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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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Eosinophil-epithelial co-culture results in increased collagen lattice contractionContraction of collagen lattices populated with 3D10 eosinophils and Caco2 cells over time. 3D10 eosinophils (5×105 cells/ml) and Caco2 cells (5×105 cells/ml) were suspended in collagen lattices either alone or in co-culture. Lattice surface area was measured at indicated time points. Contraction is reported as mean percent initial surface area ±SD. Lattice contraction was augmented when Caco2 cells were suspended with 3D10 eosinophils (triangle) compared to Caco2 cells alone (square). Lattices with 3D10 eosinophils alone (circle) and acellular collagen lattices (not shown) did not contract (p<0.02 Caco2 with eosinophils vs. Caco2).
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Figure 1: Eosinophil-epithelial co-culture results in increased collagen lattice contractionContraction of collagen lattices populated with 3D10 eosinophils and Caco2 cells over time. 3D10 eosinophils (5×105 cells/ml) and Caco2 cells (5×105 cells/ml) were suspended in collagen lattices either alone or in co-culture. Lattice surface area was measured at indicated time points. Contraction is reported as mean percent initial surface area ±SD. Lattice contraction was augmented when Caco2 cells were suspended with 3D10 eosinophils (triangle) compared to Caco2 cells alone (square). Lattices with 3D10 eosinophils alone (circle) and acellular collagen lattices (not shown) did not contract (p<0.02 Caco2 with eosinophils vs. Caco2).

Mentions: Although eosinophils are known to reside adjacent to the gastrointestinal epithelium, their potential role in remodeling of the extracellular matrix via epithelial cells has not been investigated. To address this, we hypothesized that EDGPs stimulate gastrointestinal tissue contraction and thus measured the impact of eosinophils on epithelial populated collagen lattice contraction. Co-culture of Caco2 cells with AML14.3D10 eosinophils (3D10 eosinophils) led to significant contraction compared to Caco2 cells or 3D10 eosinophils alone as shown in Figure 1 (43.0±1.0% vs. 34.3 ±2.2%, p<0.02; Caco2 with eosinophils vs. Caco2 alone). We next used the highly charged MBP analog, PA, in the collagen lattice contraction assay. PA (0.5 µM and 1µM) stimulated a concentration and time dependent contraction of Caco2 populated lattices that was significantly augmented compared to unstimulated controls (Figure 2A and 2B) (46.4±1.6 vs. 36.7±1.3, p<0.001; PA vs. media alone). Cell viability of Caco2 and AML14.3D10 cells was assured by trypan blue staining (96%+/− 2% viability in co-cultured, PA stimulated and unstimulated cells).


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)

Eosinophil-epithelial co-culture results in increased collagen lattice contractionContraction of collagen lattices populated with 3D10 eosinophils and Caco2 cells over time. 3D10 eosinophils (5×105 cells/ml) and Caco2 cells (5×105 cells/ml) were suspended in collagen lattices either alone or in co-culture. Lattice surface area was measured at indicated time points. Contraction is reported as mean percent initial surface area ±SD. Lattice contraction was augmented when Caco2 cells were suspended with 3D10 eosinophils (triangle) compared to Caco2 cells alone (square). Lattices with 3D10 eosinophils alone (circle) and acellular collagen lattices (not shown) did not contract (p<0.02 Caco2 with eosinophils vs. Caco2).
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4321999&req=5

Figure 1: Eosinophil-epithelial co-culture results in increased collagen lattice contractionContraction of collagen lattices populated with 3D10 eosinophils and Caco2 cells over time. 3D10 eosinophils (5×105 cells/ml) and Caco2 cells (5×105 cells/ml) were suspended in collagen lattices either alone or in co-culture. Lattice surface area was measured at indicated time points. Contraction is reported as mean percent initial surface area ±SD. Lattice contraction was augmented when Caco2 cells were suspended with 3D10 eosinophils (triangle) compared to Caco2 cells alone (square). Lattices with 3D10 eosinophils alone (circle) and acellular collagen lattices (not shown) did not contract (p<0.02 Caco2 with eosinophils vs. Caco2).
Mentions: Although eosinophils are known to reside adjacent to the gastrointestinal epithelium, their potential role in remodeling of the extracellular matrix via epithelial cells has not been investigated. To address this, we hypothesized that EDGPs stimulate gastrointestinal tissue contraction and thus measured the impact of eosinophils on epithelial populated collagen lattice contraction. Co-culture of Caco2 cells with AML14.3D10 eosinophils (3D10 eosinophils) led to significant contraction compared to Caco2 cells or 3D10 eosinophils alone as shown in Figure 1 (43.0±1.0% vs. 34.3 ±2.2%, p<0.02; Caco2 with eosinophils vs. Caco2 alone). We next used the highly charged MBP analog, PA, in the collagen lattice contraction assay. PA (0.5 µM and 1µM) stimulated a concentration and time dependent contraction of Caco2 populated lattices that was significantly augmented compared to unstimulated controls (Figure 2A and 2B) (46.4±1.6 vs. 36.7±1.3, p<0.001; PA vs. media alone). Cell viability of Caco2 and AML14.3D10 cells was assured by trypan blue staining (96%+/− 2% viability in co-cultured, PA stimulated and unstimulated cells).

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