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Lineage-specific expression of bestrophin-2 and bestrophin-4 in human intestinal epithelial cells.

Ito G, Okamoto R, Murano T, Shimizu H, Fujii S, Nakata T, Mizutani T, Yui S, Akiyama-Morio J, Nemoto Y, Okada E, Araki A, Ohtsuka K, Tsuchiya K, Nakamura T, Watanabe M - PLoS ONE (2013)

Bottom Line: Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells.In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells.These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3(-) or Cl(-). Bestrophin genes represent a newly identified group of calcium-activated Cl(-) channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.

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BEST4 is expressed in post-mitotic IECs other than secretory lineage cells.(A) Double-immunostaining of BEST4 with Ki-67 (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express Ki-67 (red). (B) Double-immunostaining of BEST4 with Lysozyme (Original magnification 200x). In the small intestine, BEST4-positive cells (green) did not co-express with Lysozyme (red). (C) Double immunostaining of BEST4 with Chromogranin-A (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with Chromogranin-A (red). A magnified view is shown in the lower column (Original magnification 400x). (D) Double-immunostaining of BEST4 with HPGDS (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with HPGDS (red). A magnified view is shown in the lower column (Original magnification 400x). Representative data of three independent analyses are shown.
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pone-0079693-g003: BEST4 is expressed in post-mitotic IECs other than secretory lineage cells.(A) Double-immunostaining of BEST4 with Ki-67 (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express Ki-67 (red). (B) Double-immunostaining of BEST4 with Lysozyme (Original magnification 200x). In the small intestine, BEST4-positive cells (green) did not co-express with Lysozyme (red). (C) Double immunostaining of BEST4 with Chromogranin-A (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with Chromogranin-A (red). A magnified view is shown in the lower column (Original magnification 400x). (D) Double-immunostaining of BEST4 with HPGDS (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with HPGDS (red). A magnified view is shown in the lower column (Original magnification 400x). Representative data of three independent analyses are shown.

Mentions: We performed further analysis to identify the exact lineage of IECs that are capable of expressing BEST4. Co-staining with Ki-67 showed that BEST4-positive IECs are completely negative for this cell proliferation marker, suggesting that these cells are all post-mitotic both in the small intestine and in the colon (Figure 3A). In the small intestine, BEST4-positive IECs were clearly absent within the crypt region and never co-expressed lysozyme, a specific marker for Paneth cells (Figure 3B). In addition, BEST4-positive cells never co-expressed Chromogranin-A or HPGDS, indicating that BEST4 is expressed in cells other than enteroendocrine cells or tuft cells (Figure 3C, D).


Lineage-specific expression of bestrophin-2 and bestrophin-4 in human intestinal epithelial cells.

Ito G, Okamoto R, Murano T, Shimizu H, Fujii S, Nakata T, Mizutani T, Yui S, Akiyama-Morio J, Nemoto Y, Okada E, Araki A, Ohtsuka K, Tsuchiya K, Nakamura T, Watanabe M - PLoS ONE (2013)

BEST4 is expressed in post-mitotic IECs other than secretory lineage cells.(A) Double-immunostaining of BEST4 with Ki-67 (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express Ki-67 (red). (B) Double-immunostaining of BEST4 with Lysozyme (Original magnification 200x). In the small intestine, BEST4-positive cells (green) did not co-express with Lysozyme (red). (C) Double immunostaining of BEST4 with Chromogranin-A (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with Chromogranin-A (red). A magnified view is shown in the lower column (Original magnification 400x). (D) Double-immunostaining of BEST4 with HPGDS (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with HPGDS (red). A magnified view is shown in the lower column (Original magnification 400x). Representative data of three independent analyses are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3818177&req=5

pone-0079693-g003: BEST4 is expressed in post-mitotic IECs other than secretory lineage cells.(A) Double-immunostaining of BEST4 with Ki-67 (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express Ki-67 (red). (B) Double-immunostaining of BEST4 with Lysozyme (Original magnification 200x). In the small intestine, BEST4-positive cells (green) did not co-express with Lysozyme (red). (C) Double immunostaining of BEST4 with Chromogranin-A (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with Chromogranin-A (red). A magnified view is shown in the lower column (Original magnification 400x). (D) Double-immunostaining of BEST4 with HPGDS (Original magnification 200x). Neither in the small intestine nor in the colon did BEST4-positive cells (green) co-express with HPGDS (red). A magnified view is shown in the lower column (Original magnification 400x). Representative data of three independent analyses are shown.
Mentions: We performed further analysis to identify the exact lineage of IECs that are capable of expressing BEST4. Co-staining with Ki-67 showed that BEST4-positive IECs are completely negative for this cell proliferation marker, suggesting that these cells are all post-mitotic both in the small intestine and in the colon (Figure 3A). In the small intestine, BEST4-positive IECs were clearly absent within the crypt region and never co-expressed lysozyme, a specific marker for Paneth cells (Figure 3B). In addition, BEST4-positive cells never co-expressed Chromogranin-A or HPGDS, indicating that BEST4 is expressed in cells other than enteroendocrine cells or tuft cells (Figure 3C, D).

Bottom Line: Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells.In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells.These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.

ABSTRACT
Intestinal epithelial cells (IECs) regulate the absorption and secretion of anions, such as HCO3(-) or Cl(-). Bestrophin genes represent a newly identified group of calcium-activated Cl(-) channels (CaCCs). Studies have suggested that, among the four human bestrophin-family genes, bestrophin-2 (BEST2) and bestrophin-4 (BEST4) might be expressed within the intestinal tissue. Consistently, a study showed that BEST2 is expressed by human colonic goblet cells. However, their precise expression pattern along the gastrointestinal tract, or the lineage specificity of the cells expressing these genes, remains largely unknown. Here, we show that BEST2 and BEST4 are expressed in vivo, each in a distinct, lineage-specific manner, in human IECs. While BEST2 was expressed exclusively in colonic goblet cells, BEST4 was expressed in the absorptive cells of both the small intestine and the colon. In addition, we found that BEST2 expression is significantly down-regulated in the active lesions of ulcerative colitis, where goblet cells were depleted, suggesting that BEST2 expression is restricted to goblet cells under both normal and pathologic conditions. Consistently, the induction of goblet cell differentiation by a Notch inhibitor, LY411575, significantly up-regulated the expression of not BEST4 but BEST2 in MUC2-positive HT-29 cells. Conversely, the induction of absorptive cell differentiation up-regulated the expression of BEST4 in villin-positive Caco-2 cells. In addition, we found that the up- or down-regulation of Notch activity leads to the preferential expression of either BEST4 or BEST2, respectively, in LS174T cells. These results collectively confirmed that BEST2 and BEST4 could be added to the lineage-specific genes of humans IECs due to their abilities to clearly identify goblet cells of colonic origin and a distinct subset of absorptive cells, respectively.

Show MeSH
Related in: MedlinePlus