The Human Mucin MUC4 Is Transcriptionally Regulated by Caudal-related Homeobox, Hepatocyte Nuclear Factors, Forkhead Box A, and GATA Endodermal Transcription Factors in Epithelial Cancer Cells

Abstract

The human gene MUC4 encodes a large transmembrane mucin that is developmentally regulated and expressed along the undifferentiated pseudostratified epithelium, as early as 6.5 weeks during fetal development. Immunohistochemical analysis of Muc4 expression in developing mouse lung and gastrointestinal tract showed a different spatio-temporal pattern of expression before and after cytodifferentiation. The molecular mechanisms governing MUC4 expression during development are, however, unknown. Hepatocyte nuclear factors (HNF), forkhead box A (FOXA), GATA, and caudal-related homeobox transcription factors (TFs) are known to control cell differentiation of gut endoderm derived-tissues during embryonic development. They also control the expression of cell-and tissuespecific genes and may thus control MUC4 expression. To test this hypothesis, we studied and deciphered the molecular mechanisms responsible for MUC4 transcriptional regulation by these TFs. Experiments using small interfering RNA, cell cotransfection, and site-directed mutagenesis indicated that MUC4 is regulated at the transcriptional level by CDX-1 and -2, HNF-1␣ and -1␤, FOXA1/A2, HNF-4␣ and -4␥, and GATA-4, -5, and -6 factors in a cell-specific manner. Binding of TFs was assessed by chromatin immunoprecipitation, and gel-shift assays.Altogether, these results demonstrate that MUC4 is a target gene of endodermal TFs and thus point out an important role for these TFs in regulating MUC4 expression during epithelial differentiation during development, cancer, and repair.

Combined defects in epithelial and immunoregulatory factors exacerbate the pathogenesis of inflammation: mucin 2-interleukin 10-deficient mice

“…This pattern is in agreement with our previous data recently published in WT mice. 25 Interestingly, diminished differentiation of goblet cells in the colon of Muc2 À/À and Muc2/IL-10 DKO mice was confirmed by a decrease in Muc4 expression. There was even an almost complete loss of Muc4 protein expression in the goblet cells of the proximal colon in both these types of knockout mice.…”

“…Therefore, we ascertained the presence and maturation of the goblet-cell lineage in the different mouse models by determining the expression of Tff3 and Muc4. 24,25,28,29 Muc4 and Tff3 were expressed by goblet cells, concentrated mainly in the upper portion of the crypts, in both the proximal and distal colon of WT mice (Figure 5a and c, Tff3, blue panel; Figure 5e and g, Muc4, orange panel). The staining pattern of Muc4 in the IL-10 À/À mice corresponded to the staining pattern of this marker in the WT mice (Figure 5k and l).…”

“…This mechanism needs to be further investigated, but already transcription factors, which control intestinal differentiation, may be of interest, that is, Cdx-1 and Cdx-2 were recently identified as important regulators of Muc4 expression in mice colon. 25 In addition, loss of differentiation of intestinal cells during damage, in general, has been shown in several animal models, by downregulation of distinct enterocyte-specific proteins. 14,35,36 The ABpositive cells at the crypt bases in the distal colon in the Muc2/IL-10 DKO mice could indicate transient expression of other mucins known to be expressed in the intestine or de novo expression of Muc6 mRNA as described for the Muc2 À/À mice.…”

“…24 Muc4 was stained using anti-hMUC4 polyclonal antibody (hHA-1) that recognizes a C-terminal peptidic region of MUC4a subunit (1:6000 diluted in 1% BSA, 0.1% Triton X-100 in PBS). 25 To visualize BrdU incorporation, sections were incubated with anti-BrdU (1:250 diluted in PBS; Boehringer Mannheim, Mannheim, Germany). 26 CD3 þ cells were detected using an anti-human CD3 (1:800 diluted in 1% BSA, 0.1% Triton X-100 in PBS; A0452, DAKO, Heverlee, Belgium).…”

Cdx1, a dispensable homeobox gene for gut development with limited effect in intestinal cancer

“…Thus, the massive increase of Cdx1 expression negatively regulates Cdx2. Consequently, Cdx1 may fine-tune the activity of Cdx2, as illustrated by the fact that two targets of Cdx2, Muc4 and Muc2, show a deeper expression in the colonic glands of Cdx1 À/À mice, like as Cdx2 (Jonkheere et al, 2007 78/id; see Supplementary Figure 2D). Cell transfection experiments were performed using the Cdx1-expression plasmid pCB6-HA-CDX1 in an attempt to delineate the mechanism of downregulation of Cdx2 by Cdx1.…”