Cell cycle genes down-regulated¹ through PAR2 activation by either 2f-LIGRLO-NH₂ or trypsin.

<p><i>¹Expression level expressed as fold differences of control (control  = 1.0)</i>.</p><p><i>²2f-O: 2f-LIGRLO-NH₂ 1 µM</i> .</p><p><i>³Trypsin: Human Trypsin 50 nM</i>.</p

Microarray analysis of PAR2 induced gene expression.

<p>HEK293 cells were treated with either trypsin (50 nM) or 2f-LIGRLO-NH₂ (1 µM). Total RNA was harvested at different time points after treatment (Trypsin, 6 h; Agonist, 1.5, 3, 6, 12 h). Control and treated cDNAs were hybridized to the ∼19,000 cDNA microchip and results were quantified, normalized and filtered as outlined in experimental procedures. (A) Heatmap (red, up-regulated; green, down-regulated) featuring 5 gene clusters. Three clusters show similar modulation by PAR2 peptide agonist and trypsin (1, 2, 3). Cluster 1 shows genes up-regulated by PAR2 activation, clusters 2 and 3 show genes suppressed by PAR2. Clusters 4 and 5 show opposing effects resulting from PAR2 peptide agonist versus trypsin. (B) Scatter plot of genes regulated by trypsin and PAR2 agonist peptide after 6 h treatment. Dots in right hand top quadrant are genes up-regulated by both trypsin and peptide, those in bottom left hand quadrant are genes down-regulated by both treatments. Blue lines depict regions where gene expression increased or decreased by≤3 fold.</p

Genes regulated most strongly via PAR2 activation by either 2f-LIGRLO-NH₂ (2f-O) or trypsin.

<p><i>¹Expression level expressed as fold differences of control (control  = 1.0)</i>.</p><p><i>²2f-O: 2f-LIGRLO-NH₂ 1 µM</i>.</p><p><i>³Trypsin: Human Trypsin 50 nM</i>.</p

Transcriptional regulation¹ of HDAC and sirtuin enzymes by PAR2 activation via either 2f-LIGRLO-NH₂ or Trypsin.

<p><i>¹Expression level expressed as fold differences of control (control  = 1.0)</i>.</p><p><i>²Peptide: 2f-LIGRLO-NH₂ 1 µM</i>.</p><p><i>³Trypsin: Human Trypsin 50 nM</i>.</p

Effects of PAR2 activation on complement pathways.

<p>Relationship between genes regulated by PAR2 (black arrows) and classical, alternative and lectin pathways of complement activation. Five key genes regulated by PAR2 activation were C1q, C1r, C4b, factor D and CD55 (decay accelerating factor). C1r/q subunits of C1 are in the classical pathway; C4b is essential for formation of C3 convertase in the classical and lectin pathways; Factor D is similarly important for C3 convertase in the alternative pathway; CD55 actively breaks down C3 convertase contributing to anti-complement effects of PAR2. Such regulation of all these genes will limit formation of pro-inflammatory anaphylatoxins (C3a, C5a) and membrane attack complex.</p

Genes regulated by both PAR1 and PAR2 activation.

<p><i>¹Expression level expressed as fold differences of control (control  = 1.0).</i></p><p><i>²PAR1 results extracted from McLaughlin et.al. (2005) J.Biol.Chem.280:22172-22180.</i></p><p><i>³PAR2 2f-O: 2f-LIGRLO-NH₂ 1 µM.</i></p><p><i>⁴Trypsin: Human Trypsin 50 nM.</i></p

Correlation between microarray gene expression and qRT-PCR.

<p>HEK293 cells were treated with trypsin or 2f-LIGRLO-NH₂ and total RNA was extracted 6 h later. Data represent the average of 3 independent experiments. Columns (negative control, white; 2f-LIGRLO-NH₂, grey; trypsin, black) show representative regulation of PAR2, DUSP6, WWOX, ITGB4, p73, Amphiregulin, SERPINB2, Il-8 precursor, ERK-1, COX-2, TNF-9, TNF-15, HDAC-7A, EGR-1 and EGR-2 by PAR2 activation. Results from qRT-PCR were compared to microarray data at equal times.</p

Cell cycle genes up-regulated¹ through PAR2 activation by either 2f-LIGRLO-NH₂ or Trypsin.

<p><i>¹Expression level expressed as fold differences of control (control  = 1.0)</i>.</p><p><i>²2f-O: 2f-LIGRLO-NH₂ 1 µM</i> .</p><p><i>³Trypsin: Human Trypsin 50 nM</i>.</p

PAR2 activated genes associated with inflammation induced by either 2f-LIGRLO-NH₂ or Trypsin.

<p><i>¹Expression level expressed as fold differences of control (control  = 1.0)</i>.</p><p><i>²2f-O: 2f-LIGRLO-NH₂ 1 µM</i>.</p><p><i>³Trypsin: Human Trypsin 50 nM</i>.</p

PAR2 activated genes involved in metabolism down-regulated by either 2f-LIGRLO-NH₂ or Trypsin.

<p><i>¹Expression level expressed as fold differences of control (control  = 1.0)</i>.</p><p><i>²2f-O: 2f-LIGRLO-NH₂ 1 µM</i>.</p><p><i>³Trypsin: Human Trypsin 50 nM</i>.</p