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Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage

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ABSTRACT

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt’s base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus–surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt–villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

Electronic supplementary material: The online version of this article (doi:10.1007/s00418-016-1453-4) contains supplementary material, which is available to authorized users.

No MeSH data available.


Partitioning of Guca2a and Ppib transcripts in the pyloric–duodenal region and in pancreas. a, bGuca2a expression was found in the duodenum (D), in cells at the base of the crypts of Lieberkühn, but was not detected in the gastric (G) or the duodenal glands (DG), or in the pancreas. c, d Detection of Ppib transcript in glandular epithelia in the pyloric–duodenal region and pancreas. IL islet of Langerhans
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Fig3: Partitioning of Guca2a and Ppib transcripts in the pyloric–duodenal region and in pancreas. a, bGuca2a expression was found in the duodenum (D), in cells at the base of the crypts of Lieberkühn, but was not detected in the gastric (G) or the duodenal glands (DG), or in the pancreas. c, d Detection of Ppib transcript in glandular epithelia in the pyloric–duodenal region and pancreas. IL islet of Langerhans

Mentions: We did not detect Guca2a transcript in gastric or in pancreatic epithelia (Figs. 2a, 3), nor did we detect expression of Guca2b or Gucy2c in these tissues (Fig. 2b, c). This contrasts with previous observations on guinea pig, in which GN immunoreactivity was observed in pyloric EC cells (Cetin et al. 1994), and with observations on human and rat pancreatic tissue, in which GN, UGN, and GCC transcript and protein were detected (Kulaksiz et al. 2001; Kulaksiz and Cetin 2002). RT-PCR analysis confirmed that, compared to the gut, these glandular tissues contain only very low levels of these three transcripts (not shown). Such transcripts of low abundance may not be detectable by in situ hybridization, particularly in the pancreas, which contains high levels of ribonucleases (Azevedo-Pouly et al. 2014). However, arguing against significant mRNA loss prior to tissue fixation, we could readily detect Ppib transcript, in both the stomach and the pancreas (Fig. 3c, d). Therefore, we tentatively conclude that expression of the GCC signaling axis is low in mouse, compared to human pancreas. Interestingly, this apparent difference in the pancreatic expression of the GCC signaling axis between these species mirrors a previously observed disparity in the regulation of ductal anion and fluid secretion: Whereas in the human pancreas, the GCC signaling axis is thought to control the activity of the phosphorylation-regulated CFTR anion channel, anion and fluid secretion in the murine exocrine pancreas is principally governed by Ca2+-dependent anion channels, which operate independently of the GCC pathway (Winpenny et al. 1995; Kulaksiz et al. 2001).Fig. 3


Guanylin and uroguanylin are produced by mouse intestinal epithelial cells of columnar and secretory lineage
Partitioning of Guca2a and Ppib transcripts in the pyloric–duodenal region and in pancreas. a, bGuca2a expression was found in the duodenum (D), in cells at the base of the crypts of Lieberkühn, but was not detected in the gastric (G) or the duodenal glands (DG), or in the pancreas. c, d Detection of Ppib transcript in glandular epithelia in the pyloric–duodenal region and pancreas. IL islet of Langerhans
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Fig3: Partitioning of Guca2a and Ppib transcripts in the pyloric–duodenal region and in pancreas. a, bGuca2a expression was found in the duodenum (D), in cells at the base of the crypts of Lieberkühn, but was not detected in the gastric (G) or the duodenal glands (DG), or in the pancreas. c, d Detection of Ppib transcript in glandular epithelia in the pyloric–duodenal region and pancreas. IL islet of Langerhans
Mentions: We did not detect Guca2a transcript in gastric or in pancreatic epithelia (Figs. 2a, 3), nor did we detect expression of Guca2b or Gucy2c in these tissues (Fig. 2b, c). This contrasts with previous observations on guinea pig, in which GN immunoreactivity was observed in pyloric EC cells (Cetin et al. 1994), and with observations on human and rat pancreatic tissue, in which GN, UGN, and GCC transcript and protein were detected (Kulaksiz et al. 2001; Kulaksiz and Cetin 2002). RT-PCR analysis confirmed that, compared to the gut, these glandular tissues contain only very low levels of these three transcripts (not shown). Such transcripts of low abundance may not be detectable by in situ hybridization, particularly in the pancreas, which contains high levels of ribonucleases (Azevedo-Pouly et al. 2014). However, arguing against significant mRNA loss prior to tissue fixation, we could readily detect Ppib transcript, in both the stomach and the pancreas (Fig. 3c, d). Therefore, we tentatively conclude that expression of the GCC signaling axis is low in mouse, compared to human pancreas. Interestingly, this apparent difference in the pancreatic expression of the GCC signaling axis between these species mirrors a previously observed disparity in the regulation of ductal anion and fluid secretion: Whereas in the human pancreas, the GCC signaling axis is thought to control the activity of the phosphorylation-regulated CFTR anion channel, anion and fluid secretion in the murine exocrine pancreas is principally governed by Ca2+-dependent anion channels, which operate independently of the GCC pathway (Winpenny et al. 1995; Kulaksiz et al. 2001).Fig. 3

View Article: PubMed Central - PubMed

ABSTRACT

Guanylin (GN) and uroguanylin (UGN), through activation of guanylyl cyclase C (GCC), serve to control intestinal fluid homeostasis. Both peptides are produced in the intestinal epithelium, but their cellular origin has not been fully charted. Using quantitative PCR and an improved in situ hybridization technique (RNAscope), we have assessed the expression of GN (Guca2a), UGN (Guca2b), and GCC (Gucy2c) in mouse intestine. In the crypts of Lieberkühn, expression of Guca2a and Guca2b was restricted to cells of secretory lineage, at the crypt’s base, and to a region above, previously identified as a common origin of cellular differentiation. In this compartment, comparatively uniform levels of Guca2a and Guca2b expression were observed throughout the length of the gut. In contrast, Guca2a and Guca2b expression in the villus–surface region was more variable, and reflected the distinct, but overlapping expression pattern observed previously. Accordingly, in jejunum and ileum, Guca2a and Guca2b were abundantly expressed by enterocytes, whereas in colon only Guca2a transcript was found in the surface region. In duodenum, only low levels of Guca2b transcript were observed in columnar cells, and Guca2a expression was restricted entirely to cells of the secretory lineage. Gucy2c was shown to be expressed relatively uniformly along the rostrocaudal and crypt–villus axes and was also found in the duodenal glands. Our study reveals novel aspects of the cellular localization of the GCC signaling axis that, apart from its role in the regulation of fluid balance, link it to pH regulation, cell cycle control, and host defense.

Electronic supplementary material: The online version of this article (doi:10.1007/s00418-016-1453-4) contains supplementary material, which is available to authorized users.

No MeSH data available.