Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells - PubMed (original) (raw)

. 2006 Apr 18;103(16):6326-31.

doi: 10.1073/pnas.0601223103. Epub 2006 Apr 10.

Katharina Lötzer, Steffen Jahn, Cornelia Kramer, Markus Hildner, Ellen Bretschneider, Dörte Radke, Michael Beer, Rüdiger Vollandt, Jilly F Evans, Colin D Funk, Andreas J R Habenicht

Affiliations

Cysteinyl leukotriene 2 receptor and protease-activated receptor 1 activate strongly correlated early genes in human endothelial cells

Barbara Uzonyi et al. Proc Natl Acad Sci U S A. 2006.

Abstract

Cysteinyl leukotrienes (cysLT), i.e., LTC4, LTD4, and LTE4, are lipid mediators derived from the 5-lipoxygenase pathway, and the cysLT receptors cysLT1-R/cysLT2-R mediate inflammatory tissue reactions. Although endothelial cells (ECs) predominantly express cysLT2-Rs, their role in vascular biology remains to be fully understood. To delineate cysLT2-R actions, we stimulated human umbilical vein EC with LTD4 and determined early induced genes. We also compared LTD4 effects with those induced by thrombin that binds to protease-activated receptor (PAR)-1. Stringent filters yielded 37 cysLT2-R- and 34 PAR-1-up-regulated genes (>2.5-fold stimulation). Most LTD4-regulated genes were also induced by thrombin. Moreover, LTD4 plus thrombin augmented gene expression when compared with each agonist alone. Strongly induced genes were studied in detail: Early growth response (EGR) and nuclear receptor subfamily 4 group A transcription factors; E-selectin; CXC ligand 2; IL-8; a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif 1 (ADAMTS1); Down syndrome critical region gene 1 (DSCR1); tissue factor (TF); and cyclooxygenase 2. Transcripts peaked at approximately 60 min, were unaffected by a cysLT1-R antagonist, and were superinduced by cycloheximide. The EC phenotype was markedly altered: LTD4 induced de novo synthesis of EGR1 protein and EGR1 localized in the nucleus; LTD4 up-regulated IL-8 formation and secretion; and LTD4 raised TF protein and TF-dependent EC procoagulant activity. These data show that cysLT2-R activation results in a proinflammatory EC phenotype. Because LTD4 and thrombin are likely to be formed concomitantly in vivo, cysLT2-R and PAR-1 may cooperate to augment vascular injury.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.

Fig. 1.

Early LTD4- and thrombin-regulated genes in HUVECs. HUVECs were stimulated with 100 nM LTD4 or 10 nM thrombin. (A) Heatmap of genes up-regulated >2.5-fold by LTD4; thrombin was used for comparison. (B and C) Scatterplots of LTD4- and thrombin-stimulated cells versus control; lines depict 2.5-fold change. (D) Scatterplot of LTD4- versus thrombin-stimulated cells. (E and F) Comparison of gene expression of LTD4-plus-thrombin-treated cells with cells stimulated with LTD4 or thrombin alone. Probe sets were selected as described in Supporting Text. Line separates up- from down-regulated probe sets. Columns in A indicate umbilical cord preparations; dots in scatterplots indicate means of signal intensities of four (B–D) or three (E and F) umbilical cords.

Fig. 2.

Fig. 2.

Induction of EGR transcription factor family members by LTD4. Cells were stimulated with 100 nM LTD4, and EGR QRT-PCR analyses were performed at various time points thereafter. (A) Transcript kinetics of EGR1, -2, and -3. (B) EGR1 protein is up-regulated by LTD4 or TNFα. (C) EGR1 localizes in the nucleus at 1 h.

Fig. 3.

Fig. 3.

Induction of the NR4A transcription factor family members upon LTD4 stimulation. Cells were stimulated with 100 nM LTD4, and NR4A family member transcript kinetics were performed by QRT-PCR. (Inset) NR4A1 transcript induction by 100 nM LTD4 at 1 h in the absence and presence of 10 μg/ml cycloheximide (CHX).

Fig. 4.

Fig. 4.

LTD4 induces IL-8 expression and secretion. Cells were stimulated with 100 nM LTD4 for the indicated time points. Supernatants were collected for ELISA, and cells were harvested for QRT-PCR analysis. (Inset) IL-8 protein content of control (C) and LTD4-stimulated (L) cultures at 2 h. Columns represent means of quadruplicate dishes ± SEM (P < 0.005; Student t test).

Fig. 5.

Fig. 5.

LTD4 induces TF expression and procoagulant activity. Cells were stimulated with 100 nM LTD4 for increasing periods of time. (A) TF transcript kinetics in response to LTD4 determined by QRT-PCR. (B) TF protein expression at 6 h after addition of LTD4 or TNFα. (C) TF-dependent procoagulant activity. Cells were stimulated with LTD4 for 6 h, the culture medium was removed, and TF-dependent procoagulant activity was determined in cell lysates as described in Materials and Methods. Data represent means of eight cell lysates ± SEM derived from three umbilical cords (P < 0.01; Student t test).

Similar articles

Cited by

References

    1. Funk C. D. Science. 2001;294:1871–1875. - PubMed
    1. Samuelsson B. Science. 1983;220:568–575. - PubMed
    1. Maclouf J., Murphy R. C., Henson P. M. Blood. 1989;74:703–707. - PubMed
    1. Sala A., Testa T., Folco G. FEBS Lett. 1996;388:94–98. - PubMed
    1. Brink C., Dahlen S. E., Drazen J., Evans J. F., Hay D. W., Nicosia S., Serhan C. N., Shimizu T., Yokomizo T. Pharmacol. Rev. 2003;55:195–227. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources