Anat Cell Biol.  2012 Sep;45(3):160-169. 10.5115/acb.2012.45.3.160.

Expression of Bis in the mouse gastrointestinal system

• Lee YD
• Yoon JS
• Yoon HH
• Youn HJ
• Kim J
• Lee JH

• KMID: 2046743
• DOI: http://doi.org/10.5115/acb.2012.45.3.160

Abstract

The Bcl-2 interacting death suppressor (Bis) protein is known to be involved in a variety of pathophysiological conditions. We recently generated bis-deficient mice, which exhibited early lethality with typical nutritional deprivation status. To further investigate the molecular basis for the malnutrition phenotype of bis deficient mice, we explored Bis expression in the digestive system of normal mice. Western blot analysis and quantitative real time reverse transcription polymerase chain reaction analysis indicated that Bis expression is highest in the esophagus, followed by the stomach, colon, jejunum and ileum. Immunohistochemical data indicated that Bis expression is restricted to the stratified squamous epitheliums in the esophagus and forestomach, and was not notable in the columnar epitheliums in the stomach, small intestine and colon. In addition, strong Bis immunoreactivity was detected in the striated muscles surrounding the esophagus and smooth muscles at a lesser intensity throughout the gastrointestinal (GI) tract. Ganglionated plexuses, located in submucous layers, as well as intermuscular layers, were specifically immunoreactive for Bis. Immunofluorescence studies revealed that Bis is co-localized in glial fibrillary acidic protein-expressing enteric glial cells. Immunostaining with neuron specific esterase antibodies indicate that Bis is also present in the cell bodies of ganglions in the enteric nervous system (ENS). Our findings indicate that Bis plays a role in regulating GI functions, such as motility and absorption, through modulating signal transmission between the ENS and smooth muscles or the intestinal epitheliums.

Keyword

Bag3; Enteric nervous system; Enteric glial cell; Mouse; Immunohistochemistry

MeSH Terms

Absorption
Animals
Antibodies
Blotting, Western
Colon
Digestive System
Enteric Nervous System
Epithelium
Esophagus
Fluorescent Antibody Technique
Ganglion Cysts
Ileum
Immunohistochemistry
Intestinal Mucosa
Intestine, Small
Jejunum
Malnutrition
Mice
Muscle, Smooth
Muscle, Striated
Neuroglia
Neurons
Phenotype
Polymerase Chain Reaction
Reverse Transcription
Stomach
Antibodies

Figure

• 

  Fig. 1 Expression of Bis in the mouse gastrointestinal (GI) tract. (A) Western blot analysis for Bis in the each part of GI tracts from 14-day-old mice. A representative result was provided with heat shock protein 70 (Hsp70) as a loading control. (B) A quantitative determination of Bis expression using densitometric analysis after normalizing to Hsp70 (mean±SE, n=3). The relative density of the Bis bands to that of Hsp70 from ileum was designated as 1.0. (C) Determination of the relative expression of bis mRNA in the mouse GI tract by quantitative real time polymerase chain reaction analysis. Data are normalized relative to β-actin mRNA in the same samples, and the value for the ileum was arbitrarily set as 1.0 (mean±SE, n=3). S.M., skeletal muscle; ESO, esophagus; STO, stomach; JEJ, jejunum; ILE, ileum; COL, colon. *P<0.05, **P<0.01 and ***P<0.005 compared with the value from ileum.

• 

  Fig. 2 Immunohistochemical analysis of Bis in the esophagus (A) and stomach (B-D). Intense Bis immunoreactivity was observed in the stratified squamous epithelium and striated muscles (marked as white or black asterisk, respectively) in the esophagus (A). In epithelia of the grandular stomach, only trace levels of Bis expression were detected (B, C) and weak immunostaining for Bis was detected in the smooth muscles of the stomach, while specific localization of Bis was detected in myenteric plexus (arrows) (B, D). Higher magnifications of the boxed areas in B are shown in C and D. Scale bars=50 µm (A-D).

• 

  Fig. 3 Bis immunoreactivity in the small intestine. Bis immunoreactivity was clearly localized in the ganglionated networks of the submucous plexus (arrow) and myenteric plexus (arrowhead) in jejunum (A, C) and ileum (B, D). Higher magnifications of the boxed areas in A and B are shown in C and D, respectively. Scale bars=50 µm (A, B), 20 µm (C, D).

• 

  Fig. 4 Immunofluorescence staining of Bis and glial fibrillary acidic protein (GFAP) in Jejunum. Confocal laser microscopic imaging of immunofluorescence for Bis (B), GFAP (C) and overlay (A, D) in the myenteric plexus of jejunum. Note that Bis immunoreactvity was observed in GFAP-expressing the enteric glial cells (marked as arrows).The higher magnification views of the boxed area in A are shown in B-D. Scale bars=50 µm (A), 10 µm (B-D).

• 

  Fig. 5 Comparison of immunoreactivity for Bis (A, C) and neuron specific esterase (NSE) (B, D) in adjacent sections of the jejunum. The large cell bodies, which were immunostained with Bis antibodies (A, C), were also positive for NSE (B, D) in submucosal plexus (arrows), as well as in myetneric plexus (arrowheads). Higher magnifications of the boxes in A and B were shown in C and D, respectively. Scale bars=20 µm (A-D).

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