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Epithelial calcium-sensing receptor activation by eosinophil granule protein analog stimulates collagen matrix contraction.

Ngo PD, MacLeod RJ, Mukkada V, Turki R, Furuta GT - Pediatr. Res. (2012)

Bottom Line: HEK-CaSR stimulation with PA or Ca(2+) resulted in greater pERK activation than that of stimulated HEK-WT cells.Exposure of intestinal epithelia to the EDGP analog PA stimulates CaSR-dependent ERK phosphorylation and epithelial-mediated collagen lattice contraction.We speculate that EDGP release within the epithelial layers activates the CaSR receptor, leading to matrix contraction and tissue fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Pediatric Gastroenterology, Floating Hospital for Children at Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT

Background: Eosinophils reside in normal gastrointestinal tracts and increase during disease states. Receptors for eosinophil-derived granule proteins (EDGPs) have not been identified, but highly cationic molecules, similar to eosinophil proteins, bind extracellular calcium-sensing receptors (CaSRs). We hypothesized that stimulation of CaSRs by eosinophil proteins activates epithelial cells.

Methods: Caco2 intestinal epithelial cells, AML14.3D10 eosinophils, wild-type (WT) human embryonic kidney 293 (HEK293) cells not expressing CaSRs (HEK-WT), and CaSR-transfected HEK293 cells (HEK-CaSR) were stimulated with an eosinophil protein analog poly-L-arginine (PA) and phosphorylated extracellular signal-regulated kinase (pERK)1 and pERK2 were measured. Functional activation was measured with collagen lattice contraction assays.

Results: Coculture of Caco2 cells with AML14.3D10 eosinophils augmented lattice contraction as compared with lattices containing Caco2 cells alone. PA stimulation of Caco2 lattices augmented contraction. HEK-CaSR stimulation with PA or Ca(2+) resulted in greater pERK activation than that of stimulated HEK-WT cells. PA stimulated greater HEK-CaSR lattice contraction than unstimulated lattices. Contraction of PA-stimulated and PA-unstimulated HEK-WT lattices did not differ.

Conclusion: Exposure of intestinal epithelia to the EDGP analog PA stimulates CaSR-dependent ERK phosphorylation and epithelial-mediated collagen lattice contraction. We speculate that EDGP release within the epithelial layers activates the CaSR receptor, leading to matrix contraction and tissue fibrosis.

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CaSR contributes to collagen lattice contraction. Contraction of HEK-WT (white bar) and HEK-CaSR (black bar) populated collagen lattices stimulated with PA and media control. HEK-WT and HEK-CaSR cells were suspended in collagen lattices at 5×105 cells/ml and exposed to 1µM PA or not. Percent initial lattice surface area at 24 hours is reported. Values represent means ±SD of five lattices (*p<0.001).
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Figure 6: CaSR contributes to collagen lattice contraction. Contraction of HEK-WT (white bar) and HEK-CaSR (black bar) populated collagen lattices stimulated with PA and media control. HEK-WT and HEK-CaSR cells were suspended in collagen lattices at 5×105 cells/ml and exposed to 1µM PA or not. Percent initial lattice surface area at 24 hours is reported. Values represent means ±SD of five lattices (*p<0.001).

Mentions: We next determined if gain of CaSR ligation resulted in a functional impact. PA stimulation (1 µM) significantly increased contraction of collagen lattices populated with HEK-CaSR cells compared to both unstimulated HEK-CaSR lattices and PA stimulated HEK-WT lattices at 24 hours (percent initial surface area; 58.8%±2.9 vs. 71.0%±3.9, PA stimulated HEK-CaSR vs. unstimulated HEK-CaSR lattices respectively, p<0.01, and 58.8%±2.9 vs. 71.8%±2.0, PA stimulated HEK-CaSR vs. PA stimulated HEK-WT lattices respectively, p<0.01) (Figure 6). PA stimulation did not significantly affect collagen lattice contraction in HEK-WT populated collagen lattices compared to HEK-WT unstimulated control (CTL) lattices (71.8%±2.0 vs. 69.6%±1.0, PA vs. CTL respectively, NS).


Epithelial calcium-sensing receptor activation by eosinophil granule protein analog stimulates collagen matrix contraction.

Ngo PD, MacLeod RJ, Mukkada V, Turki R, Furuta GT - Pediatr. Res. (2012)

CaSR contributes to collagen lattice contraction. Contraction of HEK-WT (white bar) and HEK-CaSR (black bar) populated collagen lattices stimulated with PA and media control. HEK-WT and HEK-CaSR cells were suspended in collagen lattices at 5×105 cells/ml and exposed to 1µM PA or not. Percent initial lattice surface area at 24 hours is reported. Values represent means ±SD of five lattices (*p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4321999&req=5

Figure 6: CaSR contributes to collagen lattice contraction. Contraction of HEK-WT (white bar) and HEK-CaSR (black bar) populated collagen lattices stimulated with PA and media control. HEK-WT and HEK-CaSR cells were suspended in collagen lattices at 5×105 cells/ml and exposed to 1µM PA or not. Percent initial lattice surface area at 24 hours is reported. Values represent means ±SD of five lattices (*p<0.001).
Mentions: We next determined if gain of CaSR ligation resulted in a functional impact. PA stimulation (1 µM) significantly increased contraction of collagen lattices populated with HEK-CaSR cells compared to both unstimulated HEK-CaSR lattices and PA stimulated HEK-WT lattices at 24 hours (percent initial surface area; 58.8%±2.9 vs. 71.0%±3.9, PA stimulated HEK-CaSR vs. unstimulated HEK-CaSR lattices respectively, p<0.01, and 58.8%±2.9 vs. 71.8%±2.0, PA stimulated HEK-CaSR vs. PA stimulated HEK-WT lattices respectively, p<0.01) (Figure 6). PA stimulation did not significantly affect collagen lattice contraction in HEK-WT populated collagen lattices compared to HEK-WT unstimulated control (CTL) lattices (71.8%±2.0 vs. 69.6%±1.0, PA vs. CTL respectively, NS).

Bottom Line: HEK-CaSR stimulation with PA or Ca(2+) resulted in greater pERK activation than that of stimulated HEK-WT cells.Exposure of intestinal epithelia to the EDGP analog PA stimulates CaSR-dependent ERK phosphorylation and epithelial-mediated collagen lattice contraction.We speculate that EDGP release within the epithelial layers activates the CaSR receptor, leading to matrix contraction and tissue fibrosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Pediatric Gastroenterology, Floating Hospital for Children at Tufts Medical Center, Tufts University School of Medicine, Boston, Massachusetts, USA.

ABSTRACT

Background: Eosinophils reside in normal gastrointestinal tracts and increase during disease states. Receptors for eosinophil-derived granule proteins (EDGPs) have not been identified, but highly cationic molecules, similar to eosinophil proteins, bind extracellular calcium-sensing receptors (CaSRs). We hypothesized that stimulation of CaSRs by eosinophil proteins activates epithelial cells.

Methods: Caco2 intestinal epithelial cells, AML14.3D10 eosinophils, wild-type (WT) human embryonic kidney 293 (HEK293) cells not expressing CaSRs (HEK-WT), and CaSR-transfected HEK293 cells (HEK-CaSR) were stimulated with an eosinophil protein analog poly-L-arginine (PA) and phosphorylated extracellular signal-regulated kinase (pERK)1 and pERK2 were measured. Functional activation was measured with collagen lattice contraction assays.

Results: Coculture of Caco2 cells with AML14.3D10 eosinophils augmented lattice contraction as compared with lattices containing Caco2 cells alone. PA stimulation of Caco2 lattices augmented contraction. HEK-CaSR stimulation with PA or Ca(2+) resulted in greater pERK activation than that of stimulated HEK-WT cells. PA stimulated greater HEK-CaSR lattice contraction than unstimulated lattices. Contraction of PA-stimulated and PA-unstimulated HEK-WT lattices did not differ.

Conclusion: Exposure of intestinal epithelia to the EDGP analog PA stimulates CaSR-dependent ERK phosphorylation and epithelial-mediated collagen lattice contraction. We speculate that EDGP release within the epithelial layers activates the CaSR receptor, leading to matrix contraction and tissue fibrosis.

Show MeSH
Related in: MedlinePlus