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RANKL regulates endothelial cell survival through the phosphatidylinositol 3'-kinase/Akt signal transduction pathway¹

HONG-HEE KIM, HYOUNG SEEK SHIN^*, HEE JIN KWAK^*, KYU YOUN AHN, JU-HYUN KIM^*, HYUEK JONG LEE^*, MI-SOOK LEE^*, ZANG HEE LEE^,2 and GOU YOUNG KOH^*,2

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; and
National Research Laboratory for Bone Metabolism and School of Dentistry, Chosun University, Gwangju 501-759, Korea

²Correspondence: National Research Laboratory for Bone Metabolism and School of Dentistry, Chosun University, Gwangju 501-759, Korea; E-mail: jhblee{at}mail.chosun.ac.kr or National Creative Research Initiatives Center for Endothelial Cells, Department of Life Science, POSTECH, San 31, Hyoja-Dong, Pohang, 790-784, Republic of Korea; E-mail: gykoh{at}postech.ac.kr

SPECIFIC AIMS

Using immunofluorescent staining, we found that receptor activator of NFB (RANK) is highly expressed in endothelial cells of normal adult arterial blood vessels, which are not actively involved in angiogenesis and vasculogenesis. Therefore, we examined whether the RANK ligand (RANKL)-RANK system may be important in regulating the survival of endothelial cells.

PRINCIPAL FINDINGS

1. 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 not detected in vascular smooth muscle cells (Fig. 1 A). Moreover, RANK was detected in freshly isolated HUVECs from umbilical cords (Fig. 1Ab ). RANK was highly expressed in endothelial cells of rat coronary artery, while it was detected as punctate spots in rat coronary vein (Fig. 1Ac, d ). 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.

View larger version (33K): [in this window] [in a new window]   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 postimmune 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 in panel (e). B) RT-PCR analysis of RANK and RANKL in primary cultured HUVECs, HUASMCs, and PBMC and in immortalized cell lines Hep3B and SaOS.

2. RANKL induces endothelial cell survival
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.

3. 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, 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. 2 A). 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. 2B ). The maximum mean increase in Akt phosphorylation was 4.8-fold. RANKL increased Akt in a dose-dependent manner (Fig. 2C ). Pretreatment with wortmannin (30 nM) completely abolished RANKL-induced Akt phosphorylation (Fig. 2D ). We further examined whether Akt is directly involved in the RANKL-induced anti-apoptotic effect by using adenoviral gene transfer methods and specific Akt inhibitor. The selective inactivation of Akt using adenoviral transfer of a dominant-negative form of Akt (Ade-Akt-AA) or Akt inhibitor almost abolished the RANKL-induced survival effect. Thus, RANKL-induced endothelial cell survival is mediated mainly by Akt activation.

View larger version (28K): [in this window] [in a new window]   Figure 2. RANKL prevents endothelial cell apoptosis in a PI 3'-kinase dependent pathway, and RANKL induces Akt (Ser473) phosphorylation in endothelial cells. A) HUVECs were changed to serum 10% (S) or serum-deprived (SD) medium and incubated with control buffer (CB), wortmannin (WT, 30 nM), or LY294002 (LY, 100 nM) in the absence or presence of RANKL (300 ng/mL). Apoptotic cells were quantified using flow cytometry. Bars represent the mean ± SD of 4 experiments. *P < 0.05 vs. S; #P < 0.05 vs. SDonly. B–D) HUVECs were incubated for 24 h in 1% serum-containing M-199 medium, then incubated with RANKL for the indicated times (B), for 30 min at the indicated concentrations (C), or in the presence of wortmannin (30 nM) (D). After treatment, cell lysates were harvested. Each lane contains 50 µg of total protein from the cell lysates. The blots were probed with anti-phospho-Akt (Ser473) antibody (upper panels). The membranes were stripped and reprobed with anti-Akt antibody (lower panels) to verify equal loading of protein in each lane. Fold: Densitometric analyses are presented as the relative ratio of phospho-Akt to Akt. The relative ratio measured at time 0, or the ratio relative to control buffer (0), is arbitrarily presented as 1. Numbers represent the mean ± SD from 3 experiments. *P< 0.05 vs. time 0 or control buffer.

4. RANKL inhibits TNF- and LPS-induced endothelial cell apoptosis
We next examined the survival effect of RANKL on endothelial cells under more clinically relevant conditions. The inflammatory cytokine TNF- and the endotoxin LPS exert potent effects on endothelial cells when bacterial infection takes place. When HUVECs were treated with TNF- (50 ng/mL) or LPS (10 µg/mL) in the presence of cyclohexamide (20 µg/mL) for 12 h, 30–40% of total cells underwent apoptosis. RANKL at 300 ng/mL inhibited 45–50% of the apoptotic events. Pretreatment with wortmannin (30 nM) almost completely blocked the RANKL-induced antiapoptotic effect. These results suggest that RANK may protect endothelial cells under infectious and inflammatory conditions through the antiapoptotic signaling molecule PI 3'-kinase.

5. TNF- induces osteoprotogerin (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- 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- or LPS treatment in either HUVECs or HUASMCs. However, TNF-, but not LPS, induced OPG in HUVECs. Neither TNF- nor LPS affected OPG expression in HUASMCs. These results suggest that regulation of OPG expression by endothelial cells may be a part of mechanism by which TNF- causes endothelial damage in vivo.

CONCLUSIONS AND SIGNIFICANCE

Maintaining a normal integrity of vascular endothelium in response to physical, biochemical, and immune-mediated damage is important to prevent vascular diseases. Damage to vascular endothelial cells can be prevented by several growth factors such as basic fibroblast growth factor, vascular endothelial growth factor, and angiopoietin-1.

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. 3 ). 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, 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-, 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. 3) . Furthermore, involvement of the tripartite system of RANKL/RANK/OPG was suggested by the finding that TNF- 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- in addition to its direct apoptosis-triggering mechanism (Fig. 3) .

View larger version (35K): [in this window] [in a new window]   Figure 3. Model for RANK/RANKL/OPG system in blood vessels. The presence of RAMK in normal endothelial cells has important implications in the maintenance of endothelial cell integrity. The PI 3'-kinase/Akt signal transduction pathway mediates the endothelial cell survival effect of RANK in response to RANKL provided in a paracrine manner by vascular smooth muscle cells. In pathological conditions, TNF--induced OPG expression in endothelial cells may be partly involved in the inhibition of RANKL/RANK-mediated endothelial cell survival through OPG binding to RANKL.

In this study, we demonstrated that RANKL induces Akt phosphorylation at Ser473 in endothelial cells and that this induction is PI 3'-kinase dependent. Interfering with Akt activation by using a dominant-negative form of Akt 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-, or LPS. Thus, the activity of PI 3'-kinase and Akt is critical for RANK to transduce its survival signal in endothelial cells.

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.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0215fje; doi: 10.1096/fj.03-0215fje

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This Article Full Text (PDF) All Versions of this Article: 17/14/2163 03-0215fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by KIM, H.-H. Articles by KOH, G. Y. Search for Related Content PubMed PubMed Citation Articles by KIM, H.-H. Articles by KOH, G. Y.