Homeodomain protein CDX2 regulates goblet-specific MUC2 gene expression

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Abstract

Intestinal mucin gene MUC2 is abundantly expressed in goblet cells. To identify the transcriptional activator that regulates goblet-specific expression of MUC2, we analyzed the interaction between the MUC2 promoter and homeodomain proteins CDX1/2, which are involved in the regulation of intestinal development and differentiation. COS-7 cells were transiently transfected with a CDX1 or CDX2 expression construct and then used for the luciferase assay, reverse transcription-polymerase chain reaction, and electrophoretic mobility shift assay (EMSA). The CDX2 expression construct activated the MUC2 promoter and increased the endogenous MUC2 mRNA level, while the CDX1 one did not. EMSA revealed that CDX2 bound to the MUC2 gene cis element, MUC2-WT. These results suggest that CDX2, but not CDX1, interacts with the MUC2 promoter and activates MUC2 transcription, and plays an important role in the differentiation of goblet cells.

Section snippets

Materials and methods

Human CDX1 and CDX2 expression constructs and a promoter–reporter construct. CDX1 and CDX2 complementary DNA (cDNA) inserts were obtained by RT-PCR using human colon mRNA as described previously [13]. The CDX1 and CDX2 PCR products were subcloned into the HindIII and BamHI restriction sites of mammalian expression vector pcDNA 3.1 (Invitrogen, Groningen, Netherlands) downstream of the cytomegalovirus promoter. We also used the pcDNA 3.1 empty vector as a negative control. The expression of CDX1

CDX2 activates transcription of the MUC2 promoter–reporter construct

To determine whether CDX1 or CDX2 activates the MUC2 promoter, preconfluent COS-7 cells were transfected with pcDNA 3.1 CDX1 or pcDNA 3.1 CDX2, pGL3 reporter constructs, and the pRL-SV40 vector. Compared with the pcDNA 3.1 empty vector and pcDNA 3.1 CDX1, transfection with pcDNA 3.1 CDX2 resulted in about 5-fold transcriptional activation of the MUC2 promoter construct (Fig. 2).

CDX2 expression induces MUC2 transcripts

To determine whether ectopic expression of the CDX1 or CDX2 protein up-regulates transcription of endogenous MUC2,

Discussion

Mucins are high-molecular-weight O-linked glycoproteins that are prominently expressed in the intestine and other epithelial organs. MUC2, one of the mucin genes, is abundantly expressed in goblet cells in the intestine [1], [2]. The promoter activity of the 5-flanking region of the MUC2 gene was examined by means of the luciferase assay [17]. According to the results, the region comprising bases −228 to −171 contains an element or elements important for specific expression from the MUC2

Acknowledgements

We wish to thank Drs. S. Miyake, Y. Akiyama, and Y. Yanagisawa for their valuable discussions. This study was supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan.

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