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Hyperplasia of interstitial cells of cajal in sprouty homolog 4 deficient mice.

Thys A, Vandenberghe P, Hague P, Klein OD, Erneux C, Vanderwinden JM - PLoS ONE (2015)

Bottom Line: Sprouty homolog 4 was upregulated both at the mRNA and protein level in these cells, suggesting that Sprouty homolog 4 is downstream of oncogenic KIT activation and potentially engaged in the negative feedback loop of ERK activation in this model.Here, we used KitK641E heterozygous and Sprouty homolog 4 knock out animals to quantify interstitial cells of Cajal in situ, using quantitative immunofluorescence for the receptor tyrosine kinase Kit and for phosphodiesterase 3a (PDE3A).We concluded that the lack of Sprouty homolog 4 expression leads to hyperplasia of the interstitial cells of Cajal and is functionally associated with a delayed transit time.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurophysiology, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium.

ABSTRACT
Gastrointestinal stromal tumors, which are thought to derive from interstitial cells of Cajal or their precursors, often harbor an oncogenic mutation of the KIT receptor tyrosine kinase. Sprouty homolog 4, a known negative regulator of ERK pathway, has been identified in the interstitial cells of Cajal in the KitK641E murine model of gastrointestinal stromal tumors. Sprouty homolog 4 was upregulated both at the mRNA and protein level in these cells, suggesting that Sprouty homolog 4 is downstream of oncogenic KIT activation and potentially engaged in the negative feedback loop of ERK activation in this model. Here, we used KitK641E heterozygous and Sprouty homolog 4 knock out animals to quantify interstitial cells of Cajal in situ, using quantitative immunofluorescence for the receptor tyrosine kinase Kit and for phosphodiesterase 3a (PDE3A). In the antrum of Sprouty homolog 4 knock out mice, hyperplasia of interstitial cells of Cajal was reminiscent of the KitK641E heterozygous mice antrum. Additionally, the density of interstitial cells of Cajal was higher in the colon of adult Sprouty homolog 4 knock out mice than in WT littermates, although hyperplasia seemed more severe in KitK641E heterozygous mice. Functional transit studies also show similarities between Sprouty homolog 4 knock out and KitK641E heterozygous mice, as the total transit time in 9 month old animals was significantly increased in both genotypes compared to WT littermates. We concluded that the lack of Sprouty homolog 4 expression leads to hyperplasia of the interstitial cells of Cajal and is functionally associated with a delayed transit time.

No MeSH data available.


Related in: MedlinePlus

ICC hyperplasia in colon of 3-month-old Spry4 KO and KitWT/K641E.A) Widefield microscopy acquisitions. PDE3A immunoreactivity (-ir) and KIT-ir (rabbit) highlight ICC in the colon of 3-month-old WT, Spry4 KO and KitWT/K641E mice. B) Quantification of PDE3A-ir, KIT-ir (rabbit) and KIT-ir (goat) ICC area in the muscularis propria of 3-month-old WT, Spry4 KO and KitWT/K641E colon (n = 5–7 animals per group). Single IF staining performed on adjacent sections. The 3 antibodies identified similarly a significant increase in ICC area in Spry4 KO (p value < 0.05 for each staining) and in KitWT/K641E colon (PDE3A a KIT goat p < 0.01, KIT rabbit p value < 0.001) compared to WT colon. No significant difference was observed between Spry4 KO and KitWT/K641E (p value PDE3A = 0.2509, p value KIT rabbit = 0.2668, p value KIT goat = 0.3572). Within each genotype, differences between the 3 antibodies were not significant. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 50μm. P-values (Kruskal-Wallis with Dunn’s post hoc) *: p<0.05, **: p<0.01
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pone.0124861.g010: ICC hyperplasia in colon of 3-month-old Spry4 KO and KitWT/K641E.A) Widefield microscopy acquisitions. PDE3A immunoreactivity (-ir) and KIT-ir (rabbit) highlight ICC in the colon of 3-month-old WT, Spry4 KO and KitWT/K641E mice. B) Quantification of PDE3A-ir, KIT-ir (rabbit) and KIT-ir (goat) ICC area in the muscularis propria of 3-month-old WT, Spry4 KO and KitWT/K641E colon (n = 5–7 animals per group). Single IF staining performed on adjacent sections. The 3 antibodies identified similarly a significant increase in ICC area in Spry4 KO (p value < 0.05 for each staining) and in KitWT/K641E colon (PDE3A a KIT goat p < 0.01, KIT rabbit p value < 0.001) compared to WT colon. No significant difference was observed between Spry4 KO and KitWT/K641E (p value PDE3A = 0.2509, p value KIT rabbit = 0.2668, p value KIT goat = 0.3572). Within each genotype, differences between the 3 antibodies were not significant. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 50μm. P-values (Kruskal-Wallis with Dunn’s post hoc) *: p<0.05, **: p<0.01

Mentions: Double immunofluorescence staining using KIT (rabbit) and PDE3A (sheep) antibodies (Table 3) indicated an increased ICC area in both Spry4 KO and KitWT/K641E colon (Fig 10A). Quantitative assessment of ICC area was performed by single IF staining using KIT-ir (two different, rabbit and goat, KIT antibodies—Table 3) and PDE3A-ir on adjacent sections of colon for WT, Spry4 KO and KitWT/K641E genotypes (n = 5–7 animals per group). Within each genotype, the 3 antibodies gave concordant results, with non-significant differences between antibodies. The 3 antibodies identified similarly a significant increase in ICC area in Spry4 KO colon (p value <0.05 for each antibody) and in KitWT/K641E colon (KIT rabbit p value <0.001, KIT goat and PDE3A p value < 0.01) compared to WT. Although ICC hyperplasia appeared more pronounced in KitWT/K641E than in Spry4 KO, differences between KitWT/K641E and Spry4 KO were not significant (p value PDE3A = 0.2509, p value KIT rabbit = 0.2668, p value KIT goat = 0.3572).


Hyperplasia of interstitial cells of cajal in sprouty homolog 4 deficient mice.

Thys A, Vandenberghe P, Hague P, Klein OD, Erneux C, Vanderwinden JM - PLoS ONE (2015)

ICC hyperplasia in colon of 3-month-old Spry4 KO and KitWT/K641E.A) Widefield microscopy acquisitions. PDE3A immunoreactivity (-ir) and KIT-ir (rabbit) highlight ICC in the colon of 3-month-old WT, Spry4 KO and KitWT/K641E mice. B) Quantification of PDE3A-ir, KIT-ir (rabbit) and KIT-ir (goat) ICC area in the muscularis propria of 3-month-old WT, Spry4 KO and KitWT/K641E colon (n = 5–7 animals per group). Single IF staining performed on adjacent sections. The 3 antibodies identified similarly a significant increase in ICC area in Spry4 KO (p value < 0.05 for each staining) and in KitWT/K641E colon (PDE3A a KIT goat p < 0.01, KIT rabbit p value < 0.001) compared to WT colon. No significant difference was observed between Spry4 KO and KitWT/K641E (p value PDE3A = 0.2509, p value KIT rabbit = 0.2668, p value KIT goat = 0.3572). Within each genotype, differences between the 3 antibodies were not significant. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 50μm. P-values (Kruskal-Wallis with Dunn’s post hoc) *: p<0.05, **: p<0.01
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Related In: Results  -  Collection

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pone.0124861.g010: ICC hyperplasia in colon of 3-month-old Spry4 KO and KitWT/K641E.A) Widefield microscopy acquisitions. PDE3A immunoreactivity (-ir) and KIT-ir (rabbit) highlight ICC in the colon of 3-month-old WT, Spry4 KO and KitWT/K641E mice. B) Quantification of PDE3A-ir, KIT-ir (rabbit) and KIT-ir (goat) ICC area in the muscularis propria of 3-month-old WT, Spry4 KO and KitWT/K641E colon (n = 5–7 animals per group). Single IF staining performed on adjacent sections. The 3 antibodies identified similarly a significant increase in ICC area in Spry4 KO (p value < 0.05 for each staining) and in KitWT/K641E colon (PDE3A a KIT goat p < 0.01, KIT rabbit p value < 0.001) compared to WT colon. No significant difference was observed between Spry4 KO and KitWT/K641E (p value PDE3A = 0.2509, p value KIT rabbit = 0.2668, p value KIT goat = 0.3572). Within each genotype, differences between the 3 antibodies were not significant. Abbreviations: LM: longitudinal muscle layer, CM: circular muscle layer, *: location of myenteric plexus, scale bar: 50μm. P-values (Kruskal-Wallis with Dunn’s post hoc) *: p<0.05, **: p<0.01
Mentions: Double immunofluorescence staining using KIT (rabbit) and PDE3A (sheep) antibodies (Table 3) indicated an increased ICC area in both Spry4 KO and KitWT/K641E colon (Fig 10A). Quantitative assessment of ICC area was performed by single IF staining using KIT-ir (two different, rabbit and goat, KIT antibodies—Table 3) and PDE3A-ir on adjacent sections of colon for WT, Spry4 KO and KitWT/K641E genotypes (n = 5–7 animals per group). Within each genotype, the 3 antibodies gave concordant results, with non-significant differences between antibodies. The 3 antibodies identified similarly a significant increase in ICC area in Spry4 KO colon (p value <0.05 for each antibody) and in KitWT/K641E colon (KIT rabbit p value <0.001, KIT goat and PDE3A p value < 0.01) compared to WT. Although ICC hyperplasia appeared more pronounced in KitWT/K641E than in Spry4 KO, differences between KitWT/K641E and Spry4 KO were not significant (p value PDE3A = 0.2509, p value KIT rabbit = 0.2668, p value KIT goat = 0.3572).

Bottom Line: Sprouty homolog 4 was upregulated both at the mRNA and protein level in these cells, suggesting that Sprouty homolog 4 is downstream of oncogenic KIT activation and potentially engaged in the negative feedback loop of ERK activation in this model.Here, we used KitK641E heterozygous and Sprouty homolog 4 knock out animals to quantify interstitial cells of Cajal in situ, using quantitative immunofluorescence for the receptor tyrosine kinase Kit and for phosphodiesterase 3a (PDE3A).We concluded that the lack of Sprouty homolog 4 expression leads to hyperplasia of the interstitial cells of Cajal and is functionally associated with a delayed transit time.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Neurophysiology, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium.

ABSTRACT
Gastrointestinal stromal tumors, which are thought to derive from interstitial cells of Cajal or their precursors, often harbor an oncogenic mutation of the KIT receptor tyrosine kinase. Sprouty homolog 4, a known negative regulator of ERK pathway, has been identified in the interstitial cells of Cajal in the KitK641E murine model of gastrointestinal stromal tumors. Sprouty homolog 4 was upregulated both at the mRNA and protein level in these cells, suggesting that Sprouty homolog 4 is downstream of oncogenic KIT activation and potentially engaged in the negative feedback loop of ERK activation in this model. Here, we used KitK641E heterozygous and Sprouty homolog 4 knock out animals to quantify interstitial cells of Cajal in situ, using quantitative immunofluorescence for the receptor tyrosine kinase Kit and for phosphodiesterase 3a (PDE3A). In the antrum of Sprouty homolog 4 knock out mice, hyperplasia of interstitial cells of Cajal was reminiscent of the KitK641E heterozygous mice antrum. Additionally, the density of interstitial cells of Cajal was higher in the colon of adult Sprouty homolog 4 knock out mice than in WT littermates, although hyperplasia seemed more severe in KitK641E heterozygous mice. Functional transit studies also show similarities between Sprouty homolog 4 knock out and KitK641E heterozygous mice, as the total transit time in 9 month old animals was significantly increased in both genotypes compared to WT littermates. We concluded that the lack of Sprouty homolog 4 expression leads to hyperplasia of the interstitial cells of Cajal and is functionally associated with a delayed transit time.

No MeSH data available.


Related in: MedlinePlus