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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online June 7, 2002 as doi:10.1096/fj.01-0844fje. |
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Vascular Biology Research Center, Institute of Molecular Medicine, and Division of Hematology, University of Texas-Houston Medical School, Houston, Texas, USA
2Correspondence: Vascular Biology Research Center and Division of Hematology, University of Texas-Houston Medical School, 6431 Fannin St., Houston, TX, USA. E-mail: KennethK.Wu{at}uth.tmc.edu
SPECIFIC AIMS
Cyclooxygenase-2 (COX-2) expression in human foreskin fibroblasts (HFF) induced by phorbol 12-myristate 13-acetate (PMA) is cell cycle dependent and its expression declines significantly when cells enter the S-phase of cell cycle. We hypothesized that proliferating fibroblasts produce a factor to control COX-2 expression. To test this hypothesis, we purified from the cultured medium of proliferating fibroblasts a factor that suppressed COX-2 expression induced by diverse stimuli in human and murine cells and blocked COX-2 induction by angiogenic factors as well as endothelial tube formation induced by angiogenic factors and colon cancer cell medium.
PRINCIPAL FINDINGS
1. Purification of COX-2 suppressing factor
Conditioned medium from proliferating fibroblasts inhibited PMA-induced COX-2 protein levels in quiescent fibroblasts and replacement of proliferating cell-conditioned medium with fresh medium restored the COX-2 protein in response to PMA induction. These results suggested the presence of a COX-2 suppressing factor in the conditioned medium. Filtration of the medium through a 10 kDa permeable membrane revealed positive activity in the fraction < 10 kDa. The < 10 kDa fraction was applied to a Superdex 30 column and separated in an FPLC system. Five peaks with absorption at 220 nm were detected and peak 2 (P2) completely inhibited PMA-induced COX-2 proteins. P2 was applied to a C18 column in a reverse-phase HPLC system eluted with gradient acetonitrile 10–50%. Five peaks were detected, and peak 4 (H4) completely blocked PMA-induced COX-2 proteins. H4 was further separated in the same HPLC system using 15–30% acetonitrile gradient. A single sharp peak was obtained. This final purified sample possessed full COX-2 suppressive activity. This purified sample was analyzed by a protein microsequencing technique. No peptides were detected. Amino acid analysis failed to detect any amino acid. Polyacrylamide gel electrophoresis also failed to detect an oligopeptide. This purified sample probably contains a small molecular weight bio-organic compound. We named this compound cytoguardin. The chemical nature of cytoguardin is being elucidated.
2. Cytoguardin inhibited COX-2 expression induced by diverse stimuli
COX-2 expression is agonist and cell specific. We determined whether cytoguardin was capable of blocking COX-2 induction by diverse agonists. We carried out experiments with P2 as sufficient quantities of P2 could be obtained for multiple experiments. P2 inhibited COX-2 protein levels in HFF induced by PMA, interleukin-1ß (IL-1ß), lipopolysaccharide (LPS), and tumor necrosis factor 
(TNF-) in a comparable concentration-dependent manner (Fig. 1
a). It suppressed LPS-induced COX-2 protein expression in human endothelial cells and murine RAW 264.7 cells comparable to that in HFF (Fig. 1b
). COX-2 protein induction by vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was similarly inhibited by P2 (Fig. 1c
). P2 inhibited COX-2 promoter activity induced by LPS in HFF, RAW 264.7, and human umbilical vein endothelial cells in a comparable manner (Fig. 2
). These results suggest that cytoguardin inhibits COX-2 expression induced by broad classes of agonists in diverse human and mouse cells at the transcriptional level.
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3. Cytoguardin blocked endothelial tube formation (angiogenesis) induced by angiogenic factors and colon cancer cells
As P2 inhibited COX-2 protein expression in endothelial cells induced by VEGF and bFGF, we evaluated the effect of P2 on endothelial tube formation on Matrigel. P2 blocked tube formation in a concentration-dependent manner comparable to COX-2 protein suppression. Colon cancer cells such as DLD-1 and HCT 116 were reported to promote angiogenesis via the COX-2 pathway. We found that conditioned medium from DLD-1 and HCT 116 induced tube formation that was blocked by treatment with P2.
CONCLUSIONS AND SIGNIFICANCE
Results from this study demonstrate a novel mechanism by which COX-2 expression in proliferating cells is controlled. We have isolated from the conditioned medium of proliferating fibroblasts a factor that suppressed COX-2 expression in human and murine cells induced by a wide variety of stimuli. It inhibited COX-2 expression induced by VEGF and bFGF and endothelial tube formation induced by angiogenic growth factors and colon cancer cell medium. We have purified this factor and named it cytoguardin. Preliminary characterization of cytoguardin indicates that it is not a protein or an oligopeptide. The chemical nature of this bio-organic compound is being determined with mass spectrometry, nuclear magnetic resonance spectrometry, and other pertinent analytic procedures. Cytoguardin is probably synthesized by proliferating cells and secreted into extracellular milieu, where it functions as an autacoid to control COX-2 expression in surrounding cells (Fig. 3
). It likely plays an important role in protecting proliferating cells against genomic and cellular damage caused by COX-2 overexpression.
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Persistent overexpression of COX-2 has been shown to induce drastic changes in cellular phenotypes in epithelial cells. The COX-2 overexpressed cells exhibit an increased adherence property, a high metastatic potential, and resistance to apoptosis. COX-2 overexpression stimulates angiogenesis that promotes cancer growth. COX-2 may cause these changes by damaging genomic DNA, which is exposed and more susceptible to oxidative damage in proliferating cells. Thus, cytoguardin represents an ingenious mechanism used by the proliferating cell to protect against genomic and cellular damage, thereby maintaining normal phenotype. It also plays an important role in controlling pathological angiogenesis involved in cancer growth and inflammation. COX-2 is recognized as a key mediator of inflammation. By controlling COX-2 expression, cytoguardin possesses potent anti-inflammatory properties.
Cytoguardin inhibits COX-2 expression induced by diverse agonists including PMA, IL-1ß, TNF-, LPS, VEGF, and bFGF, and inhibits COX-2 expression not only in human fibroblasts and endothelial cells but also in murine macrophages. Recent studies indicate that COX-2 promoter activation by diverse mitogenic and proinflammatory stimuli in human and murine cells requires different transcriptional activators. For example, PMA and IL-1ß induction of COX-2 transcription requires binding of CCAAT/enhancer binding protein (C/EBPß) to its cognate site on the enhancer element whereas TNF--induction of COX-2 promoter activity depends on binding of NF-
B to its cognate sites. VEGF, on the other hand, depends on binding of NF-AT to its site on COX-2 enhancer region. That cytoguardin is capable of blocking COX-2 expression by these diverse stimuli in a comparable concentration-dependent manner suggests that cytoguardin acts at a common site of transcriptional activation required by all the stimuli. This raises the possibility that cytoguardin may block interaction of transactivators with transcriptional coactivators or chromatin modifying proteins.
In summary, we have isolated a cellular factor that controls COX-2 transcription induced by multiple pathophysiologically relevant stimuli. It possessed a potent effect on blocking endothelial tube formation induced by angiogenic growth factors and colon cancer cell medium. Elucidation of the structure of cytoguardin and its synthetic pathway should provide valuable insight into the innate cellular defense mechanism and open a new avenue for developing novel therapeutic agents against important human diseases such as a broad spectrum of inflammatory disorders and cancer.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0844fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.01-0844fje 
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(400 u/ml) for 4 h. c) COX-2 protein levels in HUVEC stimulated with VEGF or bFGF for 4 h. P2 concentrations were estimated from dry weight after subtracting PBS. Each figure is representative of three separate experiments.
