RANKL regulates endothelial cell survival through the ...

The FASEB Journal express article10.1096/fj.03-0215fje. Published online September 18, 2003.

RANKL regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway Hong-Hee Kim,* Hyoung Seek Shin,† Hee Jin Kwak,† Kyu Youn Ahn,‡ Ju-Hyun Kim,† Hyuek Jong Lee,† Mi-Sook Lee,† Zang Hee Lee,§ and Gou Young Koh† *Department of Cell and Developmental Biology, College of Dentistry, Seoul National University, Seoul 110-749, Korea; †National Creative Research Initiatives Center for Endothelial Cells and Department of Life Science, Pohang University of Science and Technology, Pohang, 790-784, Korea ‡Research Institute of Medical Sciences, Chonnam National University, Gwangju 501-190, Korea §National Research Laboratory for Bone Metabolism and School of Dentistry, Chosun University, Gwangju 501-759, Korea Corresponding authors: Zang Hee Lee, National Research Laboratory for Bone Metabolism and School of Dentistry, Chosun University, Gwangju 501-759, Korea. E-mail: [email protected] or Gou Young Koh, National Creative Research Initiatives Center for Endothelial Cells, Department of Life Science, POSTECH San 31, Hyoja-Dong, Pohang, 790784, Republic of Korea. E-mail: [email protected] ABSTRACT The maintenance of endothelial integrity is important for prevention of vascular diseases. Several growth factors, such as bFGF and angiopoietin-1, have been shown to suppress endothelial cell apoptosis and thus help to maintain endothelial integrity. Several studies suggested that receptor activator of NF-κB (RANK) and its ligand (RANKL) could be involved in endothelial physiology. Using immunofluorescence and reverse transcriptase-polymerse chain reaction, we found that RANK was expressed by endothelial cells, and RANKL was expressed by arterial smooth muscle cells. Furthermore, RANKL suppressed apoptosis of primary cultured endothelial cells. The RANKL-induced survival appeared to be dependent on PI 3′-kinase activity, because wortmannin and LY294002, PI 3′-kinase-specific inhibitors, blocked the RANKL-induced survival effect. RANKL elicited the phosphorylation of the serine-threonine kinase Akt at Ser473 in a PI 3′-kinase-dependent manner. The expression of a dominant-negative form of Akt or pretreatment of Akt-specific inhibitor in endothelial cells reversed the RANKL-induced survival effect. Tumor necrosis factor-α, which causes endothelial cell apoptosis, induced endothelial cells to express osteoprotegerin, a decoy receptor that inhibits RANK-RANKL signaling. These findings indicate that RANK, in response to the paracrine stimulus of RANKL, may play an important role in maintaining endothelial cell integrity through the PI 3′-kinase/Akt signal transduction pathway. Key words: RANK • RANKL • endothelial cells • apoptosis

R

ANK is a member of the tumor necrosis factor receptor (TNFR) family. RANK plays an important role in osteoclastogenesis, dendritic cell survival, lymph node organogenesis, and mammary gland development (1, 2). The interaction of RANK with its ligand, RANKL (also called ODF, OPGL, and TRANCE), is regulated by the decoy receptor osteoprotegerin (OPG), a soluble TNFR family member that can bind RANKL and thereby inhibit RANKL/RANK signaling (3). The significance of this tripartite system to vascular physiology has been suggested by the phenotypes of OPG−/− mice, which develop arterial calcification and aneurysms in the large arteries (4). In addition, angiogenesis is an essential part of osteogenesis (5). During osteogenesis-associated angiogenesis, endothelial cells may receive signals from osteoblasts that express RANKL. The observation that transition of vascular smooth muscle cells to cells with osteoblastic characteristics was associated with arterial calcification also suggests the potential involvement of the RANKL/RANK/OPG system in the pathophysiology of the vasculature (6, 7). Using immunofluorescent staining, we found that RANK is highly expressed in endothelial cells of normal adult arterial blood vessels, which are not actively involved in angiogenesis and vasculogenesis. Therefore, in this study, we examined whether the RANKL/RANK system may be important in regulating the survival of endothelial cells. In addition, we examined the receptor/second messenger signal transduction pathway for the antiapoptotic effect of RANKL in primary cultured human umbilical vein endothelial cells (HUVECs). We found that the PI 3′kinase/Akt signal transduction pathway is a crucial element in the processes leading to endothelial cell survival induced by RANKL. MATERIALS AND METHODS Materials and cell culture Recombinant human RANKL and tumor necrosis factor (TNF)-α were purchased from PeproTech EC (London, UK). PI 3′-kinase inhibitors wortmannin and LY294002, and Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, a phosphatidylinositol ether analog that potently and selectively inhibits Akt, IC50=5.0 µM) were purchased from Calbiochem (San Diego, CA). Monoclonal antibodies against RANK were raised in Balb C mice (Damool, Daejeon, Korea). The extracellular domain of human RANK (amino acids 1–212) conjugated to GST was used as an antigen for immunization. Hybridoma clones were screened with the RANK extracellular domain protein generated by the inteintagged protein expression system (New England Biolabs, Beverly, MA) as per the manufacturer’s instructions. Antibodies for phospho-specific Akt (Ser473) and Akt were purchased form Cell Signaling Technology (Beverly, MA). LPS (Escherichia coli, 0111:B4), cell culture products, and other biochemical reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise specified. Human umbilical vein endothelial cells (HUVECs) and human umbilical artery vascular smooth muscle cells (HUASMCs) were prepared from human umbilical cords by collagenase digestion and maintained as described previously (8). The primary cultured cells used in this study were between passage 2 and 3. Human peripheral blood mononuclear cells (PBMCs), Hep3B, and SaOS cells were grown in Dulbecco’s modified Eagle’s medium.

Immunofluorescent staining Monoclonal antibodies against the extracellular domain of human RANK were raised in Balb/C mice (Damool). Immunoglobulin purified from the culture supernatant of one clone (BBL1-2) was conjugated to fluorescein isothiocyanate (FITC). Pig hearts were delivered within 1 h of slaughter under sterile conditions. Rat hearts and embryos (day 15) were isolated from SpragueDawley rats. The tissues were kept in 0.1 M phosphate buffer containing 30% sucrose before being embedded in an OCT compound (Shandon, Sewickley, PA). Sections (10 µm) and freshly isolated HUVECs from umbilical cords were fixed briefly in 4% paraformaldehyde in PBS and washed. The sections were incubated for 12–14 h at 4°C, with the anti-RANK-FITC diluted at 1:500 in PBS containing 0.3% bovine serum albumin (BSA). The slides were rinsed, mounted on a Universal Mount (Research Genetics, Huntsville, AL), and viewed using an inverted fluorescence microscope (Axioskop 2 plus, Zeiss, Gottingen, Germany). Slide images were captured with a digital camera (ProRes C14, Jenoptik, Jena, Germany) and were imported into Adobe Photoshop 5.0 for compilation and labeling. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis Total RNAs from the cells were prepared using TRI Reagent (Sigma-Aldrich). RNAs (100 ng) were reverse-transcribed with SuperScript II reverse transcriptase (Gibco BRL, Grand Island, NY). The reaction was stopped by heat inactivation, and the cDNA products were divided into three parts. Each part was amplified by PCR with specific primers for RANK (5′AGTTTAAGCCAGTGCTTCACG-3′ and 5′-ACGTAGACCACGATGATGTCG-3′) for RANKL (5′AGCACATCAGAGCAGAGAAAGC-3′ and 5′-CAGTAAGGAGGGGTTGGAGACC-3′), or for OPG (5′-TCTCCATTAAGTGGACCACC-3′ and 5′-AAGAATGCCTCCTCACACAG-3′). Each of the 30 PCR amplification cycles consisted of: 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The PCR products were separated on 1.5–2.0% agarose gels and stained with ethidium bromide. Akt (Ser473) phosphorylation assay To assay Akt (Ser473) phosphorylation, treated HUVECs were washed two times with PBS, lysed in sample buffer (50 mM Tris-HCl, 100 mM NaCl, 0.1% SDS, 1% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 µg/ml aprotinin, 1 µg/ml pepstatin, and 1 µg/ml leupeptin), boiled, separated by SDS-PAGE, and transferred to nitrocellulose membrane. The Akt (Ser473) phosphorylation levels were analyzed according to the manufacturer’s protocol (Cell Signaling Technology). All signals were visualized and analyzed by densitometric scanning (LAS-1000, Fuji Film, Tokyo). Endothelial cell survival assay Endothelial cell survival was assayed as described previously (9). HUVECs were plated onto gelatinized 24-well plates (7×104 cells per well) in M-199 containing 20% FBS and were incubated for 24 h. To induce serum-deprivation-induced apoptosis, the wells were washed extensively with PBS, the medium was changed to serum-free M-199 containing control buffer or the indicated amount of RANKL, and the cells were incubated for 24–30 h. To induce apoptosis, we incubated the cells in M-199 medium supplemented with 2% serum containing the indicated amount of TNF-α or LPS with cyclohexamide (20 µg/ml) for 12–24 h. Quantification of apoptosis was performed as described previously (9). Briefly, floating apoptotic cells were

collected with two washes in PBS. Adherent cells were collected by trypsinization. All cells were stained with the annexin-V-fluos staining kit (Roche Molecular Biochemicals, Mannheim, Germany) for 15 min at 20°C. Following staining of annexin-V and propidium iodide (PI), the cells were analyzed on a flow cytometer and data were analyzed with CellQuest software (Becton-Dickinson, Franklin Lakes, NJ). Adenoviral gene transfer Two adenoviral vectors, one encoding only β-galactosidase (Ade-β-gal) and one encoding a hemagglutinin (HA)-tagged dominant-negative mutant Akt (Ade-Akt-AA), were kindly provided by Dr. Kenneth Walsh (Boston University School of Medicine) and used as described previously (10). HUVECs were infected at a 100 multiplicity of infection (MOI) with Ade-β-gal or AdeAkt-AA for 12 h. The virus was removed, and cells were left to recover for 12 h in complete medium. These conditions resulted in uniform expression of the transgenes in nearly 100% of the cells, as determined by infection with Ade-β-gal followed by staining for β-gal activity (data not shown). Statistics Data are expressed as mean ± SD. Statistical significance between two groups was tested using the unpaired Student’s t test. Statistical significance between more than two groups was tested using one-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at P < 0.05. RESULTS RANK is predominantly present in blood vessel endothelial cells Immunofluorescent staining showed that RANK was mainly detected in endothelial cells of pig coronary artery but was not detected in vascular smooth muscle cells (Fig. 1Aa). Moreover, RANK was also detected in freshly isolated HUVECs from umbilical cords (Fig. 1Ab). RANK was highly expressed in endothelial cells of rat coronary artery, whereas it was detected as punctate spots in rat coronary vein (Fig. 1Ac, 1Ad). In addition, RANK was also detected in endothelial cells of developing blood vessels of rat embryo (Fig. 1Ae). Thus, expression of RANK in endothelial cells is distinct and heterogeneous. In agreement with the immunofluorescent staining, RT-PCR analyses showed that RANK mRNA was expressed in HUVECs, whereas RANK mRNA was not detected in HUASMCs, Hep3B, or PBMC (Fig. 1B). In comparison, RANKL mRNA was clearly observed in HUASMC, but not in HUVECs (Fig. 1C). Thus, it appears that RANKL may be secreted by arterial smooth muscle cells and act in a paracrine manner on RANK expressed on endothelial cells. RANKL induces endothelial cell survival Serum deprivation caused apoptosis in HUVECs, evidenced by more floating and less adherent cells seen with phase-contrast microscopy (Fig. 2A), fragmented DNA detected with TUNEL assay (Fig. 2B), and FACS analysis of cells that stained with PI staining (dead cells) and annexin V (apoptotic cells) (Fig. 2C, 2D). Approximately 30–35% of total cells undergo apoptosis at 24 h

during serum deprivation. RANKL at 300 ng/ml inhibited ~45–50% of the apoptotic events (Fig. 2E). Activation of PI 3′-kinase/Akt is the main pathway in RANKL-induced endothelial cell survival Because activation of PI 3′kinase/Akt pathway is a common feature in the transduction of other cell survival signals in endothelial cells (8, 11–13), we examined the effect of specific PI 3′kinase inhibitors in RANKL-induced endothelial cell survival. Both wortmannin (30 nM) and LY294002 (100 nM) almost completely blocked the RANKL-induced survival effect from serum-deprivation-induced apoptosis (Fig. 3A). Both reagents slightly enhanced the degree of apoptosis observed in the absence of RANKL, possibly because of inhibition of basal PI 3′kinase activity present in serum-deprived cells. We next examined whether RANKL induced Akt activation in HUVECs. To assay Akt activation, we examined Akt phosphorylation at Ser473 in whole-cell lysates of HUVECs by probing an immunoblot with a phospho-specific antibody. In initial time course experiments, RANKL caused maximal activation of Akt in 30 min (Fig. 3B). The maximum mean increase in Akt phosphorylation was 4.8-fold. RANKL increased Akt in a dose-dependent manner (Fig. 3C). Pretreatment with wortmannin (30 nM) completely abolished RANKL-induced Akt phosphorylation (Fig. 3D). We further examined whether Akt is directly involved in the RANKL-induced antiapoptotic effect by using adenoviral gene transfer methods. When HUVECs were transfected with a control adenovirus expressing only Ade-β-gal, addition of RANKL inhibited ~40–45% of the serum deprivation-induced apoptosis (Fig. 4). This result agrees perfectly with the previous experiments (Fig. 2E, 3A), indicating that adenoviral gene transfer did not affect the rate of apoptosis. The selective inactivation of Akt using adenoviral transfer of a dominant-negative form of Akt (Ade-Akt-AA) almost abolished the RANKL-induced survival effect. Thus, RANKL-induced endothelial cell survival is mediated mainly by Akt activation. RANKL suppresses TNF-α-, LPS-, or serum deprivation-induced endothelial cell apoptosis in a PI 3′-kinase-dependent manner We next examined the survival effect of RANKL on endothelial cells under more clinically relevant conditions. The inflammatory cytokine TNF-α and the endotoxin LPS exert potent effects on endothelial cells when bacterial infection takes place. When HUVECs were treated with TNF-α (50 ng/ml) or LPS (10 µg/ml) for 12 h, ~30–40% of total cells underwent apoptosis. RANKL at 300 ng/ml inhibited ~45–50% of the apoptotic events (Fig. 5A). Pretreatment with wortmannin (30 nM) almost completely blocked the RANKL-induced antiapoptotic effect (Fig. 5A). These results suggest that RANK may protect endothelial cells under infectious and inflammatory conditions through the antiapoptotic signaling molecule PI 3′-kinase. In addition, addition of strong PI 3′-kinase/Akt activating growth factor, angiopoietin-1 (Ang1), to RANKL produced an additive antiapoptotic effect (Fig. 5B). Pretreatment with wortmannin (30 nM) almost completely blocked the additive antiapoptotic effect (Fig. 5B). These data suggest that the both agents enhance activation of PI 3′-kinase/Akt for endothelial cell survival. Pretreatment of NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC, 30 µg/ml) did not produce significant change in the RANKL-induced antiapoptotic effect, whereas pretreatment with wortmannin (30

nM) or Akt inhibitor (5 µM) almost completely blocked the RANKL-induced antiapoptotic effect (Fig. 5A). Taken together, PI 3′-kinase/Akt, but not NF-κB, is the main signaling cascade for RANKL-induced survival effect in endothelial cells. TNF-α induces OPG expression in HUASMCs To explore the potential involvement of the regulation of the RNAKL-RANK-OPG system in the death-eliciting mechanisms of TNF-α or LPS, we examined the expression levels of RANKL, RANK, and OPG by RT-PCR. The expression levels of RANKL and RANK were not changed by either TNF-α or LPS treatment in either HUVECs or HUASMCs (data not shown). However, TNF-α, but not LPS, induced OPG in HUVECs (Fig. 6A). Neither TNF-α nor LPS affected OPG expression in HUASMCs (Fig. 6B). These results suggest that regulation of OPG expression by endothelial cells may be part of a mechanism by which TNF-α causes endothelial damage in vivo. DISCUSSION The vascular endothelium consists of a monolayer of endothelial cells, and it is involved in a variety of functions, including maintenance of normal vascular tone, prevention of thrombosis, and pathologic remodeling (14, 15). Damage to the vascular endothelium can initiate thrombosis formation, neointimal hyperplasia, and atherogenesis. Therefore, maintaining normal integrity of vascular endothelium in response to physical, biochemical, and immune-mediated damage is important to prevent vascular diseases (14–16). Damage to vascular endothelial cells can be prevented by several growth factors, such as basic fibroblast growth factor (17), vascular endothelial growth factor (VEGF) (18), and Ang1 (19). Interestingly, the Ang1 receptor, Tie2, is selectively expressed as an activated form in the endothelial cells of normal adult vessels, whereas VEGF receptors, Flk1 and Flt1, are only expressed in endothelial cells that are actively involved in vasculogenesis and angiogenesis (20–22). Recently, we reported that the angiopoietin/Tie2 system in normal adult blood vessels may be important in maintaining the integrity of nonproliferating endothelial cells (23). Our immunofluorescent staining analyses indicate that RANK is selectively expressed in the endothelial cells of normal adult vessels, in which vasculogenesis and angiogenesis do not normally occur. RT-PCR analysis also provides evidence that RANK is expressed in endothelial cells, whereas RANKL is expressed in vascular smooth muscle cells. These data suggest that RANK and RANKL may interact in a paracrine manner between the smooth muscle and endothelial cells of normal blood vessels (Fig. 7). What does the RANKL/RANK system do in endothelial cells that are not engaged in vasculogenesis or angiogenesis? Given the antiapoptotic effect of RANKL on mature osteoclasts (24), we thought that constitutive expression of RANK might function to maintain endothelial cell integrity by playing a role in endothelial cell survival, like the angiopoietin/Tie2 system. Therefore, we examined the survival effect of RANKL in primary cultured endothelial cells. Indeed, RANKL protects strongly against apoptosis induced by serum deprivation, TNF-α, and LPS. Thus, we conclude that the RANKL/RANK system could be an additional component of a protection system for maintaining the normal integrity of nonproliferating endothelial cells (Fig. 7). Furthermore, the involvement of the tripartite system of RANKL/RANK/OPG was suggested by the finding that TNF-α induced OPG expression in endothelial cells. It is likely that the inhibition of the RANKL/RANK-mediated survival by OPG

contributes to the endothelial damage by TNF-α, in addition to its direct apoptosis-triggering mechanism (Fig. 7). Our understanding of the RANKL/RANK system in endothelial cells is still very limited. RANKL has an angiogenic effect through activation of the Src-PLC-Ca2+ signaling cascade, mitogen-activated protein kinase ERK1/2, and focal adhesion kinase p125FAK (25). Interestingly, the expression of RANKL and OPG is increased by proinflammatory cytokines TNF-α and IL-1 in human microvascular endothelial cells derived from normal adult dermal tissue (26). TGF-β has also been reported to induce RANKL in endothelial cells and a mouse bone marrow-derived endothelial cell line (27). Thus, the RANKL/RANK system could play an active role in pathologic angiogenesis and inflammation in addition to its cell survival function under normal physiological conditions (Fig. 7). The serine-threonine protein kinase Akt (28) is a downstream effector of PI 3′-kinase. Activation of PI 3′-kinase increases the intracellular amount of phosphatidylinositol-4,5-biphosphate and phosphatidylinositol-3,4,5-triphosphate, which positively regulate Akt. Thus, Akt is activated by phospholipid binding and phosphorylation within the activation loop at Thr308 and within the COOH-terminus at Ser473 (29). The PI 3′-kinase and Akt pathways are common features in the transduction of survival effects in endothelial cells (8, 11–13). In this study, we demonstrated that RANKL induces Akt phosphorylation at Ser473 in endothelial cells, and this induction is PI 3′-kinase dependent. In addition, interfering with Akt activation by using a dominant-negative form of Akt or pretreament of specific Akt inhibitor abolished the increase in endothelial cell survival stimulated by RANKL. Moreover, blocking the PI 3′-kinase activity with specific inhibitors completely reversed the RANKL-induced survival effect on endothelial cells undergoing apoptosis in response to serum deprivation, TNF-α, or LPS. Thus, the activity of PI 3′-kinase and Akt is critical for RANK to transduce its survival signal in endothelial cells. We previously demonstrated that PI 3′-kinase/Akt activation follows several kinds of insults to endothelial cells and is a crucial step in Ang1-induced survival (8, 19, 23). In this study, we also showed that RANKL and Ang1 produced additive survival effects in a PI 3′-kinase-dependent manner. Thus, RANKL and Ang1 have a common signaling pathway for preventing apoptosis in endothelial cells (Fig. 7). Taken together, our findings suggest that the presence of RANK in normal endothelial cells is important for the maintenance of endothelial cell integrity and that the PI 3′-kinase/Akt signal transduction pathway mediates the endothelial cell survival effect of RANK in response to RANKL, which is secreted as a paracrine factor by vascular smooth muscle cells (Fig. 7). ACKNOWLEDGMENTS We thank Dr. Kenneth Walsh for providing critical adenoviral vectors Ade-β-gal and Ade-AktAA. We thank Jennifer Macke for help in preparing the manuscript. This work was supported by the National Research Laboratory and Creative Research Initiatives of the Korean Ministry of Science and Technology.

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Fig. 1

Figure 1. RANK is predominantly expressed in endothelial cells. A) Immunofluorescent staining of RANK in pig coronary artery (a), freshly isolated HUVECs from umbilical cords (b), rat coronary artery (c), rat coronary vein (d), and rat dorsal aorta at embryonic day 15 (e). The slides were incubated with post-immune polyclonal antibodies, and antibody binding was visualized with FITC-labeled anti-rabbit antibody. Red blood cells were visualized by the red fluorescence emitted by using a red filter (e). B, C) RT-PCR analysis of RANK and RANKL in primary cultured HUVECs, HUASMCs, and PBMC and in immortalized cell lines Hep3B and SaOS.

Fig. 2

Figure 2. RANKL enhances endothelial cell survival. HUVECs were changed to serum-deprived (SD) medium and incubated with RANKL (300 ng/ml) or control buffer for 30 h. A) Representative phase-contrast images. Note that there are fewer adherent cells and more floating cells in the SD culture than in the control with 10% serum (S). The cells exposed to RANKL (SD + RANKL) are more adherent than cells in the SD culture. Magnifications: 200×. B) Light microscopy of TUNEL assay. Arrowheads indicate brown apoptotic cells with fragmented or condensed DNA. Magnifications: 200×. C) Representative dot plots of flow cytometric analyses. The cells were stained with annexin V and propidium iodide (PI) and were analyzed by flow cytometry. D) Representative histogram of flow cytometric analyses. The cells were stained with annexin V and were analyzed by flow cytometry. E) Apoptotic cells were quantified using flow cytometry. Bars represent the mean ± SD of five experiments. *P