Original Paper Pharmacology 2013;92:238–244 DOI: 10.1159/000355837
Received: June 14, 2013 Accepted after revision: September 17, 2013 Published online: November 12, 2013
Heparin Inhibits Activation of Latent Transforming Growth Factor-β1 Mee Jeong Lee Department of Pediatrics, College of Medicine, Dankook University, Cheonan, Korea
Abstract Aim: Besides acting as an anticoagulant, heparin has antifibrotic effects. Transforming growth factor-β1 (TGF-β1) is secreted from cells as latent TGF-β1 (LTGF-β1). LTGF-β1 consists of TGF-β1 and latency-associated peptide (LAP). To be biologically active, TGF-β1 has to be released from LAP. Heparin binds to LAP as well as TGF-β1. This study was performed to explore the biological effect of the interaction of heparin with LTGF-β1. Materials and Methods: TGF-β1 was measured by ELISA. Furin-like proprotein convertase activity was assayed using the fluorogenic substrate, Pyr-Arg-Thr-LysArg-AMC. Results: Heparin did not interfere with the receptor binding of TGF-β1, but inhibited furin-like proprotein convertase-mediated activation of platelet LTGF-β1. This was not by inhibition of the enzyme because heparin did not inhibit the activity of furin-like proprotein convertase. In addition, heparin inhibited acid activations of recombinant small LTGF-β1, platelet LTGF-β1 and LTGF-β1s secreted in the supernatant of cultured cells. Low-molecular-weight heparins, including dalteparin, enoxaparin and nadroparin, also had inhibitory effects on furin-like proprotein convertasemediated or acid activation of platelet LTGF-β1. Conclusion: The findings suggest that heparin renders LTGF-β1 resistant
© 2013 S. Karger AG, Basel 0031–7012/13/0926–0238$38.00/0 E-Mail [email protected] www.karger.com/pha
to activation, possibly by binding simultaneously to TGF-β1 and LAP. Inhibition of LTGF-β1 activation by heparin may in part account for its antifibrotic effects. © 2013 S. Karger AG, Basel
Introduction
Transforming growth factor-β1 (TGF-β1) stimulates the production of extracellular matrices and has a pivotal role in the pathogenesis of fibrosis. TGF-β1 is encoded as a large precursor protein which undergoes intracellular processing. After processing, TGF-β1 remains noncovalently bound to latency-associated peptide (LAP), forming small latent TGF-β1 (LTGF-β1). In the cytoplasm, the small LTGF-β1 binds to latent TGF-β binding protein with a disulfide bond, forming large LTGF-β1 [1, 2]. TGF-β1 is secreted from cells as small LTGF-β1, or more commonly as large LTGF-β1 [2]. LTGF-β1 itself does not bind to TGF-β receptor and thus has no biological activity of TGF-β1. To be biologically active, TGF-β1 has to be cleaved and released from LAP of the latent complex [1]. After that, it can bind to TGF-β receptor type II (TGFβRII) on the cell surface, and subsequently induces the biological responses [1]. The mechanism of activation of LTGF-β1 in vivo is still poorly understood, but it may occur by proteases including plasmin, matrix metalloproteinase and furin-like proprotein convertase or by conformational changes of LAP Mee Jeong Lee, MD Department of Pediatrics, Dankook University Hospital Anseo-dong San 16-5 Cheonan, Chungcheongnam-do 330-715 (Korea) E-Mail LMJPED @ dankook.ac.kr
Downloaded by: University of Florida, Gainesville and Jacksonville 198.143.41.97 - 1/27/2016 2:43:29 PM
Key Words Furin-like proprotein convertase · Heparin · Latent transforming growth factor-β1 · Low-molecular-weight heparin
Materials and Methods Materials Recombinant human TGF-β1 (rTGF-β1) and recombinant human small LTGF-β1 were purchased from R&D systems, Inc. (Minneapolis, Minn., USA). Unfractionated heparin (from porcine intestinal mucosa) was from Sigma-Aldrich Co. (St. Louis, Mo., USA). Nadroparin calcium (Fraxiparin®), enoxaparin sodium (Clexane®, 10 μg = anti-Xa activity 1.0 IU) and dalteparin sodium (Fragmin®) were from Sanofi-Synthelabo Korea Co., Ltd. (Seoul, Korea), Sanofi-Aventis Korea Co., Ltd. (Seoul, Korea) and Pfizer Korea Co., Ltd. (Seoul, Korea), respectively. Purified furin was from New England BioLabs Inc. (Ipswich, Mass., USA). Furin inhibitors (Dec-RVKR-CMK, hexa-D-arginine) and furin substrate (Pyr-Arg-Thr-Lys-Arg-AMC) were from EMD Chemicals (Darmstadt, Germany).
Heparin Inhibits LTGF-β1 Activation
Cell Culture and Collection of Conditioned Medium HK-2 cells (human renal proximal tubular epithelial cells) were obtained from the American Type Culture Collection (Manassas, Va., USA). Normal human mesangial cells were obtained from Lonza (Walkersville, Md., USA). To obtain conditioned medium, the cells confluent in 100-mm culture dishes were rinsed with phosphate-buffered saline and rested on serum-free M199 media or RPMI media (Life Technologies, Paisley, UK) for 48 h. Thereafter, the medium was replaced with fresh medium and the cells were stimulated with 5 ng/ml of interleukin-1β to obtain a high concentration of LTGF-β1. After incubation for 72 h, the conditioned medium was collected and centrifuged to remove cell debris, aliquoted, and stored at –70 ° C for subsequent activation studies.
Preparation of Platelet Lysate Blood samples were obtained from healthy adult volunteers after informed consent was obtained. Peripheral venous blood was drawn by venipuncture of the antecubital vein and collected in the citrate tube. Blood samples were centrifuged at 300 g for 10 min, and the supernatant (platelet-rich plasma, PRP) was harvested. PRP was centrifuged at 6,000 g for 5 min, resulting in platelet pellet. The pellet was washed with M199 media to remove citrate. To induce hypotonic lysis, the platelet pellet was resuspended in cold Milli-Q water (7/10 the original volume of PRP) and placed on ice for 30 min. Thereafter, 1% bovine serum albumin (BSA) in Milli-Q water (1/5 the original volume of PRP) and M199 media (11/10 the original volume of PRP) were added to make the final volume 2 times the original volume of PRP. The platelet solution was centrifuged at 6,000 g for 5 min at 4 ° C to eliminate lysed platelets, and the supernatant was obtained as platelet lysate. For the experiment of acid activation, the platelet pellet was lysed in cold Milli-Q water (1/2 the original volume of PRP) and then diluted with M199 media to be 10 times the original volume of PRP.
Enzymatic Activation of Platelet LTGF-β1 Platelet lysates prepared on ice were preincubated with different concentrations of heparin, LMWHs, furin inhibitors or furin substrate for 30 min on ice. Thereafter, the samples were placed at room temperature (approx. 25 ° C) for 1 h to allow enzymatic activation of LTGF-β1. After the incubation, the samples were subjected to enzyme-linked immunosorbent assay (ELISA) to measure the active form of TGF-β1. Total TGF-β1s were measured after acid activation of platelet lysates, which were diluted 10 times with M199 media/0.1% BSA to be suitable for the assay range of the ELISA kit.
TGF-β1 ELISA The binding of TGF-β1 to its receptor was measured by ELISA kit (Quantikine® human TGF-β1 immunoassay; R&D systems, Inc.), in which the wells are coated with TGF-βRII. Acid activation of LTGF-β1 was performed by incubation of the samples (200 μl) with 1 N HCl (40 μl) for 10 min, followed by neutralization with 1.2 N NaOH/0.5 mol/l HEPES (40 μl). Assay of Furin-Like Proprotein Convertase Activity Furin-like proprotein convertase activity was assayed in black 96-well plates using the fluorogenic substrate, Pyr-Arg-Thr-LysArg-AMC, for 4 h at room temperature. The enzyme activity was
Pharmacology 2013;92:238–244 DOI: 10.1159/000355837
239
Downloaded by: University of Florida, Gainesville and Jacksonville 198.143.41.97 - 1/27/2016 2:43:29 PM
[3, 4]. In vitro, LTGF-β1 can be activated by transient acidification which dissociates TGF-β1 from LAP by disrupting the hydrogen bond and other noncovalent bonds through changes in ionic strength [5]. Heparin is a sulfated polysaccharide belonging to the family of glycosaminoglycans. Due to sulfo- or carboxyl groups on the heparin chain, it has high negative charge density and can interact with clusters of positively charged basic amino acids on proteins [6]. By these ionic interactions, heparin binds to cytokines and modulates their activity [6]. TGF-β1 is one of the cytokines known to bind heparin. Binding of heparin potentiates TGF-β1 activity, but this occurs only in the presence of α2-macroglobulin (α2M), as in plasma or serum [7]. α2M is abundant in the plasma [8], and is the major binding protein for TGF-β1 [9]. TGF-β1 bound to α2M does not bind to the receptor, thus becoming an inactive form. Binding of heparin does not change the biological activity of TGF-β1 itself, but interferes with its association with α2M, and thereby potentiates TGF-β1 activity [7]. Studies in human [10] and animal models [11, 12], however, have shown that heparin has antifibrotic effects. Like heparin, fucoidan is a sulfated polysaccharide. In contrast to heparin, fucoidan binding renders TGF-β1 unable to interact with its receptor [13]. Additionally, fucoidan binds to small and large LTGF-β1 and renders them resistant to activation [13]. Heparin binds to LAP [14] as well as to TGF-β1, the two components of LTGF-β1. However, the biological effects of the interaction of heparin with LTGF-β1 are not yet known. The present study was therefore performed to determine whether heparin alters the activation of LTGF-β1 and whether low-molecular-weight heparins (LMWHs) have similar effects on LTGF-β1 activation.
determined as the release of the fluorescent AMC moiety, which was measured by SPECTRmax© GEMINI XS system (Molecular Devices, Sunnyvale, Calif., USA).
800 TGF-DŽ1 (pg/ml)
Statistical Analysis Data are presented as the mean ± SE. An analysis of variance followed by the Bonferroni multiple comparison test was used to compare mean values among three or more groups. A p value of
Received: June 14, 2013 Accepted after revision: September 17, 2013 Published online: November 12, 2013
Heparin Inhibits Activation of Latent Transforming Growth Factor-β1 Mee Jeong Lee Department of Pediatrics, College of Medicine, Dankook University, Cheonan, Korea
Abstract Aim: Besides acting as an anticoagulant, heparin has antifibrotic effects. Transforming growth factor-β1 (TGF-β1) is secreted from cells as latent TGF-β1 (LTGF-β1). LTGF-β1 consists of TGF-β1 and latency-associated peptide (LAP). To be biologically active, TGF-β1 has to be released from LAP. Heparin binds to LAP as well as TGF-β1. This study was performed to explore the biological effect of the interaction of heparin with LTGF-β1. Materials and Methods: TGF-β1 was measured by ELISA. Furin-like proprotein convertase activity was assayed using the fluorogenic substrate, Pyr-Arg-Thr-LysArg-AMC. Results: Heparin did not interfere with the receptor binding of TGF-β1, but inhibited furin-like proprotein convertase-mediated activation of platelet LTGF-β1. This was not by inhibition of the enzyme because heparin did not inhibit the activity of furin-like proprotein convertase. In addition, heparin inhibited acid activations of recombinant small LTGF-β1, platelet LTGF-β1 and LTGF-β1s secreted in the supernatant of cultured cells. Low-molecular-weight heparins, including dalteparin, enoxaparin and nadroparin, also had inhibitory effects on furin-like proprotein convertasemediated or acid activation of platelet LTGF-β1. Conclusion: The findings suggest that heparin renders LTGF-β1 resistant
© 2013 S. Karger AG, Basel 0031–7012/13/0926–0238$38.00/0 E-Mail [email protected] www.karger.com/pha
to activation, possibly by binding simultaneously to TGF-β1 and LAP. Inhibition of LTGF-β1 activation by heparin may in part account for its antifibrotic effects. © 2013 S. Karger AG, Basel
Introduction
Transforming growth factor-β1 (TGF-β1) stimulates the production of extracellular matrices and has a pivotal role in the pathogenesis of fibrosis. TGF-β1 is encoded as a large precursor protein which undergoes intracellular processing. After processing, TGF-β1 remains noncovalently bound to latency-associated peptide (LAP), forming small latent TGF-β1 (LTGF-β1). In the cytoplasm, the small LTGF-β1 binds to latent TGF-β binding protein with a disulfide bond, forming large LTGF-β1 [1, 2]. TGF-β1 is secreted from cells as small LTGF-β1, or more commonly as large LTGF-β1 [2]. LTGF-β1 itself does not bind to TGF-β receptor and thus has no biological activity of TGF-β1. To be biologically active, TGF-β1 has to be cleaved and released from LAP of the latent complex [1]. After that, it can bind to TGF-β receptor type II (TGFβRII) on the cell surface, and subsequently induces the biological responses [1]. The mechanism of activation of LTGF-β1 in vivo is still poorly understood, but it may occur by proteases including plasmin, matrix metalloproteinase and furin-like proprotein convertase or by conformational changes of LAP Mee Jeong Lee, MD Department of Pediatrics, Dankook University Hospital Anseo-dong San 16-5 Cheonan, Chungcheongnam-do 330-715 (Korea) E-Mail LMJPED @ dankook.ac.kr
Downloaded by: University of Florida, Gainesville and Jacksonville 198.143.41.97 - 1/27/2016 2:43:29 PM
Key Words Furin-like proprotein convertase · Heparin · Latent transforming growth factor-β1 · Low-molecular-weight heparin
Materials and Methods Materials Recombinant human TGF-β1 (rTGF-β1) and recombinant human small LTGF-β1 were purchased from R&D systems, Inc. (Minneapolis, Minn., USA). Unfractionated heparin (from porcine intestinal mucosa) was from Sigma-Aldrich Co. (St. Louis, Mo., USA). Nadroparin calcium (Fraxiparin®), enoxaparin sodium (Clexane®, 10 μg = anti-Xa activity 1.0 IU) and dalteparin sodium (Fragmin®) were from Sanofi-Synthelabo Korea Co., Ltd. (Seoul, Korea), Sanofi-Aventis Korea Co., Ltd. (Seoul, Korea) and Pfizer Korea Co., Ltd. (Seoul, Korea), respectively. Purified furin was from New England BioLabs Inc. (Ipswich, Mass., USA). Furin inhibitors (Dec-RVKR-CMK, hexa-D-arginine) and furin substrate (Pyr-Arg-Thr-Lys-Arg-AMC) were from EMD Chemicals (Darmstadt, Germany).
Heparin Inhibits LTGF-β1 Activation
Cell Culture and Collection of Conditioned Medium HK-2 cells (human renal proximal tubular epithelial cells) were obtained from the American Type Culture Collection (Manassas, Va., USA). Normal human mesangial cells were obtained from Lonza (Walkersville, Md., USA). To obtain conditioned medium, the cells confluent in 100-mm culture dishes were rinsed with phosphate-buffered saline and rested on serum-free M199 media or RPMI media (Life Technologies, Paisley, UK) for 48 h. Thereafter, the medium was replaced with fresh medium and the cells were stimulated with 5 ng/ml of interleukin-1β to obtain a high concentration of LTGF-β1. After incubation for 72 h, the conditioned medium was collected and centrifuged to remove cell debris, aliquoted, and stored at –70 ° C for subsequent activation studies.
Preparation of Platelet Lysate Blood samples were obtained from healthy adult volunteers after informed consent was obtained. Peripheral venous blood was drawn by venipuncture of the antecubital vein and collected in the citrate tube. Blood samples were centrifuged at 300 g for 10 min, and the supernatant (platelet-rich plasma, PRP) was harvested. PRP was centrifuged at 6,000 g for 5 min, resulting in platelet pellet. The pellet was washed with M199 media to remove citrate. To induce hypotonic lysis, the platelet pellet was resuspended in cold Milli-Q water (7/10 the original volume of PRP) and placed on ice for 30 min. Thereafter, 1% bovine serum albumin (BSA) in Milli-Q water (1/5 the original volume of PRP) and M199 media (11/10 the original volume of PRP) were added to make the final volume 2 times the original volume of PRP. The platelet solution was centrifuged at 6,000 g for 5 min at 4 ° C to eliminate lysed platelets, and the supernatant was obtained as platelet lysate. For the experiment of acid activation, the platelet pellet was lysed in cold Milli-Q water (1/2 the original volume of PRP) and then diluted with M199 media to be 10 times the original volume of PRP.
Enzymatic Activation of Platelet LTGF-β1 Platelet lysates prepared on ice were preincubated with different concentrations of heparin, LMWHs, furin inhibitors or furin substrate for 30 min on ice. Thereafter, the samples were placed at room temperature (approx. 25 ° C) for 1 h to allow enzymatic activation of LTGF-β1. After the incubation, the samples were subjected to enzyme-linked immunosorbent assay (ELISA) to measure the active form of TGF-β1. Total TGF-β1s were measured after acid activation of platelet lysates, which were diluted 10 times with M199 media/0.1% BSA to be suitable for the assay range of the ELISA kit.
TGF-β1 ELISA The binding of TGF-β1 to its receptor was measured by ELISA kit (Quantikine® human TGF-β1 immunoassay; R&D systems, Inc.), in which the wells are coated with TGF-βRII. Acid activation of LTGF-β1 was performed by incubation of the samples (200 μl) with 1 N HCl (40 μl) for 10 min, followed by neutralization with 1.2 N NaOH/0.5 mol/l HEPES (40 μl). Assay of Furin-Like Proprotein Convertase Activity Furin-like proprotein convertase activity was assayed in black 96-well plates using the fluorogenic substrate, Pyr-Arg-Thr-LysArg-AMC, for 4 h at room temperature. The enzyme activity was
Pharmacology 2013;92:238–244 DOI: 10.1159/000355837
239
Downloaded by: University of Florida, Gainesville and Jacksonville 198.143.41.97 - 1/27/2016 2:43:29 PM
[3, 4]. In vitro, LTGF-β1 can be activated by transient acidification which dissociates TGF-β1 from LAP by disrupting the hydrogen bond and other noncovalent bonds through changes in ionic strength [5]. Heparin is a sulfated polysaccharide belonging to the family of glycosaminoglycans. Due to sulfo- or carboxyl groups on the heparin chain, it has high negative charge density and can interact with clusters of positively charged basic amino acids on proteins [6]. By these ionic interactions, heparin binds to cytokines and modulates their activity [6]. TGF-β1 is one of the cytokines known to bind heparin. Binding of heparin potentiates TGF-β1 activity, but this occurs only in the presence of α2-macroglobulin (α2M), as in plasma or serum [7]. α2M is abundant in the plasma [8], and is the major binding protein for TGF-β1 [9]. TGF-β1 bound to α2M does not bind to the receptor, thus becoming an inactive form. Binding of heparin does not change the biological activity of TGF-β1 itself, but interferes with its association with α2M, and thereby potentiates TGF-β1 activity [7]. Studies in human [10] and animal models [11, 12], however, have shown that heparin has antifibrotic effects. Like heparin, fucoidan is a sulfated polysaccharide. In contrast to heparin, fucoidan binding renders TGF-β1 unable to interact with its receptor [13]. Additionally, fucoidan binds to small and large LTGF-β1 and renders them resistant to activation [13]. Heparin binds to LAP [14] as well as to TGF-β1, the two components of LTGF-β1. However, the biological effects of the interaction of heparin with LTGF-β1 are not yet known. The present study was therefore performed to determine whether heparin alters the activation of LTGF-β1 and whether low-molecular-weight heparins (LMWHs) have similar effects on LTGF-β1 activation.
determined as the release of the fluorescent AMC moiety, which was measured by SPECTRmax© GEMINI XS system (Molecular Devices, Sunnyvale, Calif., USA).
800 TGF-DŽ1 (pg/ml)
Statistical Analysis Data are presented as the mean ± SE. An analysis of variance followed by the Bonferroni multiple comparison test was used to compare mean values among three or more groups. A p value of
