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Engagement of CD44 modulates cyclooxygenase induction, VEGF generation, and proliferation in human vascular endothelial cells

Joseph F. Murphy^*,1, Frances Lennon, Christopher Steele^*, Dermot Kelleher, Desmond Fitzgerald^* and Aideen C. Long

Departments of
^* Clinical Pharmacology and
Biochemistry, Royal College of Surgeons in Ireland, Dublin, Ireland; and
Department of Clinical Medicine, Trinity College and Dublin Molecular Medicine Centre, St. James’s Hospital, Dublin, Ireland.

¹Correspondence: Department of Clinical Pharmacology, RCSI, Dublin 02, Ireland. E-mail: joseph.murphy{at}emmerex.com

SPECIFIC AIMS

We investigated the relationship between the hyaluronic receptor CD44 and the enzyme cyclooxygenase (COX). We sought to determine whether CD44 ligation could modulate COX activity in human vascular endothelial cells (EC) and whether COX was a downstream effector of endothelial cell (EC) proliferation to this interaction.

PRINCIPAL FINDINGS

1. CD44 ligation induces COX-2
Endothelial cells were incubated with monoclonal antibodies (mAbs) to CD44, native hyaluronic acid (HA), or HA fragments for varying periods, then assayed for 6-keto-PGF₁ production and COX protein expression. After 3 h incubation, COX-2 protein expression was increased, accompanied by an increase in 6-keto-PGF₁ generation. COX-1 induction was not observed. Blocking antibodies that have been shown to inhibit signaling via CD44-inhibited COX-2 induction. Smaller, HA fragments did not induce COX-2 expression in EC.

2. CD44 ligation induces VEGF generation
HUVEC were grown to 50–60% confluence in 96-well tissue culture plates, serum starved (2.5% FBS) overnight, and added with anti-CD44 mAbs or native HA for 8–10 h. Analysis of the media by EIA demonstrated an increase in VEGF generation by cells stimulated with anti-CD44 mAbs (Fig. 1 A) and cells treated with HA (Fig. 1B ). The positive control, phorbol myristate acetate PMA, also induced VEGF production. Furthermore, HA-induced VEGF generation was completely inhibited in the presence of the two blocking anti-CD44 blocking antibodies (Fig. 1B ). A neutralizing anti-VEGF antibody blocked PGI₂ formation in response to CD44 ligation with stimulating antibody or HA ligand (Fig. 2 ).

View larger version (14K): [in this window] [in a new window]   Figure 1. Effect of CD44 mAbs and HA on VEGF production: EC were plated onto 6- or 24-well plates and serum starved overnight in M199/2.5% FBS. Aspirin (200 µM) was added for 45 min, washed, then treated with mAbs, HA, HA fragments (Frags) or control agonist for 3–4 h. After incubation, supernatant samples were removed and assayed for VEGF by ELISA. A) Stimulation of VEGF production after ligation of EC with stimulating CD44 antibodies (L3D1 and D2.1). PMA was used as a positive control with the nonstimulating IgM anti-CD44, Bric 238, and an isotype Ig (anti-IE) as negative controls. B) HA induces VEGF production, which is inhibited by blocking mAbs. Blocking mAbs (IM7 or KM201) were added 30 min before addition of HA. Data represent mean ±SE from 3 independent experiments. ***P<0.005; ****P<0.001

View larger version (14K): [in this window] [in a new window]   Figure 2. A neutralizing antibody to VEGF inhibits anti-CD44 mAb and high molecular weight HA induction of COX-2: EC were plated onto 6- or 24-well plates and serum starved overnight in M199/2.5% FBS. Aspirin (200 µM) was added for 45 min, washed, then treated with CD44 mAb or HA for 3–4 h. Neutralizing anti-VEGF was added 30 min before addition of anti-CD44 antibody or HA. Arachidonic acid was used as substrate to measure enzyme activity. A) 6-keto-PGF1 generation after ligation of EC with stimulating CD44 antibody (D2.1) in the presence and absence of neutralizing anti-VEGF. B) 6-Keto-PGF1 generation after ligation of EC with HA in the presence and absence of neutralizing anti-VEGF mAb. Data represent mean ±SE from 3 independent experiments. ***P<0.005, **P <0.01, +P <0.05.

3. CD44 ligation induces COX-2-dependent EC proliferation
HA-induced COX-2 induction and VEGF generation were accompanied by an increase in EC proliferation. Proliferation was stimulated by CD44 antibodies and high molecular weight HA. HA-stimulated EC proliferation was attenuated by the COX-2 (and not COX-1) -selective inhibitor NS398. Figure 3 outlines a diagrammatic representation of our findings.

View larger version (9K): [in this window] [in a new window]   Figure 3. Schematic diagram.

CONCLUSIONS

This study has demonstrated that signaling through the adhesion molecule CD44 on endothelial cells results in the induction of COX-2 gene expression and prostacyclin production, generation of VEGF, and subsequent stimulation of EC proliferation. These biological effects are mediated via stimulating CD44 antibodies or the natural CD44 ligand HA. Though CD44 has been well characterized as a molecule that mediates cellular adhesion and motility, its role in cellular activation is not as well described. Antibodies to CD44 have been shown to be costimulatory for T cell activation and thymocyte apoptosis, whereas HA fragment-induced signaling through CD44 results in transcription of proinflammatory genes such as chemokines, cytokines, cell adhesion molecules, and inducible NO synthase.

Our previous results demonstrating COX induction by the vitronectin receptor _vß₃ show that receptor/ligand interactions mediate COX induction and subsequent downstream effects. In the present study, high molecular weight HA, the physiological ligand of the CD44 receptor, was a potent inducer of COX-2 and prostacyclin production in EC. Stimulation of cells with fragments of HA did not result in stimulation of COX-2 expression. These differences between the role of native HA and HA fragments in COX-2 activation and prostacyclin production correlate with their effect on VEGF production and induction of EC proliferation. This would suggest that the COX-2 induction, prostacyclin formation, VEGF production, and cellular proliferation induced by engagement of CD44 is dependent on a high avidity interaction that can only be achieved with high molecular weight HA and antibody cross-linking. However, fragments of HA have been shown to have a potent signaling capacity mediated through CD44 in many cellular systems, resulting in activation of NF-B and transcription of proinflammatory genes. HA degradation products or "angiogenic oligosaccharides of hyaluronan" have been shown to stimulate endothelial cell proliferation and transient up-regulation of immediate early genes including c-fos, c-jun, and jun-B. It has become clear, however, that the term HA fragment is heterogenic and covers a multitude of sizes. In a study by Deed et al. (1997), only fragments from 1.35 to 4.35 kDa were angiogenic whereas the size of our fragments were 200 kDa. HA fragments are generated at sites of inflammation and are increased with carcinogenesis, which may correlate with increased hyaluronidase activity. Although native HA has not been closely associated with induction of proinflammatory gene expression, it does have distinct signaling properties. Our results support the observations that HA and HA fragments transduce distinct signals.

COX-2 and CD44 display a parallel expression pattern in many physiological and pathological conditions. Expression of CD44 and COX-2 can be induced by cytokines, growth factors, and tumor promoters and COX-2 is a source of PG formation during inflammation, embryogenesis, and tumor growth, processes in which CD44 plays major roles. CD44 and COX-2 are overexpressed in a variety of malignancies and have been linked to matrix metalloproteinase expression/activity, cell migration, invasion, and tumor metastasis. COX-2 and CD44 (particularly variant CD44) have been implicated in the pathogenesis of colon cancer. Earlier studies investigated the impact of COX-2 expression in lung cancer invasiveness, demonstrating that non-small cell lung cancer cells transduced with COX-2 cDNA showed enhanced invasive capacity and overexpressed CD44.

In this study, ligation of CD44 by mAbs or its natural ligand HA induced VEGF secretion by EC. HA was a potent stimulus for VEGF production. This HA-driven VEGF secretion was abrogated by blocking CD44 antibodies that inhibit HA-mediated signaling. CD44-mediated COX-2 induction was significantly inhibited by a neutralizing VEGF antibody, indicating that the VEGF pathway is driving COX-2 activation. VEGF mediates its effects through two receptors, VEGFR-1 and -2. VEGFR-2 mediates much of the signaling in EC, which leads to actin reorganization, membrane ruffling, and proliferation, resulting in vascular growth and angiogenesis. EC proliferation stimulated via CD44 (via mAbs and HA) was completely inhibited by blocking CD44 mAbs that attenuate HA signaling. This HA-stimulated EC proliferation was blocked by a COX-2 (but not a COX-1) -specific inhibitor. The effect of high molecular weight HA on EC proliferation and, ultimately, vessel formation are complex and are believed to be mediated by multiple HA receptors. It has been demonstrated that CD44 is the major determinant of EC adhesion to HA and EC proliferation; another HA receptor, receptor for HA-mediated motility, regulates EC migration through the basement membrane substrate, matrigel. Our finding that HA-CD44 mediated signaling in EC stimulates proliferation is consistent with this.

Earlier studies observed individual roles for CD44, COX, and VEGF in embryogenesis, angiogenesis, cellular proliferation, and wound healing and in pathophysiological conditions such as cancer and inflammation. However, this is the first study to demonstrate a mechanistic link between engagement of CD44, induction of COX-2 gene expression, and the production of VEGF in endothelial cell signaling. This novel pathway and its products are potentially central to all of the above processes and may be a target used in the design of therapeutic strategies to treat tumor growth, diabetic retinopathy, and inflammatory conditions such as rheumatoid arthritis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1376fje;

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