). The arrows indicate the thickness of epidermis (H&E (white), M-T (white), involucrin (black)). Scale bars indicate 100 μm in the H&E and M-T stained specimens, 200 μm in those stained using anti-involucrin. (D) Quantitative analysis of the thickness of the regenerated epidermis. (E) Immunohistochemical staining of smooth muscle α-actin (SMA) in the mid-wound healing region for each treatment group and quantitative data for arterioles in healed regions. The arrows point to arterioles (SMA, red). Scale bars indicate 50 μm. (F) Schematic summary of PTD-UQCRB function. When PTD-UQCRB permeates cells, the mitochondrial complex III-derived ROS production is induced. Increased mROS stabilize HIF-1α. The stable heterodimer complex, containing HIF-1, translocates to the nucleus where it initiates the transcription of the target gene, VEGF, which leads to angiogenesis and cutaneous wound healing. Quantified data are presented as mean (±s.e.m.) compared to control (** indicates p < 0.005, *** indicates p < 0.0001). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 5. (continued)
investigate its biological role, UQCRB was overexpressed by DNA transfection. However, direct delivery of UQCRB protein into cells by using PTD enabled efficient transduction of UQCRB in vitro and in vivo. In addition, PTD-UQCRB treatment had a strong proangiogenic effect as well as effects on wound healing, suggesting that PTD-UQCRB has great potential as a potent pro-angiogenic agent. Unlike a similar mitochondrial inner membrane protein, prohibitin-1, which was also identified as an angiogenic factor [17], PTD-UQCRB enhanced angiogenesis by inducing mROS production. Although high mROS levels are known to have harmful effects on cell growth, resulting in apoptosis [18], mROS, at low levels, are thought to act as signaling molecules to regulate various cellular processes, including differentiation, metabolic adaptation, and immune cell activation [19]. Consistent with this report, PTDUQCRB treatment did not show cytotoxicity (Fig. 3D). However, the mechanism by which PTD-UQCRB transduction increases mROS and results in angiogenesis will be investigated in a future study. HIF-1α induction is commonly implemented as a strategy to enhance wound healing [14,20–22]. Consistent with previous studies, PTD-UQCRB significantly increased HIF-1α stability, resulting in an increase in VEGF expression, followed by induction of angiogenesis. Notably, however, PTD-UQCRB is different from previous agents in that it can be applied topically. Recently, our group reported the biological effect of UQCRB mutation, originated from a patient with lactic acidosis, in angiogenesis
[1]. The mutant has 7 modified amino acids as well as 14 additional amino acids on the C-terminal end. We constructed the UQCRB mutant stable cell lines, which exhibited glycolytic, proliferative, and pro-angiogenic activities through the mROS-induced HIF-1 signaling pathway. It is surprising that the biological traits of the UQCRB mutant and UQCRB supplementation present highly relevant similarities in angiogenesis. Presumably, similarities resulted from changes in complex III conformation. However, the unsolved mechanism should be examined in a future study. In this study, we showed that PTD-UQCRB presents a proangiogenic activity in vitro and in vivo. To our best knowledge, this is the first report on the application of UQCRB in vivo. UQCRB, a component of the mitochondrial complex III, plays a critical role in angiogenesis. These results provide new insights into the biological role of UQCRB in angiogenesis by demonstrating that it plays a role in mROS production and leads to HIF-1α activation (Fig. 5F). In addition, these data provide the basis for the medical application of PTD-UQCRB as a pro-angiogenic agent. Acknowledgements This work was partly supported by grants from the National Research Foundation of Korea grant funded by the Korean government (MSIP; 2010-0017984, 2012M3A9D1054520), Translational Research Center for Protein Function Control, NRF (2009-0083522),
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Ministry of Health and Welfare (0620360-1), and Brain Korea 21 Plus Project, Republic of Korea.
Conflict of interest The authors declare that they have no conflict of interest.
Appendix: Supplementary material Supplementary data to this article can be found online at doi:10.1016/j.canlet.2015.06.013.
References [1] J. Chang, H.J. Jung, S.H. Jeong, H.K. Kim, J. Han, H.J. Kwon, A mutation in the mitochondrial protein UQCRB promotes angiogenesis through the generation of mitochondrial reactive oxygen species, Biochem. Biophys. Res. Commun. 455 (2014) 290–297. [2] H.J. Jung, J.S. Shim, J. Lee, Y.M. Song, K.C. Park, S.H. Choi, et al., Terpestacin inhibits tumor angiogenesis by targeting UQCRB of mitochondrial complex III and suppressing hypoxia-induced reactive oxygen species production and cellular oxygen sensing, J. Biol. Chem. 285 (2010) 11584–11595. [3] K.H. Kim, D. Kim, J.Y. Park, H.J. Jung, Y.H. Cho, H.K. Kim, et al., NNC 55-0396, a T-type Ca channel inhibitor, inhibits angiogenesis via suppression of hypoxiainducible factor-1alpha signal transduction, J. Mol. Med. (Berl) 93 (2015) 499–509. [4] R. Solito, F. Corti, C.H. Chen, D. Mochly-Rosen, A. Giachetti, M. Ziche, et al., Mitochondrial aldehyde dehydrogenase-2 activation prevents beta-amyloidinduced endothelial cell dysfunction and restores angiogenesis, J. Cell Sci. 126 (2013) 1952–1961. [5] H.J. Jung, Y. Kim, J. Chang, S.W. Kang, J.H. Kim, H.J. Kwon, Mitochondrial UQCRB regulates VEGFR2 signaling in endothelial cells, J. Mol. Med. 91 (2013) 1117– 1128. [6] L.N. Patel, J.L. Zaro, W.C. Shen, Cell penetrating peptides: intracellular pathways and pharmaceutical perspectives, Pharm. Res. 24 (2007) 1977–1992. [7] F. Heitz, M.C. Morris, G. Divita, Twenty years of cell-penetrating peptides: from molecular mechanisms to therapeutics, Br. J. Pharmacol. 157 (2009) 195– 206.
[8] M.C. Morris, J. Depollier, J. Mery, F. Heitz, G. Divita, A peptide carrier for the delivery of biologically active proteins into mammalian cells, Nat. Biotechnol. 19 (2001) 1173–1176. [9] J.M. Choi, M.H. Ahn, W.J. Chae, Y.G. Jung, J.C. Park, H.M. Song, et al., Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation, Nat. Med. 12 (2006) 574–579. [10] H. Kato, S. Tamamizu-Kato, F. Shibasaki, Histone deacetylase 7 associates with hypoxia-inducible factor 1alpha and increases transcriptional activity, J. Biol. Chem. 279 (2004) 41966–41974. [11] H. Suzuki, Y. Hosokawa, H. Toda, M. Nishikimi, T. Ozawa, Cloning and sequencing of a cDNA for human mitochondrial ubiquinone-binding protein of complex III, Biochem. Biophys. Res. Commun. 156 (1988) 987–994. [12] T. Klimova, N.S. Chandel, Mitochondrial complex III regulates hypoxic activation of HIF, Cell Death Differ. 15 (2008) 660–666. [13] J.A. Forsythe, B.H. Jiang, N.V. Iyer, F. Agani, S.W. Leung, R.D. Koos, et al., Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1, Mol. Cell. Biol. 16 (1996) 4604–4613. [14] A. Ahluwalia, A.S. Tarnawski, Critical role of hypoxia sensor – HIF-1alpha in VEGF gene activation. Implications for angiogenesis and tissue injury healing, Curr. Med. Chem. 19 (2012) 90–97. [15] M. Gruber, M.C. Simon, Hypoxia-inducible factors, hypoxia, and tumor angiogenesis, Curr. Opin. Hematol. 13 (2006) 169–174. [16] G.L. Wang, B.H. Jiang, E.A. Rue, G.L. Semenza, Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 5510–5514. [17] M. Schleicher, B.R. Shepherd, Y. Suarez, C. Fernandez-Hernando, J. Yu, Y. Pan, et al., Prohibitin-1 maintains the angiogenic capacity of endothelial cells by regulating mitochondrial function and senescence, J. Cell Biol. 180 (2008) 101–112. [18] N. Zamzami, P. Marchetti, M. Castedo, D. Decaudin, A. Macho, T. Hirsch, et al., Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death, J. Exp. Med. 182 (1995) 367–377. [19] L.A. Sena, N.S. Chandel, Physiological roles of mitochondrial reactive oxygen species, Mol. Cell 48 (2012) 158–167. [20] B.D. Kelly, S.F. Hackett, K. Hirota, Y. Oshima, Z. Cai, S. Berg-Dixon, et al., Cell type-specific regulation of angiogenic growth factor gene expression and induction of angiogenesis in nonischemic tissue by a constitutively active form of hypoxia-inducible factor 1, Circ. Res. 93 (2003) 1074–1081. [21] L. Liu, G.P. Marti, X. Wei, X. Zhang, H. Zhang, Y.V. Liu, et al., Age-dependent impairment of HIF-1alpha expression in diabetic mice: correction with electroporation-facilitated gene therapy increases wound healing, angiogenesis, and circulating angiogenic cells, J. Cell. Physiol. 217 (2008) 319–327. [22] E. Andrikopoulou, X. Zhang, R. Sebastian, G. Marti, L. Liu, S.M. Milner, et al., Current Insights into the role of HIF-1 in cutaneous wound healing, Curr. Mol. Med. 11 (2011) 218–235.