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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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BEST2 expression is significantly up-regulated in HT-29 cells by the induction of goblet cell differentiation.(A) LY411575 induces the expression of BEST2 in HT-29 cells. The HT-29 cells were treated with either LY411575 (1μM) or DMSO for the indicated period of time and then subjected to quantitative RT-PCR analysis or immunostaining. The quantitative data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO of the corresponding time period, as determined by Student’s t-test. The immunostaining of MUC2 (red, left panel) and BEST2 (green, left panel) after 120 h of treatment showed a clear increase in the number of MUC2- or BEST2- positive cells by LY411575 (Original magnification 200x). (B) The expression of absorptive cell marker genes, as well as BEST4, was not induced in HT-29 cells by LY411575. HT-29 cells were treated with either LY411575 or DMSO for 72 h and subjected for quantitative RT-PCR analysis. Data were normalized to expression levels of β-actin. Error bars represent S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO, determined by Student’s t-test. (C) LY411575 induces BEST2 expression in MUC2-positive cells. HT-29 cells were treated with either LY411575 or DMSO for 120 h and subjected to double-immunostaining. The analysis of LY411575-treated cells revealed that BEST2-positive (green) cells clearly co-localize within MUC2-positive (red) cells (Original magnification 200x for upper series, 400x for lower series).
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pone-0079693-g006: BEST2 expression is significantly up-regulated in HT-29 cells by the induction of goblet cell differentiation.(A) LY411575 induces the expression of BEST2 in HT-29 cells. The HT-29 cells were treated with either LY411575 (1μM) or DMSO for the indicated period of time and then subjected to quantitative RT-PCR analysis or immunostaining. The quantitative data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO of the corresponding time period, as determined by Student’s t-test. The immunostaining of MUC2 (red, left panel) and BEST2 (green, left panel) after 120 h of treatment showed a clear increase in the number of MUC2- or BEST2- positive cells by LY411575 (Original magnification 200x). (B) The expression of absorptive cell marker genes, as well as BEST4, was not induced in HT-29 cells by LY411575. HT-29 cells were treated with either LY411575 or DMSO for 72 h and subjected for quantitative RT-PCR analysis. Data were normalized to expression levels of β-actin. Error bars represent S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO, determined by Student’s t-test. (C) LY411575 induces BEST2 expression in MUC2-positive cells. HT-29 cells were treated with either LY411575 or DMSO for 120 h and subjected to double-immunostaining. The analysis of LY411575-treated cells revealed that BEST2-positive (green) cells clearly co-localize within MUC2-positive (red) cells (Original magnification 200x for upper series, 400x for lower series).

Mentions: Our in vivo results suggested that the expression of BEST2 is restricted to goblet cells. However, whether the expression of BEST2 is a part of goblet cell differentiation or a phenotype that is acquired after the goblet cells have matured remains uncertain. Therefore, we used an in vitro goblet cell differentiation model to examine the expression levels of BEST2. In our previous study, we showed that a γ-secretase inhibitor, LY411575, can promote the goblet cell differentiation of HT-29 cells through the inhibition of Notch signaling [24]. Using the same model, we found that LY411575 can not only up-regulate the expression of MUC2 but can also up-regulate the expression of BEST2 at both the mRNA and protein levels (Figure 6A). Further analysis of the cells treated by LY411575 for 72 h confirmed that BEST2 expression is induced when Notch signaling has been down-regulated, as shown by the down-regulation of Hes1 mRNA expression (Figure 6B). In sharp contrast to BEST2 and MUC2, the mRNA expression of absorptive cell-specific genes, such as DPP4, Villin and BEST4, showed no significant change (Figure 6B). Double immunostaining confirmed that BEST2 expression is induced exclusively within MUC2-positive cells by the LY411575 treatment of HT-29 cells (Figure 6C). However, the concomitant expression of MUC2 is not necessarily required for BEST2 expression, as the siRNA-mediated knockdown of MUC2 had no effect on LY411575-induced BEST2 expression in HT-29 cells (Figure S2). These findings collectively show that BEST2 is one of the genes that is up-regulated during the process of human intestinal goblet cell differentiation.


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)

BEST2 expression is significantly up-regulated in HT-29 cells by the induction of goblet cell differentiation.(A) LY411575 induces the expression of BEST2 in HT-29 cells. The HT-29 cells were treated with either LY411575 (1μM) or DMSO for the indicated period of time and then subjected to quantitative RT-PCR analysis or immunostaining. The quantitative data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO of the corresponding time period, as determined by Student’s t-test. The immunostaining of MUC2 (red, left panel) and BEST2 (green, left panel) after 120 h of treatment showed a clear increase in the number of MUC2- or BEST2- positive cells by LY411575 (Original magnification 200x). (B) The expression of absorptive cell marker genes, as well as BEST4, was not induced in HT-29 cells by LY411575. HT-29 cells were treated with either LY411575 or DMSO for 72 h and subjected for quantitative RT-PCR analysis. Data were normalized to expression levels of β-actin. Error bars represent S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO, determined by Student’s t-test. (C) LY411575 induces BEST2 expression in MUC2-positive cells. HT-29 cells were treated with either LY411575 or DMSO for 120 h and subjected to double-immunostaining. The analysis of LY411575-treated cells revealed that BEST2-positive (green) cells clearly co-localize within MUC2-positive (red) cells (Original magnification 200x for upper series, 400x for lower series).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3818177&req=5

pone-0079693-g006: BEST2 expression is significantly up-regulated in HT-29 cells by the induction of goblet cell differentiation.(A) LY411575 induces the expression of BEST2 in HT-29 cells. The HT-29 cells were treated with either LY411575 (1μM) or DMSO for the indicated period of time and then subjected to quantitative RT-PCR analysis or immunostaining. The quantitative data were normalized to the expression levels of β-actin. Error bars represent the S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO of the corresponding time period, as determined by Student’s t-test. The immunostaining of MUC2 (red, left panel) and BEST2 (green, left panel) after 120 h of treatment showed a clear increase in the number of MUC2- or BEST2- positive cells by LY411575 (Original magnification 200x). (B) The expression of absorptive cell marker genes, as well as BEST4, was not induced in HT-29 cells by LY411575. HT-29 cells were treated with either LY411575 or DMSO for 72 h and subjected for quantitative RT-PCR analysis. Data were normalized to expression levels of β-actin. Error bars represent S.D. of triplicate experiments. * represents P< 0.05 compared to DMSO, determined by Student’s t-test. (C) LY411575 induces BEST2 expression in MUC2-positive cells. HT-29 cells were treated with either LY411575 or DMSO for 120 h and subjected to double-immunostaining. The analysis of LY411575-treated cells revealed that BEST2-positive (green) cells clearly co-localize within MUC2-positive (red) cells (Original magnification 200x for upper series, 400x for lower series).
Mentions: Our in vivo results suggested that the expression of BEST2 is restricted to goblet cells. However, whether the expression of BEST2 is a part of goblet cell differentiation or a phenotype that is acquired after the goblet cells have matured remains uncertain. Therefore, we used an in vitro goblet cell differentiation model to examine the expression levels of BEST2. In our previous study, we showed that a γ-secretase inhibitor, LY411575, can promote the goblet cell differentiation of HT-29 cells through the inhibition of Notch signaling [24]. Using the same model, we found that LY411575 can not only up-regulate the expression of MUC2 but can also up-regulate the expression of BEST2 at both the mRNA and protein levels (Figure 6A). Further analysis of the cells treated by LY411575 for 72 h confirmed that BEST2 expression is induced when Notch signaling has been down-regulated, as shown by the down-regulation of Hes1 mRNA expression (Figure 6B). In sharp contrast to BEST2 and MUC2, the mRNA expression of absorptive cell-specific genes, such as DPP4, Villin and BEST4, showed no significant change (Figure 6B). Double immunostaining confirmed that BEST2 expression is induced exclusively within MUC2-positive cells by the LY411575 treatment of HT-29 cells (Figure 6C). However, the concomitant expression of MUC2 is not necessarily required for BEST2 expression, as the siRNA-mediated knockdown of MUC2 had no effect on LY411575-induced BEST2 expression in HT-29 cells (Figure S2). These findings collectively show that BEST2 is one of the genes that is up-regulated during the process of human intestinal goblet cell differentiation.

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