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Identification of KRAP-expressing cells and the functional relevance of KRAP to the subcellular localization of IP3R in the stomach and kidney.

Fujimoto T, Shirasawa S - Int. J. Mol. Med. (2012)

Bottom Line: We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas.Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs.These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.

ABSTRACT
KRAS-induced actin-interacting protein (KRAP), originally identified as one of the deregulated genes expressed in colorectal cancer, participates under physiological conditions in the regulation of systemic energy homeostasis and of the exocrine system. We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas. However, the expression of KRAP and its precise function in other tissues remain elusive. In this study, we aimed to identify the KRAP-expressing cells in mouse stomach and kidneys and to examine the relevance of KRAP expression in the regulation of IP(3)R localization in these tissues. In the stomach, double immunohistochemical staining for KRAP and IP(3)R demonstrated that KRAP was expressed along with the apical regions in the mucous cells and the chief cells, and IP(3)R3 was dominantly co-localized with KRAP in these cells. Furthermore, IP(3)R2 was also co-localized with IP(3)R3 in the chief cells. It is of note that the proper localization of IP(3)R3 and IP(3)R2 in the chief cells and of IP(3)R3 in the mucous cells were significantly abrogated in KRAP-deficient mice. In the kidneys, KRAP was expressed in both the apical and the basal regions of the proximal tubular cells. Intriguingly, KRAP deficiency abrogated the localization of IP(3)R1 in the proximal tubular cells. Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs. These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

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KRAP expression in the mucous cells and the chief cells of the mouse stomach.(A–D) Fluorescent confocal images of stomach sections for KRAP (red),filamentous actin (F-actin) with phalloidin (green), and the merged photo. Lowmagnification images from the pit region to the base region of gastric glands fromwild-type (A) or KRAP-deficient (B) mice. Asterisk and arrows indicategastric lumen and muscularis mucosae beneath the base region, respectively. (C) Highmagnification images of the pit region of gastric glands. Asterisk indicates gastriclumen. (D) High magnification images of the base regions of gastric glands. Asterisksand arrowheads indicate the parietal cells and the apical membranes of the chief cells,respectively. (E) Fluorescent confocal images of the base regions of gastric glands forKRAP (red), ZO-1 (green), and the merged photo. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 50μm.
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f1-ijmm-30-06-1287: KRAP expression in the mucous cells and the chief cells of the mouse stomach.(A–D) Fluorescent confocal images of stomach sections for KRAP (red),filamentous actin (F-actin) with phalloidin (green), and the merged photo. Lowmagnification images from the pit region to the base region of gastric glands fromwild-type (A) or KRAP-deficient (B) mice. Asterisk and arrows indicategastric lumen and muscularis mucosae beneath the base region, respectively. (C) Highmagnification images of the pit region of gastric glands. Asterisk indicates gastriclumen. (D) High magnification images of the base regions of gastric glands. Asterisksand arrowheads indicate the parietal cells and the apical membranes of the chief cells,respectively. (E) Fluorescent confocal images of the base regions of gastric glands forKRAP (red), ZO-1 (green), and the merged photo. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 50μm.

Mentions: To examine the cellular distribution of KRAP protein in the adult mouse tissues, weperformed immunohistochemical staining by using anti-KRAP antibody. In the stomach, strongKRAP immunoreactivity was restricted to the pit regions of gastric glands (Fig. 1A), whereas significant expressionof KRAP was not detected in the muscularis mucosae beneath the gastric glands (Fig. 1A, arrows). The specificity of KRAPexpression in the stomach was confirmed by using KRAP-KO tissue as acontrol (Fig. 1B). In the pit regionof the gastric gland, where columnar surface mucous cells mainly exist (22), KRAP was localized beneath theapical membranes of the mucous cells (Fig.1C). In the base region of the gastric glands, where zymogenic chief cells mainlyexist, coronal plane of deeper gastric glands showed that KRAP was restricted to theapical regions of the chief cells (Fig. 1D,arrowheads), whereas KRAP was not detected in the parietal cells (Fig. 1D, asterisks). The distinctionbetween the chief and the parietal cells was validated by ZO-1 staining as described(23), indicating that KRAP wasexpressed in the ZO-1-positive chief cells but not in the ZO-1-negative parietal cells(Fig. 1E).


Identification of KRAP-expressing cells and the functional relevance of KRAP to the subcellular localization of IP3R in the stomach and kidney.

Fujimoto T, Shirasawa S - Int. J. Mol. Med. (2012)

KRAP expression in the mucous cells and the chief cells of the mouse stomach.(A–D) Fluorescent confocal images of stomach sections for KRAP (red),filamentous actin (F-actin) with phalloidin (green), and the merged photo. Lowmagnification images from the pit region to the base region of gastric glands fromwild-type (A) or KRAP-deficient (B) mice. Asterisk and arrows indicategastric lumen and muscularis mucosae beneath the base region, respectively. (C) Highmagnification images of the pit region of gastric glands. Asterisk indicates gastriclumen. (D) High magnification images of the base regions of gastric glands. Asterisksand arrowheads indicate the parietal cells and the apical membranes of the chief cells,respectively. (E) Fluorescent confocal images of the base regions of gastric glands forKRAP (red), ZO-1 (green), and the merged photo. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 50μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042864&req=5

f1-ijmm-30-06-1287: KRAP expression in the mucous cells and the chief cells of the mouse stomach.(A–D) Fluorescent confocal images of stomach sections for KRAP (red),filamentous actin (F-actin) with phalloidin (green), and the merged photo. Lowmagnification images from the pit region to the base region of gastric glands fromwild-type (A) or KRAP-deficient (B) mice. Asterisk and arrows indicategastric lumen and muscularis mucosae beneath the base region, respectively. (C) Highmagnification images of the pit region of gastric glands. Asterisk indicates gastriclumen. (D) High magnification images of the base regions of gastric glands. Asterisksand arrowheads indicate the parietal cells and the apical membranes of the chief cells,respectively. (E) Fluorescent confocal images of the base regions of gastric glands forKRAP (red), ZO-1 (green), and the merged photo. Blue,4′,6-diamidino-2-phenylindole (DAPI) staining; scale bar, 50μm.
Mentions: To examine the cellular distribution of KRAP protein in the adult mouse tissues, weperformed immunohistochemical staining by using anti-KRAP antibody. In the stomach, strongKRAP immunoreactivity was restricted to the pit regions of gastric glands (Fig. 1A), whereas significant expressionof KRAP was not detected in the muscularis mucosae beneath the gastric glands (Fig. 1A, arrows). The specificity of KRAPexpression in the stomach was confirmed by using KRAP-KO tissue as acontrol (Fig. 1B). In the pit regionof the gastric gland, where columnar surface mucous cells mainly exist (22), KRAP was localized beneath theapical membranes of the mucous cells (Fig.1C). In the base region of the gastric glands, where zymogenic chief cells mainlyexist, coronal plane of deeper gastric glands showed that KRAP was restricted to theapical regions of the chief cells (Fig. 1D,arrowheads), whereas KRAP was not detected in the parietal cells (Fig. 1D, asterisks). The distinctionbetween the chief and the parietal cells was validated by ZO-1 staining as described(23), indicating that KRAP wasexpressed in the ZO-1-positive chief cells but not in the ZO-1-negative parietal cells(Fig. 1E).

Bottom Line: We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas.Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs.These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Faculty of Medicine, Fukuoka University, Fukuoka 814-0180, Japan.

ABSTRACT
KRAS-induced actin-interacting protein (KRAP), originally identified as one of the deregulated genes expressed in colorectal cancer, participates under physiological conditions in the regulation of systemic energy homeostasis and of the exocrine system. We have recently found that KRAP is a molecule associated with inositol 1,4,5-trisphosphate receptor (IP(3)R) and is critical for the proper subcellular localization of IP(3)R in the liver and the pancreas. However, the expression of KRAP and its precise function in other tissues remain elusive. In this study, we aimed to identify the KRAP-expressing cells in mouse stomach and kidneys and to examine the relevance of KRAP expression in the regulation of IP(3)R localization in these tissues. In the stomach, double immunohistochemical staining for KRAP and IP(3)R demonstrated that KRAP was expressed along with the apical regions in the mucous cells and the chief cells, and IP(3)R3 was dominantly co-localized with KRAP in these cells. Furthermore, IP(3)R2 was also co-localized with IP(3)R3 in the chief cells. It is of note that the proper localization of IP(3)R3 and IP(3)R2 in the chief cells and of IP(3)R3 in the mucous cells were significantly abrogated in KRAP-deficient mice. In the kidneys, KRAP was expressed in both the apical and the basal regions of the proximal tubular cells. Intriguingly, KRAP deficiency abrogated the localization of IP(3)R1 in the proximal tubular cells. Finally, co-immunoprecipitation study in the stomachs and the kidneys validated the physical association of KRAP with IP(3)Rs. These findings demonstrate that KRAP physically associates with IP(3)Rs and regulates the proper localization of IP(3)Rs in the mucous cells and the chief cells of the stomach and in the proximal tubular cells of the kidneys.

Show MeSH
Related in: MedlinePlus