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IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation
Microvascular Research 78 (2009) 25–32
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Microvascular Research 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 / y m v r e
Regular Article
IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation Troels Hammer a, Katerina Tritsaris a, Martin V. Hübschmann a, Josefine Gibson a, Riccardo E. Nisato b, Michael S. Pepper b, Steen Dissing a,⁎ a b
Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Center for healthy Ageing, University of Copenhagen, Denmark Department of Cell Physiology and Metabolism, University hospitals of Geneva, Switzerland
a r t i c l e
i n f o
Article history: Received 22 October 2008 Revised 16 February 2009 Accepted 17 February 2009 Available online 9 March 2009 Keywords: IL-20 Lymphangiogenesis Interleukins Endothelium
a b s t r a c t IL-20 is an arteriogenic cytokine that remodels collateral networks in vivo, and plays a role in cellular organization. Here, we investigate its role in lymphangiogenesis using a lymphatic endothelial cell line, hTERT-HDLEC, which expresses the lymphatic markers LYVE-1 and podoplanin. Upon stimulation of hTERT-HDLEC with IL-20, we found an increase in the intracellular free calcium concentration, in Akt and eNOS phosphorylations as well as in perinuclear NO production. We found that eNOS phosphorylation and NO synthesis are highly dependent on the PI3K/Akt signalling pathway. We also found an IL-20 induced phosphorylation of Erk1/2 and mTOR, and using the MEK inhibitor PD98059 and mTOR complex inhibitor rapamycin we demonstrated the importance of these signalling pathways in IL-20-mediated proliferation. IL-20 triggered actin polymerization and morphological changes resulting in elongated cell structures, and in matrigels, IL-20 caused tube formations of hTERT-HDLEC in a PI3K- and mTOR dependent way. In a sprouting assay we found that IL-20 caused cell migration within 24 h at a rate comparable to VEGF-C, and this migration could be inhibited by wortmannin and rapamycin. These data show that IL-20 activates cell signalling resulting in lymphangiogenic processes including migration, proliferation and tube formation. Thus, IL20 is a cytokine that has the potential of activating or modulating the formation of lymphatic vessels. © 2009 Elsevier Inc. All rights reserved.
Introduction Lymphangiogenesis – the growth of new lymphatic vessels – is implicated in both normal developing tissues and in a number of pathological processes such as lymphoedema, inflammation, cancer lymphatic metastasis, psoriasis and diabetic wound healing (Cao, 2005; Kunstfeld et al., 2004; Maruyama et al., 2007). The identification of specific lymphatic markers including LYVE-1 and podoplanin (Breiteneder-Geleff et al., 1999; Fournier et al., 1999; Nisato et al., 2004; Podgrabinska et al., 2002) has set in motion the research in lymphangiogenesis, and clinical implications are quickly emerging (Karkkainen et al., 2004; Stacker et al., 2007). The embryonic lymphatic differentiation is relatively well described (Schacht et al., 2003; Wigle et al., 2002; Wilting et al., 2002) with focus on vascular endothelial growth factor C (VEGF-C) and its primary receptor VEGF receptor 3 (VEGFR-3) (Shibuya and Claesson-Welsh, 2006). However, in the adult organism, lymphangiogenesis – as in angiogenesis – is an intricate process with multiple components. One group of factors that regulate lymphangiogenesis is the inflammatory cytokines. The pro-inflammatory cytokine interleukin 20 (IL-20) belongs to the IL-10 cytokine ⁎ Corresponding author. Panum Institute, Building 12.6, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. Fax: +45 35 32 75 26. 0026-2862/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2009.02.007
family that also includes IL-10, 19, 22, 24, 26, 28, and 29 (Sabat et al., 2007). IL-20 is expressed in monocytes, epithelial, endothelial and maturing dendritic cells (Hsing et al., 2006; Wolk et al., 2008) and exerts its biological functions through activation of the receptor complexes IL-20Rα/IL-20Rβ or IL-22Rα/IL-20Rβ, which consist of transmembrane glycoproteins belonging to the family of class II cytokine receptors (Hsieh et al., 2006; Sabat et al., 2007). IL-20 is involved in the pathogenesis of several angiogenesisdependent inflammatory diseases including psoriasis and rheumatoid arthritis (Blumberg et al., 2001; Sabat et al., 2007; Kragstrup et al., 2008), and recently the cytokine has been shown to induce nitric oxide (NO) synthesis in blood vessel endothelial cells (BVECs) and to promote angiogenesis (Hsieh et al., 2006; Tritsaris et al., 2007). NO is known as a key regulator of several processes in lymphangiogenesis and mediates lymphatic vessel activation (Kajiya et al., 2008). The endothelial NO production is dependent on Akt-activation, which is also a central mediator of endothelial cell survival, migration and proliferation (Dormond et al., 2007). The mammalian target of rapamycin (mTOR)–rictor complex plays a pivotal role in the activation of Akt, by phosphorylating Ser473, while PDK1 phosphorylates Thr308 in the activation loop. Downstream of Akt, mTOR exists in another protein complex together with raptor (Sarbassov et al., 2005). Recent studies using the selective inhibitor of mTOR, rapamycin, has highlighted
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the importance of mTOR for numerous processes essential for lymphangiogenesis, including cell growth, proliferation, motility, and survival, in part by enhancing the production of VEGF-A and VEGF-C (Blei, 2007). NO is synthesized by endothelial nitric oxide synthase (eNOS) through phosphorylation of eNOS by several Ser/Thr kinases such as Akt, as well as by interactions with the Ca2+/calmodulin complex (Dudzinski and Michel, 2007). Signal transduction coupled to the IL-20 receptor in lymphatic endothelial cells has not previously been characterized, and we used the newly developed cell line hTERT-HDLEC (human Telomerase Reverse Transcriptase Human Dermal Lymphatic Endothelial Cells) that expresses the recognized lymphatic markers Prox1, LYVE-1 and podoplanin (Nisato et al., 2004) to investigate the IL-20-induced signalling in lymphatic endothelial cells. We show that this inflammatory cytokine is a modulator of lymphangiogenic processes. Materials and methods Reagents Recombinant human (rh)IL-20 was obtained from Biosource (Belgium) and rhVEGF-C was obtained from R&D systems (Germany). Rapamycin, wortmannin and PD98059 were purchased from SigmaAldrich (USA). Lipofectamin was obtained from Invitrogen (USA) and calyculin from Calbiochem (USA). BKAR (B kinase activity reporter) plasmid was kindly provided by Prof. Alexandra Newton (UCSD, USA) and was amplified as described in Kunkel et al., 2005. Cell lines and cell culture hTERT-HDLEC is a human lymphatic endothelial cell line displaying longer life-span (Nisato et al., 2004). Cells were cultured in gelatine-coated tissue culture flasks in EGM MV2 (Promocell, Germany) supplemented with penicillin (200 U/ml)–streptomycin (50 μg/ml) and L-Glutamine (0.29 mg/ml). Signal transduction assays hTERT-HDLECs were grown to 90% confluency in gelatine-coated 60-mm dishes, washed with PBS and incubated for 60 min in serumfree medium (RPMI medium 1640) before stimulation with growth factor or interleukin. Cellular activity was stopped by adding 100 μl 1% Igepal lysis buffer (Sigma-Aldrich, USA) containing 1.2 μg/ml aprotinin, pepstatin, and leupeptin and 1.25 mmol/L NaF, PMSF, and sodium orthovanadate. Samples were mixed for 1 min and centrifuged for 10 min at 11,000 ×g. Pellets were discarded and equal amounts of protein samples were separated by SDS-PAGE (10% BIS–Tris gel). Proteins were transferred to nitrocellulose membranes, and membranes were blocked with 5% bovine serum albumin (BSA) in PBS with 0.1% Tween. Membranes were probed overnight at 4 °C with antibodies diluted in PBS with 5% BSA and 0.1% Tween for detecting P-Akt (Ser473), P-mTOR (Ser2448), P-p70S6K (Thr389), P-eNOS (Ser1177) (Cell Signalling Technology, USA), P-Erk 1/2 (Tyr204) (Santa Cruz Biotechnology, USA) or GAPDH (Millipore, USA) as recommended by the manufacturer. This was followed by incubation for 1 h in PBS with 1% BSA and 0.1% Tween with peroxidase-conjugated goat anti-rabbit immunoglobulin antibody diluted 1:1000 for P-Akt, 1:500 for P-mTOR, and 1:1000 for P-eNOS and with peroxidase-conjugated goat antimouse immunoglobulin antibody diluted 1:5000 for P-Erk1/2, 1:1000 for P-p70S6K and 1:10000 for GAPDH. Protein bands were visualized by enhanced chemiluminiscence using a LAS3000 Lumi-Imager. Phalloidin staining hTERT-HDLECs were grown on chambered gelatine-coated coverglasses (Lab-Tek; Nalge Nunc International, USA) to 90% confluency.
Cells were serum-starved for 1 h in RPMI 1640 medium before stimulation. Activation was stopped by removal of the media and cells were fixed, permeabilized, and stained using F-actin visualization Biochem Kit according to the instructions provided by the supplier (Cytoskeleton, USA). Measurements of [Ca2+]i, nitric oxide and BKAR emission The intracellular free calcium concentration ([Ca2+]i) was measured in hTERT-HDLECs grown to 90% confluency in chambered gelatine-coated cover-glasses (Lab-Tek; Nalge Nunc International, USA). Cells were loaded with 3 μM fura-2/AM (Invitrogen, USA) in RPMI medium 1640 for 30 min and washed with Krebs–Ringer buffer. Images were acquired by means of a Zeiss Axiovert 135 microscope equipped with a Zeiss Achrostigmat 40 × 1.3 NA objective. Excitation was obtained by a Polychrome V illuminator from Till Photonics (Germany) and images were acquired using a Cool Snap CCD camera (Photometrics, USA) from Robert Scientific (Malaysia). For measurements of [Ca2+]i, the excitation wavelengths were 338 and 380 nm, measuring emission above 510 nm using a cut-off filter. Calculations of [Ca2+]i were done by using MetaFluor software from Molecular Devices and using a Kd of 160 nM (Dissing et al., 1990). Measurements of Akt activity using BKAR was performed by measuring CFP and YFP emissions using a Dual View from Optical Insights (USA). After background subtraction, ratio images were formed using MetaFluor software. For measurements of NO production, hTERT-HDLECs were grown to 90% confluency in chambered gelatine-coated cover-glasses and pre-loaded with 5 μM DAF-2 DA (Merck, Germany) in RPMI 1640 medium for 30 min. Cells were washed in Krebs–Ringer buffer, and images were acquired by exciting at 440 nm and measuring emission above 510 nm using MetaFluor. Cells were seeded in chambered gelatine-coated cover-glasses (Lab-Tek; Nalge Nunc International, USA) and transfected with BKAR plasmid for 24 h using lipofectamin (100 μl of 5% lipofectamin) with 1 μg/ml of BKAR DNA added to 1 ml of 2% EGM MV2 in which FCS was reduced to 2%. Prior to measurements cells were incubated for 4 h in a medium containing 2% EGM MV2. RT-PCR Reverse transcription was performed by using total RNA from hTERT-HDLEC and Omniscript Reverse Transcriptase kit (Qiagen, DK). Complementary cDNAs were amplified by PCR using the following primer pairs: (I) a 20-mer 5′-GGGCTTCCTCGTCGCAGTAC-3′ (forward) oligo and (II) a 22-mer GGGCGTAGAATGGGAATTGAGC-3′ (reverse) oligo to generate an IL-22Rα fragment of 419 bp; (III) a 24-mer 5′-CCGAACACTCTTTACTGCGTACAC-3′ (forward) oligo and (IV) a 22-mer 5′-GCTGCCTGCGACTCCAATAATG-3′ (reverse) oligo to generate an IL-20Rα fragment of 683 bp; (V) a 21-mer 5′-CAGACCTCAGCCTGGAGCATC-3′ (forward) oligo and (VI) a 24-mer 5′-AGGATCAGCATGAAGCCAACAAAG-3′ (reverse) oligo to generate an IL-20Rβ fragment of 383 bp;(VII) a 22-mer 5′-TGGATGGAGACACACAGACAAC3′ (forward) oligo and (VIII) a 19-mer 5′-TCAGGGACAGGGCACAGAG-3′ (reverse) oligo to generate a Podoplanin fragment of 226 bp;(IX) a 22mer 5′-GGCTCTGCTAGTGCTTGCTCTC-3′ (forward) oligo and (X) a 24mer 5′-GCTTGGACTCTTGGACTCTTCTGG-3′ (reverse) oligo to generate a LYVE-1 fragment of 220 bp. Spheroid in vitro lymphangiogenesis assay An in vitro lymphangiogenesis assay using hTERT-HDLEC spheroids was performed principally as described in Korff and Augustin (1999). Briefly, spheroids were generated by seeding 750 hTERT-HDLEC cells in each well of a 96-well nonadherent round-bottomed plate, in a
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reduced MV2-medium (containing no growth factors, 1% FCS and pen./strep.) with 0.24% high viscosity methyl cellulose (SigmaAldrich, USA), incubated at 37 °C and 5% CO2. Under these conditions, all cells in each well formed a single spheroid, and after 1 day the spheroids were collected and embedded into collagen gels. The collagen stock solution was prepared by mixing rat tail collagen I (3.13 mg/ml) (BD Biosciences, DK) with 10% (vol.) 10× Earle's balanced salt solution (Sigma-Aldrich, USA) and 1 M NaOH to adjust pH to 7.4. The collagen stock solution was mixed 1:1 with 1.2% methyl cellulose in reduced medium and IL-20 or VEGF-C as indicated, and added to the collected spheroids. Spheroids in collagen were plated in a 24 well plate and incubated at 37 °C in 5% CO2 for 24 h, and spheroid sprouting/migration was visualized by using a Leitz Labovert phasecontrast microscope (Leica Microsystems, Germany) and a digital camera (CoolPix 990; Nikon, Japan). Quantification was done by measuring the total area covered by each spheroid, using the ImageJ software. (http://rsb.info.nih.gov/ij/index.html). Matrigel tube formation assay Growth factor-reduced matrigel matrix (BD Biosciences, Denmark) was added to cover-glasses (Lab-Tek; Nalge Nunc International, USA)
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(250 μl to each chamber) and the matrigel was allowed to polymerize for 30 min at 37 °C. hTERT-HDLECs (50,000 cells) in reduced MV2medium (no growth factors, 0.5% FCS, pen./strep.) were plated on the matrigel and stimulated. At appropriate time intervals tube formation was observed with a Leitz Labovert phase-contrast microscope × 150 magnification (Leica Microsystems, Denmark), and images were acquired by using a digital camera (CoolPix 990; Nikon, Japan). Tube length was quantified using the ImageJ software. Proliferation assay hTERT-HDLECs were seeded into 96-well plates at 1000 cells per well in a total volume of 200 μl and grown in FGF- and VEGF-A depleted EGM-MV2 medium (reduced medium) containing 5% FCS and pen./strep. for 4 or 5 days; the cells were left untreated or were treated with IL-20 (50 ng/ml or 100 ng/ml) at day 1. Inhibition of cell proliferation was conducted with rapamycin (100 nM), wortmannin (100 nM) or PD98059 (10 μM). Reduced medium with cytokines/ inhibitors was renewed at day 3. After 4 or 5 days, proliferation was measured using the EZ4U-proliferation Kit (Biomedica Gruppe, Austria) based on the method of reduction of tetrazolium salt to coloured formazan. Samples were incubated 3 h with substrate before
Fig. 1. (A) RT-PCR was performed and the amplified fragments of IL-20Rβ, IL-20Rα, IL-22Rα, podoplanin and LYVE-1 cDNA were visualized on an agarose gel. NTC is non template control. (B) hTERT-HDLECs were stimulated with IL-20 (10 ng/ml) at time zero. Western blot analysis was performed using an antibody specific for phosphorylation of Akt at Ser473 (P-Akt). Phosphorylation levels are shown in arbitrary units (AU) with control samples normalized to 100. Membranes were reprobed with an anti-GAPDH antibody. (C) Statistical significant dephosphorylation of Akt at 1 and 5 min, and phosphorylation of Akt at 15 min after IL-20 stimulation; n = 6, ⁎⁎, P b 0.01. (D) P-Akt levels at 15 min after IL-20 stimulation with or without 30 min pre-incubation with wortmannin (100 nM); n = 3 in one experiment, ⁎⁎, P b 0.01; ⁎, P b 0.05. (E) Measurement of Akt activity by use of BKAR. IL-20 stimulation (20 ng/ml) was followed by addition of calyculin (100 nM). The trace represents ratio values of CFP/YFP (±SEM) obtained from a perinuclear area as indicated in (F); n = 3. (F) CFP emission image. Arrow points to the area from which data in E was measured; n = 3. (G, H) Measurement of intracellular calcium after IL-20 (10 ng/ml) stimulation of hTERT-HDLECs. Cells were stimulated with IL-20 at the indicated time points in Krebs–Ringer; n = 4 (G) or in calcium-free medium; n = 3 (H). Each trace represents the recorded calcium level in a single cell.
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measurement of absorbance at 450 nm with 630 nm as background. Results represent the mean of 16 replications. Statistical evaluations Data are presented as means ± SEM. Statistical evaluation of the results was made by two-tailed Student's t test, using Microsoft (Redmond, USA) Excel and Origin® 6.1 (Northampton, USA) software. Results IL-20 receptor expression and activation of intracellular signalling leading to NO synthesis in hTERT-HDLECs We found transcripts of all three IL-20 receptor subunits, IL-20Rα, IL-20Rβ and IL-22Rα, as well as the LEC markers LYVE-1 and podoplanin in hTERT-HDLECs by RT-PCR (Fig. 1A), which led us to investigate cellular signalling. One of the mediators of lymphatic vessel activation is NO (Kajiya et al., 2008) and we studied whether IL-20 stimulation of the lymphatic endothelial cells was coupled to NO synthesis. This was done by
studying the phosphatidylinositol 3-kinase (PI3K)/Akt and calcium signalling pathways, which lead to activation of eNOS (Dudzinski and Michel, 2007). To investigate a direct effect of IL-20 on Akt activity we performed a time-course experiment measuring phosphorylation of Akt at Ser473. As shown in Figs. 1B, C incubation of hTERT-HDLECs with IL-20 (10 ng/ml) resulted in an initial dephosphorylation of Akt within the first 0–5 min after stimulation followed by a phosphorylation process causing elevated levels of phosphorylated Akt reaching a maximum after 15 min and gradually declining there after. Wortmannin (100 nM), a PI3K inhibitor, significantly inhibited the IL-20-induced phosphorylation of Akt (Fig. 1D). To further clarify the spatio-temporal dynamics of Akt phosphorylation we transfected hTERT-HDLEC with BKAR plasmid where the transcript contains two fluorescent moieties, CFP and YFP, in close proximity (Kunkel et al., 2005). Translated BKAR contains a consensus phosphorylation site for Akt and upon phosphorylation the BKAR protein changes its conformation. That leads to a reduced energy transfer from CFP to YFP that can be detected as a change in CFP/YFP ratio. Fig. 1E shows that upon IL-20 stimulation there was a rapid, localized phosphorylation of BKAR revealing localized Akt activity which was specific for a perinuclear area (Fig. 1F). At other locations far from the nucleus we
Fig. 2. (A) hTERT-HDLECs were stimulated with IL-20 (10 ng/ml) at time zero. Western blot analysis was performed using an antibody specific for phosphorylation of eNOS at Ser1177 (P-eNOS). Phosphorylation levels are shown in arbitrary units (AU) with control samples normalized to 100. Membranes were reprobed with an anti-GAPDH antibody. (B) Statistical significant phosphorylation of eNOS at 5 and 10 min after IL-20 stimulation, and 5 min after stimulation with VEGF-A (20 ng/ml); n = 4, ⁎⁎, P b 0.01; ⁎, P b 0.05. (C) P-eNOS levels at 5 min after IL-20 stimulation with or without 30 min pre-incubation with wortmannin (100 nM); n = 3 in one experiment, ⁎⁎, P b 0.01. (D, E) Measurements of NO production before and after stimulation with either IL-20 (50 ng/ml) (left), VEGF-A (100 ng/ml) (middle) or IL-20 + wortmannin (100 nM) (right); n = 3. Images show the localized NO synthesis in the cytoplasm. Measurements of NO production in the presence of wortmannin confirmed the absence of NO synthesis when cells were not stimulated. (E) Traces were continuously recorded and represent NO production in single cells.
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did not observe a significant increase in Akt activity within this time frame. Following addition of the serine/threonine phosphatase inhibitor calyculin, we observed a strong increase in BKAR phosphorylation revealing the rapid kinetics of phosphorylation and dephosphorylation processes in the cytoplasm. Hence, despite an apparent initial dephosphorylation of Akt seen in western blots (Figs. 1B, C), there is in fact a localized increase in Akt activity within 5 min upon IL20 stimulation at a perinuclear area. Treatment of the lymphatic endothelial cells with IL-20 (10 ng/ml) also rapidly increased the intracellular free calcium concentration ([Ca2+]i). This increase in [Ca2+]i was not dependent on the presence of calcium in the extracellular medium revealing that IL-20 activates calcium release from intracellular stores (Figs. 1G, H). The elevated levels of [Ca2+]i induced by IL-20 occurred immediately after addition of IL-20 in most cells, whereas a subset of cells displayed a delayed response. VEGF-A (50 ng/ml) was used as a positive control and as expected induced a rapid calcium release, whereas VEGF-C (50 ng/ml)
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did not increase the intracellular free calcium concentration (data not shown). These data show that IL-20 stimulates both signalling pathways that are capable of activating eNOS, that is a rise in [Ca2+]i and activation of Akt. We therefore tested to which extent IL-20 could induce phosphorylation of eNOS at the activation site Ser1177, and show that IL-20 mediates phosphorylation of eNOS after 5 min (Figs. 2A, B). This IL-20-mediated phosphorylation of eNOS could be significantly reduced by wortmannin (Fig. 2C), revealing that the phosphorylation is primarily dependent on Akt activity. These observations prompted us to investigate NO synthesis in real-time in single DAF-2 loaded cells taking advantage of DAF-2 being converted to a highly fluorescent derivative appearing as bright spots in the cytoplasm when reacting with NO. We found NO production in most of the cells stimulated with IL-20 and the NO production occurred primarily in the perinuclear area (Fig. 2D). When IL-20 stimulation was performed on cells in the presence of wortmannin (100 nM) the NO
Fig. 3. (A–C) hTERT-HDLECs were stimulated with IL-20 (10 ng/ml) at time zero. Western blot analysis was performed using an antibody specific for mTOR phosphorylated at Ser2448 (P-mTOR) (A), p70S6K phosphorylated at Thr389 (P-p70S6K) (B) and Erk1/2 phosphorylated at Tyr204 (P-Erk1/2) (C). (B) P-p70S6K levels at 15 min after IL-20 stimulation with or without 10 min pre-incubation with rapamycin (100 nM); n = 3 in one experiment, ⁎⁎, P b 0.01. Phosphorylation levels are shown in arbitrary units (AU) with control samples normalized to 100. Membranes were reprobed with an anti-GAPDH antibody. (D) Statistical significant phosphorylation of Erk2 at 30 min after IL-20 stimulation; n = 5, ⁎, P b 0.05. (E) P-Erk2 levels at 30 min after IL-20 stimulation with or without 20 min pre-incubation with PD98059 (10 μM); n = 3 in one experiment, ⁎⁎, P b 0.01. (F) Proliferation assay. hTERTHDLECs were grown in 5% FCS in reduced media and stimulated with IL-20 (50 ng/ml or 100 ng/ml) for 4 days (n = 16). (G) Proliferation of unstimulated or IL-20 stimulated (50 ng/ ml) hTERT-HDLECs, pre-incubated with or without rapamycin (100 nM), wortmannin (100 nM) or PD98059 (10 μM) were measured after 5 days of incubation. Proliferation measurements are shown in arbitrary units (AU), n = 16 in one experiment, ⁎⁎, P b 0.01.
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synthesis was abolished (Figs. 2D, E). There was no detectable NO production in non-stimulated control cells (Fig. S2 supplemental data). The time pattern of the IL-20-induced NO production was similar to that observed with VEGF-A (Fig. 2E). Proliferation of hTERT-HDLEC is Akt, Erk and mTOR dependent In hTERT-HDLEC IL-20 caused a rapid dephosphorylation and subsequently an increased phosphorylation of mTOR (Ser2448) that reached a maximum after 15 min and gradually declined towards 60 min (Fig. 3A). This IL-20-induced mTOR phosphorylation occurred simultaneously with the observed activation of Akt (Figs. 1B, C). Pre-
incubation with the selective inhibitor of mTOR rapamycin significantly reduced the IL-20-induced phosphorylation level of the mTORdownstream protein p70S6K (Fig. 3B). Another signalling pathway known to be essential for angiogenic proliferation is the Mitogen-Activated Protein Kinase (MAPK) signalling cascade in which the Extracellular signal-Regulated Kinases (ERKs) play a major role (Huang et al., 2008; Johnson and Lapadat, 2002). After 1 min of stimulation with IL-20, levels of phosphorylated Erk1/2 kinases were significantly higher than those of non-treated cells reaching maximum phosphorylation after 20 min and maintaining elevated levels of phosphorylation until 30 min (Figs. 3C, D). Preincubation with the specific MEK inhibitor, PD98059, significantly
Fig. 4. (A, B) Stimulation of hTERT-HDLECs with IL-20 (10 ng/ml) for 5 h. (A) Cells were visualized by light microscopy before and after stimulation with IL-20. Arrows point to elongated cells after IL-20 stimulation. (B) IL-20-induced stimulation of actin polymerization was visualized by staining hTERT-HDLECs with phalloidin–rhodamin; n = 3. (C, D) Inhibition of IL-20-induced tube formation by wortmannin 100 nM (W) or rapamycin 100 nM (R). hTERT-HDLECs (30,000 cells/chamber) were seeded on matrigel, incubated with IL-20 (10 ng/ml) and inhibitors as indicated. Matrigels were visualized 8 h after incubation. n = 3, ⁎⁎, P b 0.01. (E) Spheroid migration assay. Stimulation of spheroids with IL20 (20 ng/ml) or VEGF-C (100 ng/ml). Cell migration was imaged 24 h after incubation and spheroid area, shown in arbitrary units (AU), was quantified. Control spheroids (n = 5), IL-20 stimulated spheroids (n = 7) or VEGF-C stimulated spheroids (n = 7) ⁎, P b 0.05. (F) Inhibition of IL-20-induced cell migration by wortmannin 100 nM (W) or rapamycin 100 nM (R); n = 3, ⁎⁎, P b 0.01.
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reduced the phosphorylation level of Erk1/2 in IL-20 stimulated cells (Fig. 3E). In accordance with this activation of two central proliferative pathways in hTERT-HDLEC, we observed a significant proliferative effect of IL-20 (50 ng/ml and 100 ng/ml) on the lymphatic endothelial cells (Fig. 3F). We show that pre-incubation of hTERT-HDLEC with rapamycin (100 nM) significantly inhibited proliferation of control cells and the IL-20 treated cells (Fig. 3G) verifying the importance of mTOR activity for lymphatic endothelial cell proliferation. Also, wortmannin (100 nM) and PD98059 (10 μM) significantly inhibited the IL-20-induced proliferation of hTERT-HDLEC (Fig. 3G). Rapamycin and wortmannin inhibit IL-20-induced tube formation and cell migration Regulated changes in morphology are essential for initial lymphangiogenic processes such as migration, cellular sprouting and formation of tubes (Folkman, 2003). We investigated the effect of IL20 on hTERT-HDLEC morphology and show that IL-20 stimulation induced cell shape changes resulting in elongated cell structures and actin polymerization (Figs. 4A, B). In line with these morphology changes we demonstrate that IL-20 (10 ng/ml) promoted tube formation of hTERT-HDLECs in a matrigel assay with VEGF-C (100 ng/ml) as a positive control (Fig. 4D and Fig. S1 supplemental data). This effect of IL-20 on tube formation in matrigel could be inhibited by wortmannin (100 nM) and rapamycin (100 nM) (Figs. 4C, D). To further test the lymphangiogenic potential of IL-20 we investigated the effect of the cytokine on hTERT-HDLECs in a spheroid sprouting assay. We show that IL-20 (20 ng/ml) is able to induce significant cellular migration in a magnitude comparable to VEGF-C (100 ng/ml) (Fig. 4E). In accordance with the rapamycin-, and wortmannin-induced inhibition of tube formation in the matrigel assay, we also observed an inhibitory effect of these compounds on the IL-20-induced cell migration in the spheroid assay (Fig. 4F). Whereas wortmannin only inhibited the IL-20-induced migration, rapamycin inhibited both the IL-20-induced migration and the migration of the control cells (Fig. 4F). Discussion In this study we show that the inflammatory cytokine IL-20 initiates signalling processes and causes morphological changes which are part of the lymphangiogenic process. hTERT-HDLECs express mRNA for the IL-20 receptor subunits IL-20Rα, IL-20Rβ and IL-22Rα and we accordingly found that IL-20 induces tube formation of hTERT-HDLECs in matrigel at a magnitude similar to that of VEGFC. Furthermore, in the spheroid sprouting assay we observed an IL20-induced migration of the cells compared to control cells, however, the lymphatic endothelial cells did not form complete chord-like structures as seen with blood vessel endothelial cells in response to IL-20 (Tritsaris et al., 2007). Both IL-20-induced cell migration and tube formation were dependent on mTOR activity, consistent with IL20 inducing mTOR phosphorylation as well as phosphorylation of the mTOR target p70S6K. Furthermore, inhibition of IL-20-induced proliferation by rapamycin demonstrates the pivotal role of the mTOR–Akt signalling axis in lymphatic endothelial cells, which is in line with other experimental evidence for an anti-lymphangiogenic activity of rapamycin (Blei, 2007; Huber et al., 2007; Kobayashi et al., 2007). Furthermore, we observe a MEK-dependent transient increase of P-Erk1/2 after IL-20 stimulation, and using a specific inhibitor we demonstrate that inhibition of this signalling pathway inhibits IL-20induced proliferation. These observations are in accordance with MAPKs as important components in LEC proliferation (Jila et al., 2007). We demonstrate that IL-20 stimulates phosphorylation of Akt at Ser473 in a PI3K-dependent manner, and that inhibition of PI3K/Akt reduced IL-20-induced cell migration, proliferation and tube forma-
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tion. The observed initial IL-20-induced dephosphorylation process of Akt (Figs. 1B, C) appears to be due to a strong phosphatase activity throughout the cytoplasm following stimulation. When this activity is inhibited by the phosphatase inhibitor calyculin, the concomitant strong kinase activity is revealed as shown by the BKAR reporting (Fig. 1E). Thus, there is a continuous activity of both kinases and phosphatases, and the ratio of those activities can be regulated by stimulation. From BKAR reporting we demonstrate that IL-20 induces Akt activity at a perinuclear area within the initial 10 min following stimulation. eNOS has been shown to be associated with Golgistructures that are positioned at a perinuclear location (Fulton et al., 2004; Sanchez et al., 2006), and our observations are in accordance with the observed IL-20-induced NO formation in this area. These findings are also consistent with previous findings in pig aorta endothelial cells where IL-20-induced NO formation was observed predominantly in the perinuclear area (Tritsaris et al., 2007). The PI3K inhibitor wortmannin strongly reduced the phosphorylation level of eNOS at Ser1179 (Fig. 2C) as well as the IL-20-induced NO synthesis as measured by fluorescence emission from DAF-2. Thus, although a small contribution from Ca2+/calmodulin signalling might participate in IL-20-induced NO synthesis, our data reveal that the observed IL-20-induced NO synthesis occur primarily in the perinuclear area and is dependent on the PI3K/Akt signalling pathway. Accordingly, we demonstrate that IL-20 induces a PI3Kdependent eNOS-activation and subsequent NO production in hTERTHDLEC. Previous findings have shown that the eNOS/NO signalling pathway is involved in a range of lymphangiogenic processes such as proliferation, migration and regulation of lymphatic permeability (Hagendoorn et al., 2005; Kajiya et al., 2008; Ohhashi et al., 2005). In addition, NO signalling has been linked to carcinogenesis during chronic inflammation, and NO synthase is implicated in lymphatic vascular neoformation in melanoma (Jaiswal et al., 2001; Massi et al., 2008). Dual inhibition of the PI3K/mTOR signalling pathways has recently been shown to inhibit VEGF-induced angiogenesis and NO synthesis (Schnell et al., 2008) and our results are consistent with such an approach being effective in reducing inflammatory lymphangiogenic processes. Our findings, along with other results that link inflammatory cytokines to lymphangiogenesis, reveal that interleukins play an important role in regulating vessel growth and in maintaining homeostasis of the lymphatic vasculature (Al-Rawi et al., 2005b; Al-Rawi et al., 2005a; Groger et al., 2004). Functional improvement of the lymphatic vessels could greatly help patients suffering from lymphoedema, and inhibiting lymphangiogenesis is proving to be an important step in hindering metastasis in cancer (Blei, 2007; Kobayashi et al., 2007). Furthermore, lymphangiogenesis and the cytokine environment are emerging as key issues in carcinogenesis and in the immunopathogenesis of several inflammatory diseases such as psoriasis and inflammatory bowel disease (Angelo and Kurzrock, 2007; Cliff et al., 1999; Nickoloff, 2007). Our present study identifies the pro-inflammatory cytokine IL-20 as a lymphangiogenic modulator, but further research is needed to elucidate the complex network of growth factors and cytokines that govern the lymphangiogenic processes in vivo. Acknowledgments We thank Lene Grønne Pedersen for technical assistance. This study was supported by grants from Købmand M. Kristian Kjær og hustru Margrethe Kjær, født la Cour-Holmens Fond, Martha Margrethe og Christian Hermansens legat, Fonden til Lægevidenskabens Fremme (A.P.-Møller), Vera og Carl Johan Michaelsens legat, Novo Nordisk Fonden and Center for Healthy Aging Fonden (Nordea Fonden). BKAR plasmid was a generous gift from Prof. Alexandra Newton, University of California, San Diego, USA.
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Microvascular Research 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 / y m v r e
Regular Article
IL-20 activates human lymphatic endothelial cells causing cell signalling and tube formation Troels Hammer a, Katerina Tritsaris a, Martin V. Hübschmann a, Josefine Gibson a, Riccardo E. Nisato b, Michael S. Pepper b, Steen Dissing a,⁎ a b
Department of Cellular and Molecular Medicine, Faculty of Health Sciences, Center for healthy Ageing, University of Copenhagen, Denmark Department of Cell Physiology and Metabolism, University hospitals of Geneva, Switzerland
a r t i c l e
i n f o
Article history: Received 22 October 2008 Revised 16 February 2009 Accepted 17 February 2009 Available online 9 March 2009 Keywords: IL-20 Lymphangiogenesis Interleukins Endothelium
a b s t r a c t IL-20 is an arteriogenic cytokine that remodels collateral networks in vivo, and plays a role in cellular organization. Here, we investigate its role in lymphangiogenesis using a lymphatic endothelial cell line, hTERT-HDLEC, which expresses the lymphatic markers LYVE-1 and podoplanin. Upon stimulation of hTERT-HDLEC with IL-20, we found an increase in the intracellular free calcium concentration, in Akt and eNOS phosphorylations as well as in perinuclear NO production. We found that eNOS phosphorylation and NO synthesis are highly dependent on the PI3K/Akt signalling pathway. We also found an IL-20 induced phosphorylation of Erk1/2 and mTOR, and using the MEK inhibitor PD98059 and mTOR complex inhibitor rapamycin we demonstrated the importance of these signalling pathways in IL-20-mediated proliferation. IL-20 triggered actin polymerization and morphological changes resulting in elongated cell structures, and in matrigels, IL-20 caused tube formations of hTERT-HDLEC in a PI3K- and mTOR dependent way. In a sprouting assay we found that IL-20 caused cell migration within 24 h at a rate comparable to VEGF-C, and this migration could be inhibited by wortmannin and rapamycin. These data show that IL-20 activates cell signalling resulting in lymphangiogenic processes including migration, proliferation and tube formation. Thus, IL20 is a cytokine that has the potential of activating or modulating the formation of lymphatic vessels. © 2009 Elsevier Inc. All rights reserved.
Introduction Lymphangiogenesis – the growth of new lymphatic vessels – is implicated in both normal developing tissues and in a number of pathological processes such as lymphoedema, inflammation, cancer lymphatic metastasis, psoriasis and diabetic wound healing (Cao, 2005; Kunstfeld et al., 2004; Maruyama et al., 2007). The identification of specific lymphatic markers including LYVE-1 and podoplanin (Breiteneder-Geleff et al., 1999; Fournier et al., 1999; Nisato et al., 2004; Podgrabinska et al., 2002) has set in motion the research in lymphangiogenesis, and clinical implications are quickly emerging (Karkkainen et al., 2004; Stacker et al., 2007). The embryonic lymphatic differentiation is relatively well described (Schacht et al., 2003; Wigle et al., 2002; Wilting et al., 2002) with focus on vascular endothelial growth factor C (VEGF-C) and its primary receptor VEGF receptor 3 (VEGFR-3) (Shibuya and Claesson-Welsh, 2006). However, in the adult organism, lymphangiogenesis – as in angiogenesis – is an intricate process with multiple components. One group of factors that regulate lymphangiogenesis is the inflammatory cytokines. The pro-inflammatory cytokine interleukin 20 (IL-20) belongs to the IL-10 cytokine ⁎ Corresponding author. Panum Institute, Building 12.6, Blegdamsvej 3B, 2200 Copenhagen N, Denmark. Fax: +45 35 32 75 26. 0026-2862/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2009.02.007
family that also includes IL-10, 19, 22, 24, 26, 28, and 29 (Sabat et al., 2007). IL-20 is expressed in monocytes, epithelial, endothelial and maturing dendritic cells (Hsing et al., 2006; Wolk et al., 2008) and exerts its biological functions through activation of the receptor complexes IL-20Rα/IL-20Rβ or IL-22Rα/IL-20Rβ, which consist of transmembrane glycoproteins belonging to the family of class II cytokine receptors (Hsieh et al., 2006; Sabat et al., 2007). IL-20 is involved in the pathogenesis of several angiogenesisdependent inflammatory diseases including psoriasis and rheumatoid arthritis (Blumberg et al., 2001; Sabat et al., 2007; Kragstrup et al., 2008), and recently the cytokine has been shown to induce nitric oxide (NO) synthesis in blood vessel endothelial cells (BVECs) and to promote angiogenesis (Hsieh et al., 2006; Tritsaris et al., 2007). NO is known as a key regulator of several processes in lymphangiogenesis and mediates lymphatic vessel activation (Kajiya et al., 2008). The endothelial NO production is dependent on Akt-activation, which is also a central mediator of endothelial cell survival, migration and proliferation (Dormond et al., 2007). The mammalian target of rapamycin (mTOR)–rictor complex plays a pivotal role in the activation of Akt, by phosphorylating Ser473, while PDK1 phosphorylates Thr308 in the activation loop. Downstream of Akt, mTOR exists in another protein complex together with raptor (Sarbassov et al., 2005). Recent studies using the selective inhibitor of mTOR, rapamycin, has highlighted
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the importance of mTOR for numerous processes essential for lymphangiogenesis, including cell growth, proliferation, motility, and survival, in part by enhancing the production of VEGF-A and VEGF-C (Blei, 2007). NO is synthesized by endothelial nitric oxide synthase (eNOS) through phosphorylation of eNOS by several Ser/Thr kinases such as Akt, as well as by interactions with the Ca2+/calmodulin complex (Dudzinski and Michel, 2007). Signal transduction coupled to the IL-20 receptor in lymphatic endothelial cells has not previously been characterized, and we used the newly developed cell line hTERT-HDLEC (human Telomerase Reverse Transcriptase Human Dermal Lymphatic Endothelial Cells) that expresses the recognized lymphatic markers Prox1, LYVE-1 and podoplanin (Nisato et al., 2004) to investigate the IL-20-induced signalling in lymphatic endothelial cells. We show that this inflammatory cytokine is a modulator of lymphangiogenic processes. Materials and methods Reagents Recombinant human (rh)IL-20 was obtained from Biosource (Belgium) and rhVEGF-C was obtained from R&D systems (Germany). Rapamycin, wortmannin and PD98059 were purchased from SigmaAldrich (USA). Lipofectamin was obtained from Invitrogen (USA) and calyculin from Calbiochem (USA). BKAR (B kinase activity reporter) plasmid was kindly provided by Prof. Alexandra Newton (UCSD, USA) and was amplified as described in Kunkel et al., 2005. Cell lines and cell culture hTERT-HDLEC is a human lymphatic endothelial cell line displaying longer life-span (Nisato et al., 2004). Cells were cultured in gelatine-coated tissue culture flasks in EGM MV2 (Promocell, Germany) supplemented with penicillin (200 U/ml)–streptomycin (50 μg/ml) and L-Glutamine (0.29 mg/ml). Signal transduction assays hTERT-HDLECs were grown to 90% confluency in gelatine-coated 60-mm dishes, washed with PBS and incubated for 60 min in serumfree medium (RPMI medium 1640) before stimulation with growth factor or interleukin. Cellular activity was stopped by adding 100 μl 1% Igepal lysis buffer (Sigma-Aldrich, USA) containing 1.2 μg/ml aprotinin, pepstatin, and leupeptin and 1.25 mmol/L NaF, PMSF, and sodium orthovanadate. Samples were mixed for 1 min and centrifuged for 10 min at 11,000 ×g. Pellets were discarded and equal amounts of protein samples were separated by SDS-PAGE (10% BIS–Tris gel). Proteins were transferred to nitrocellulose membranes, and membranes were blocked with 5% bovine serum albumin (BSA) in PBS with 0.1% Tween. Membranes were probed overnight at 4 °C with antibodies diluted in PBS with 5% BSA and 0.1% Tween for detecting P-Akt (Ser473), P-mTOR (Ser2448), P-p70S6K (Thr389), P-eNOS (Ser1177) (Cell Signalling Technology, USA), P-Erk 1/2 (Tyr204) (Santa Cruz Biotechnology, USA) or GAPDH (Millipore, USA) as recommended by the manufacturer. This was followed by incubation for 1 h in PBS with 1% BSA and 0.1% Tween with peroxidase-conjugated goat anti-rabbit immunoglobulin antibody diluted 1:1000 for P-Akt, 1:500 for P-mTOR, and 1:1000 for P-eNOS and with peroxidase-conjugated goat antimouse immunoglobulin antibody diluted 1:5000 for P-Erk1/2, 1:1000 for P-p70S6K and 1:10000 for GAPDH. Protein bands were visualized by enhanced chemiluminiscence using a LAS3000 Lumi-Imager. Phalloidin staining hTERT-HDLECs were grown on chambered gelatine-coated coverglasses (Lab-Tek; Nalge Nunc International, USA) to 90% confluency.
Cells were serum-starved for 1 h in RPMI 1640 medium before stimulation. Activation was stopped by removal of the media and cells were fixed, permeabilized, and stained using F-actin visualization Biochem Kit according to the instructions provided by the supplier (Cytoskeleton, USA). Measurements of [Ca2+]i, nitric oxide and BKAR emission The intracellular free calcium concentration ([Ca2+]i) was measured in hTERT-HDLECs grown to 90% confluency in chambered gelatine-coated cover-glasses (Lab-Tek; Nalge Nunc International, USA). Cells were loaded with 3 μM fura-2/AM (Invitrogen, USA) in RPMI medium 1640 for 30 min and washed with Krebs–Ringer buffer. Images were acquired by means of a Zeiss Axiovert 135 microscope equipped with a Zeiss Achrostigmat 40 × 1.3 NA objective. Excitation was obtained by a Polychrome V illuminator from Till Photonics (Germany) and images were acquired using a Cool Snap CCD camera (Photometrics, USA) from Robert Scientific (Malaysia). For measurements of [Ca2+]i, the excitation wavelengths were 338 and 380 nm, measuring emission above 510 nm using a cut-off filter. Calculations of [Ca2+]i were done by using MetaFluor software from Molecular Devices and using a Kd of 160 nM (Dissing et al., 1990). Measurements of Akt activity using BKAR was performed by measuring CFP and YFP emissions using a Dual View from Optical Insights (USA). After background subtraction, ratio images were formed using MetaFluor software. For measurements of NO production, hTERT-HDLECs were grown to 90% confluency in chambered gelatine-coated cover-glasses and pre-loaded with 5 μM DAF-2 DA (Merck, Germany) in RPMI 1640 medium for 30 min. Cells were washed in Krebs–Ringer buffer, and images were acquired by exciting at 440 nm and measuring emission above 510 nm using MetaFluor. Cells were seeded in chambered gelatine-coated cover-glasses (Lab-Tek; Nalge Nunc International, USA) and transfected with BKAR plasmid for 24 h using lipofectamin (100 μl of 5% lipofectamin) with 1 μg/ml of BKAR DNA added to 1 ml of 2% EGM MV2 in which FCS was reduced to 2%. Prior to measurements cells were incubated for 4 h in a medium containing 2% EGM MV2. RT-PCR Reverse transcription was performed by using total RNA from hTERT-HDLEC and Omniscript Reverse Transcriptase kit (Qiagen, DK). Complementary cDNAs were amplified by PCR using the following primer pairs: (I) a 20-mer 5′-GGGCTTCCTCGTCGCAGTAC-3′ (forward) oligo and (II) a 22-mer GGGCGTAGAATGGGAATTGAGC-3′ (reverse) oligo to generate an IL-22Rα fragment of 419 bp; (III) a 24-mer 5′-CCGAACACTCTTTACTGCGTACAC-3′ (forward) oligo and (IV) a 22-mer 5′-GCTGCCTGCGACTCCAATAATG-3′ (reverse) oligo to generate an IL-20Rα fragment of 683 bp; (V) a 21-mer 5′-CAGACCTCAGCCTGGAGCATC-3′ (forward) oligo and (VI) a 24-mer 5′-AGGATCAGCATGAAGCCAACAAAG-3′ (reverse) oligo to generate an IL-20Rβ fragment of 383 bp;(VII) a 22-mer 5′-TGGATGGAGACACACAGACAAC3′ (forward) oligo and (VIII) a 19-mer 5′-TCAGGGACAGGGCACAGAG-3′ (reverse) oligo to generate a Podoplanin fragment of 226 bp;(IX) a 22mer 5′-GGCTCTGCTAGTGCTTGCTCTC-3′ (forward) oligo and (X) a 24mer 5′-GCTTGGACTCTTGGACTCTTCTGG-3′ (reverse) oligo to generate a LYVE-1 fragment of 220 bp. Spheroid in vitro lymphangiogenesis assay An in vitro lymphangiogenesis assay using hTERT-HDLEC spheroids was performed principally as described in Korff and Augustin (1999). Briefly, spheroids were generated by seeding 750 hTERT-HDLEC cells in each well of a 96-well nonadherent round-bottomed plate, in a
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reduced MV2-medium (containing no growth factors, 1% FCS and pen./strep.) with 0.24% high viscosity methyl cellulose (SigmaAldrich, USA), incubated at 37 °C and 5% CO2. Under these conditions, all cells in each well formed a single spheroid, and after 1 day the spheroids were collected and embedded into collagen gels. The collagen stock solution was prepared by mixing rat tail collagen I (3.13 mg/ml) (BD Biosciences, DK) with 10% (vol.) 10× Earle's balanced salt solution (Sigma-Aldrich, USA) and 1 M NaOH to adjust pH to 7.4. The collagen stock solution was mixed 1:1 with 1.2% methyl cellulose in reduced medium and IL-20 or VEGF-C as indicated, and added to the collected spheroids. Spheroids in collagen were plated in a 24 well plate and incubated at 37 °C in 5% CO2 for 24 h, and spheroid sprouting/migration was visualized by using a Leitz Labovert phasecontrast microscope (Leica Microsystems, Germany) and a digital camera (CoolPix 990; Nikon, Japan). Quantification was done by measuring the total area covered by each spheroid, using the ImageJ software. (http://rsb.info.nih.gov/ij/index.html). Matrigel tube formation assay Growth factor-reduced matrigel matrix (BD Biosciences, Denmark) was added to cover-glasses (Lab-Tek; Nalge Nunc International, USA)
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(250 μl to each chamber) and the matrigel was allowed to polymerize for 30 min at 37 °C. hTERT-HDLECs (50,000 cells) in reduced MV2medium (no growth factors, 0.5% FCS, pen./strep.) were plated on the matrigel and stimulated. At appropriate time intervals tube formation was observed with a Leitz Labovert phase-contrast microscope × 150 magnification (Leica Microsystems, Denmark), and images were acquired by using a digital camera (CoolPix 990; Nikon, Japan). Tube length was quantified using the ImageJ software. Proliferation assay hTERT-HDLECs were seeded into 96-well plates at 1000 cells per well in a total volume of 200 μl and grown in FGF- and VEGF-A depleted EGM-MV2 medium (reduced medium) containing 5% FCS and pen./strep. for 4 or 5 days; the cells were left untreated or were treated with IL-20 (50 ng/ml or 100 ng/ml) at day 1. Inhibition of cell proliferation was conducted with rapamycin (100 nM), wortmannin (100 nM) or PD98059 (10 μM). Reduced medium with cytokines/ inhibitors was renewed at day 3. After 4 or 5 days, proliferation was measured using the EZ4U-proliferation Kit (Biomedica Gruppe, Austria) based on the method of reduction of tetrazolium salt to coloured formazan. Samples were incubated 3 h with substrate before
Fig. 1. (A) RT-PCR was performed and the amplified fragments of IL-20Rβ, IL-20Rα, IL-22Rα, podoplanin and LYVE-1 cDNA were visualized on an agarose gel. NTC is non template control. (B) hTERT-HDLECs were stimulated with IL-20 (10 ng/ml) at time zero. Western blot analysis was performed using an antibody specific for phosphorylation of Akt at Ser473 (P-Akt). Phosphorylation levels are shown in arbitrary units (AU) with control samples normalized to 100. Membranes were reprobed with an anti-GAPDH antibody. (C) Statistical significant dephosphorylation of Akt at 1 and 5 min, and phosphorylation of Akt at 15 min after IL-20 stimulation; n = 6, ⁎⁎, P b 0.01. (D) P-Akt levels at 15 min after IL-20 stimulation with or without 30 min pre-incubation with wortmannin (100 nM); n = 3 in one experiment, ⁎⁎, P b 0.01; ⁎, P b 0.05. (E) Measurement of Akt activity by use of BKAR. IL-20 stimulation (20 ng/ml) was followed by addition of calyculin (100 nM). The trace represents ratio values of CFP/YFP (±SEM) obtained from a perinuclear area as indicated in (F); n = 3. (F) CFP emission image. Arrow points to the area from which data in E was measured; n = 3. (G, H) Measurement of intracellular calcium after IL-20 (10 ng/ml) stimulation of hTERT-HDLECs. Cells were stimulated with IL-20 at the indicated time points in Krebs–Ringer; n = 4 (G) or in calcium-free medium; n = 3 (H). Each trace represents the recorded calcium level in a single cell.
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measurement of absorbance at 450 nm with 630 nm as background. Results represent the mean of 16 replications. Statistical evaluations Data are presented as means ± SEM. Statistical evaluation of the results was made by two-tailed Student's t test, using Microsoft (Redmond, USA) Excel and Origin® 6.1 (Northampton, USA) software. Results IL-20 receptor expression and activation of intracellular signalling leading to NO synthesis in hTERT-HDLECs We found transcripts of all three IL-20 receptor subunits, IL-20Rα, IL-20Rβ and IL-22Rα, as well as the LEC markers LYVE-1 and podoplanin in hTERT-HDLECs by RT-PCR (Fig. 1A), which led us to investigate cellular signalling. One of the mediators of lymphatic vessel activation is NO (Kajiya et al., 2008) and we studied whether IL-20 stimulation of the lymphatic endothelial cells was coupled to NO synthesis. This was done by
studying the phosphatidylinositol 3-kinase (PI3K)/Akt and calcium signalling pathways, which lead to activation of eNOS (Dudzinski and Michel, 2007). To investigate a direct effect of IL-20 on Akt activity we performed a time-course experiment measuring phosphorylation of Akt at Ser473. As shown in Figs. 1B, C incubation of hTERT-HDLECs with IL-20 (10 ng/ml) resulted in an initial dephosphorylation of Akt within the first 0–5 min after stimulation followed by a phosphorylation process causing elevated levels of phosphorylated Akt reaching a maximum after 15 min and gradually declining there after. Wortmannin (100 nM), a PI3K inhibitor, significantly inhibited the IL-20-induced phosphorylation of Akt (Fig. 1D). To further clarify the spatio-temporal dynamics of Akt phosphorylation we transfected hTERT-HDLEC with BKAR plasmid where the transcript contains two fluorescent moieties, CFP and YFP, in close proximity (Kunkel et al., 2005). Translated BKAR contains a consensus phosphorylation site for Akt and upon phosphorylation the BKAR protein changes its conformation. That leads to a reduced energy transfer from CFP to YFP that can be detected as a change in CFP/YFP ratio. Fig. 1E shows that upon IL-20 stimulation there was a rapid, localized phosphorylation of BKAR revealing localized Akt activity which was specific for a perinuclear area (Fig. 1F). At other locations far from the nucleus we
Fig. 2. (A) hTERT-HDLECs were stimulated with IL-20 (10 ng/ml) at time zero. Western blot analysis was performed using an antibody specific for phosphorylation of eNOS at Ser1177 (P-eNOS). Phosphorylation levels are shown in arbitrary units (AU) with control samples normalized to 100. Membranes were reprobed with an anti-GAPDH antibody. (B) Statistical significant phosphorylation of eNOS at 5 and 10 min after IL-20 stimulation, and 5 min after stimulation with VEGF-A (20 ng/ml); n = 4, ⁎⁎, P b 0.01; ⁎, P b 0.05. (C) P-eNOS levels at 5 min after IL-20 stimulation with or without 30 min pre-incubation with wortmannin (100 nM); n = 3 in one experiment, ⁎⁎, P b 0.01. (D, E) Measurements of NO production before and after stimulation with either IL-20 (50 ng/ml) (left), VEGF-A (100 ng/ml) (middle) or IL-20 + wortmannin (100 nM) (right); n = 3. Images show the localized NO synthesis in the cytoplasm. Measurements of NO production in the presence of wortmannin confirmed the absence of NO synthesis when cells were not stimulated. (E) Traces were continuously recorded and represent NO production in single cells.
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did not observe a significant increase in Akt activity within this time frame. Following addition of the serine/threonine phosphatase inhibitor calyculin, we observed a strong increase in BKAR phosphorylation revealing the rapid kinetics of phosphorylation and dephosphorylation processes in the cytoplasm. Hence, despite an apparent initial dephosphorylation of Akt seen in western blots (Figs. 1B, C), there is in fact a localized increase in Akt activity within 5 min upon IL20 stimulation at a perinuclear area. Treatment of the lymphatic endothelial cells with IL-20 (10 ng/ml) also rapidly increased the intracellular free calcium concentration ([Ca2+]i). This increase in [Ca2+]i was not dependent on the presence of calcium in the extracellular medium revealing that IL-20 activates calcium release from intracellular stores (Figs. 1G, H). The elevated levels of [Ca2+]i induced by IL-20 occurred immediately after addition of IL-20 in most cells, whereas a subset of cells displayed a delayed response. VEGF-A (50 ng/ml) was used as a positive control and as expected induced a rapid calcium release, whereas VEGF-C (50 ng/ml)
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did not increase the intracellular free calcium concentration (data not shown). These data show that IL-20 stimulates both signalling pathways that are capable of activating eNOS, that is a rise in [Ca2+]i and activation of Akt. We therefore tested to which extent IL-20 could induce phosphorylation of eNOS at the activation site Ser1177, and show that IL-20 mediates phosphorylation of eNOS after 5 min (Figs. 2A, B). This IL-20-mediated phosphorylation of eNOS could be significantly reduced by wortmannin (Fig. 2C), revealing that the phosphorylation is primarily dependent on Akt activity. These observations prompted us to investigate NO synthesis in real-time in single DAF-2 loaded cells taking advantage of DAF-2 being converted to a highly fluorescent derivative appearing as bright spots in the cytoplasm when reacting with NO. We found NO production in most of the cells stimulated with IL-20 and the NO production occurred primarily in the perinuclear area (Fig. 2D). When IL-20 stimulation was performed on cells in the presence of wortmannin (100 nM) the NO
Fig. 3. (A–C) hTERT-HDLECs were stimulated with IL-20 (10 ng/ml) at time zero. Western blot analysis was performed using an antibody specific for mTOR phosphorylated at Ser2448 (P-mTOR) (A), p70S6K phosphorylated at Thr389 (P-p70S6K) (B) and Erk1/2 phosphorylated at Tyr204 (P-Erk1/2) (C). (B) P-p70S6K levels at 15 min after IL-20 stimulation with or without 10 min pre-incubation with rapamycin (100 nM); n = 3 in one experiment, ⁎⁎, P b 0.01. Phosphorylation levels are shown in arbitrary units (AU) with control samples normalized to 100. Membranes were reprobed with an anti-GAPDH antibody. (D) Statistical significant phosphorylation of Erk2 at 30 min after IL-20 stimulation; n = 5, ⁎, P b 0.05. (E) P-Erk2 levels at 30 min after IL-20 stimulation with or without 20 min pre-incubation with PD98059 (10 μM); n = 3 in one experiment, ⁎⁎, P b 0.01. (F) Proliferation assay. hTERTHDLECs were grown in 5% FCS in reduced media and stimulated with IL-20 (50 ng/ml or 100 ng/ml) for 4 days (n = 16). (G) Proliferation of unstimulated or IL-20 stimulated (50 ng/ ml) hTERT-HDLECs, pre-incubated with or without rapamycin (100 nM), wortmannin (100 nM) or PD98059 (10 μM) were measured after 5 days of incubation. Proliferation measurements are shown in arbitrary units (AU), n = 16 in one experiment, ⁎⁎, P b 0.01.
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synthesis was abolished (Figs. 2D, E). There was no detectable NO production in non-stimulated control cells (Fig. S2 supplemental data). The time pattern of the IL-20-induced NO production was similar to that observed with VEGF-A (Fig. 2E). Proliferation of hTERT-HDLEC is Akt, Erk and mTOR dependent In hTERT-HDLEC IL-20 caused a rapid dephosphorylation and subsequently an increased phosphorylation of mTOR (Ser2448) that reached a maximum after 15 min and gradually declined towards 60 min (Fig. 3A). This IL-20-induced mTOR phosphorylation occurred simultaneously with the observed activation of Akt (Figs. 1B, C). Pre-
incubation with the selective inhibitor of mTOR rapamycin significantly reduced the IL-20-induced phosphorylation level of the mTORdownstream protein p70S6K (Fig. 3B). Another signalling pathway known to be essential for angiogenic proliferation is the Mitogen-Activated Protein Kinase (MAPK) signalling cascade in which the Extracellular signal-Regulated Kinases (ERKs) play a major role (Huang et al., 2008; Johnson and Lapadat, 2002). After 1 min of stimulation with IL-20, levels of phosphorylated Erk1/2 kinases were significantly higher than those of non-treated cells reaching maximum phosphorylation after 20 min and maintaining elevated levels of phosphorylation until 30 min (Figs. 3C, D). Preincubation with the specific MEK inhibitor, PD98059, significantly
Fig. 4. (A, B) Stimulation of hTERT-HDLECs with IL-20 (10 ng/ml) for 5 h. (A) Cells were visualized by light microscopy before and after stimulation with IL-20. Arrows point to elongated cells after IL-20 stimulation. (B) IL-20-induced stimulation of actin polymerization was visualized by staining hTERT-HDLECs with phalloidin–rhodamin; n = 3. (C, D) Inhibition of IL-20-induced tube formation by wortmannin 100 nM (W) or rapamycin 100 nM (R). hTERT-HDLECs (30,000 cells/chamber) were seeded on matrigel, incubated with IL-20 (10 ng/ml) and inhibitors as indicated. Matrigels were visualized 8 h after incubation. n = 3, ⁎⁎, P b 0.01. (E) Spheroid migration assay. Stimulation of spheroids with IL20 (20 ng/ml) or VEGF-C (100 ng/ml). Cell migration was imaged 24 h after incubation and spheroid area, shown in arbitrary units (AU), was quantified. Control spheroids (n = 5), IL-20 stimulated spheroids (n = 7) or VEGF-C stimulated spheroids (n = 7) ⁎, P b 0.05. (F) Inhibition of IL-20-induced cell migration by wortmannin 100 nM (W) or rapamycin 100 nM (R); n = 3, ⁎⁎, P b 0.01.
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reduced the phosphorylation level of Erk1/2 in IL-20 stimulated cells (Fig. 3E). In accordance with this activation of two central proliferative pathways in hTERT-HDLEC, we observed a significant proliferative effect of IL-20 (50 ng/ml and 100 ng/ml) on the lymphatic endothelial cells (Fig. 3F). We show that pre-incubation of hTERT-HDLEC with rapamycin (100 nM) significantly inhibited proliferation of control cells and the IL-20 treated cells (Fig. 3G) verifying the importance of mTOR activity for lymphatic endothelial cell proliferation. Also, wortmannin (100 nM) and PD98059 (10 μM) significantly inhibited the IL-20-induced proliferation of hTERT-HDLEC (Fig. 3G). Rapamycin and wortmannin inhibit IL-20-induced tube formation and cell migration Regulated changes in morphology are essential for initial lymphangiogenic processes such as migration, cellular sprouting and formation of tubes (Folkman, 2003). We investigated the effect of IL20 on hTERT-HDLEC morphology and show that IL-20 stimulation induced cell shape changes resulting in elongated cell structures and actin polymerization (Figs. 4A, B). In line with these morphology changes we demonstrate that IL-20 (10 ng/ml) promoted tube formation of hTERT-HDLECs in a matrigel assay with VEGF-C (100 ng/ml) as a positive control (Fig. 4D and Fig. S1 supplemental data). This effect of IL-20 on tube formation in matrigel could be inhibited by wortmannin (100 nM) and rapamycin (100 nM) (Figs. 4C, D). To further test the lymphangiogenic potential of IL-20 we investigated the effect of the cytokine on hTERT-HDLECs in a spheroid sprouting assay. We show that IL-20 (20 ng/ml) is able to induce significant cellular migration in a magnitude comparable to VEGF-C (100 ng/ml) (Fig. 4E). In accordance with the rapamycin-, and wortmannin-induced inhibition of tube formation in the matrigel assay, we also observed an inhibitory effect of these compounds on the IL-20-induced cell migration in the spheroid assay (Fig. 4F). Whereas wortmannin only inhibited the IL-20-induced migration, rapamycin inhibited both the IL-20-induced migration and the migration of the control cells (Fig. 4F). Discussion In this study we show that the inflammatory cytokine IL-20 initiates signalling processes and causes morphological changes which are part of the lymphangiogenic process. hTERT-HDLECs express mRNA for the IL-20 receptor subunits IL-20Rα, IL-20Rβ and IL-22Rα and we accordingly found that IL-20 induces tube formation of hTERT-HDLECs in matrigel at a magnitude similar to that of VEGFC. Furthermore, in the spheroid sprouting assay we observed an IL20-induced migration of the cells compared to control cells, however, the lymphatic endothelial cells did not form complete chord-like structures as seen with blood vessel endothelial cells in response to IL-20 (Tritsaris et al., 2007). Both IL-20-induced cell migration and tube formation were dependent on mTOR activity, consistent with IL20 inducing mTOR phosphorylation as well as phosphorylation of the mTOR target p70S6K. Furthermore, inhibition of IL-20-induced proliferation by rapamycin demonstrates the pivotal role of the mTOR–Akt signalling axis in lymphatic endothelial cells, which is in line with other experimental evidence for an anti-lymphangiogenic activity of rapamycin (Blei, 2007; Huber et al., 2007; Kobayashi et al., 2007). Furthermore, we observe a MEK-dependent transient increase of P-Erk1/2 after IL-20 stimulation, and using a specific inhibitor we demonstrate that inhibition of this signalling pathway inhibits IL-20induced proliferation. These observations are in accordance with MAPKs as important components in LEC proliferation (Jila et al., 2007). We demonstrate that IL-20 stimulates phosphorylation of Akt at Ser473 in a PI3K-dependent manner, and that inhibition of PI3K/Akt reduced IL-20-induced cell migration, proliferation and tube forma-
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tion. The observed initial IL-20-induced dephosphorylation process of Akt (Figs. 1B, C) appears to be due to a strong phosphatase activity throughout the cytoplasm following stimulation. When this activity is inhibited by the phosphatase inhibitor calyculin, the concomitant strong kinase activity is revealed as shown by the BKAR reporting (Fig. 1E). Thus, there is a continuous activity of both kinases and phosphatases, and the ratio of those activities can be regulated by stimulation. From BKAR reporting we demonstrate that IL-20 induces Akt activity at a perinuclear area within the initial 10 min following stimulation. eNOS has been shown to be associated with Golgistructures that are positioned at a perinuclear location (Fulton et al., 2004; Sanchez et al., 2006), and our observations are in accordance with the observed IL-20-induced NO formation in this area. These findings are also consistent with previous findings in pig aorta endothelial cells where IL-20-induced NO formation was observed predominantly in the perinuclear area (Tritsaris et al., 2007). The PI3K inhibitor wortmannin strongly reduced the phosphorylation level of eNOS at Ser1179 (Fig. 2C) as well as the IL-20-induced NO synthesis as measured by fluorescence emission from DAF-2. Thus, although a small contribution from Ca2+/calmodulin signalling might participate in IL-20-induced NO synthesis, our data reveal that the observed IL-20-induced NO synthesis occur primarily in the perinuclear area and is dependent on the PI3K/Akt signalling pathway. Accordingly, we demonstrate that IL-20 induces a PI3Kdependent eNOS-activation and subsequent NO production in hTERTHDLEC. Previous findings have shown that the eNOS/NO signalling pathway is involved in a range of lymphangiogenic processes such as proliferation, migration and regulation of lymphatic permeability (Hagendoorn et al., 2005; Kajiya et al., 2008; Ohhashi et al., 2005). In addition, NO signalling has been linked to carcinogenesis during chronic inflammation, and NO synthase is implicated in lymphatic vascular neoformation in melanoma (Jaiswal et al., 2001; Massi et al., 2008). Dual inhibition of the PI3K/mTOR signalling pathways has recently been shown to inhibit VEGF-induced angiogenesis and NO synthesis (Schnell et al., 2008) and our results are consistent with such an approach being effective in reducing inflammatory lymphangiogenic processes. Our findings, along with other results that link inflammatory cytokines to lymphangiogenesis, reveal that interleukins play an important role in regulating vessel growth and in maintaining homeostasis of the lymphatic vasculature (Al-Rawi et al., 2005b; Al-Rawi et al., 2005a; Groger et al., 2004). Functional improvement of the lymphatic vessels could greatly help patients suffering from lymphoedema, and inhibiting lymphangiogenesis is proving to be an important step in hindering metastasis in cancer (Blei, 2007; Kobayashi et al., 2007). Furthermore, lymphangiogenesis and the cytokine environment are emerging as key issues in carcinogenesis and in the immunopathogenesis of several inflammatory diseases such as psoriasis and inflammatory bowel disease (Angelo and Kurzrock, 2007; Cliff et al., 1999; Nickoloff, 2007). Our present study identifies the pro-inflammatory cytokine IL-20 as a lymphangiogenic modulator, but further research is needed to elucidate the complex network of growth factors and cytokines that govern the lymphangiogenic processes in vivo. Acknowledgments We thank Lene Grønne Pedersen for technical assistance. This study was supported by grants from Købmand M. Kristian Kjær og hustru Margrethe Kjær, født la Cour-Holmens Fond, Martha Margrethe og Christian Hermansens legat, Fonden til Lægevidenskabens Fremme (A.P.-Møller), Vera og Carl Johan Michaelsens legat, Novo Nordisk Fonden and Center for Healthy Aging Fonden (Nordea Fonden). BKAR plasmid was a generous gift from Prof. Alexandra Newton, University of California, San Diego, USA.
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