ABSTRACT
Mammalian male fertility depends on the epididymis, a highly segmented organ that promotes sperm maturation and protects sperm from oxidative damage. Remarkably little is known about how gene expression is controlled in the epididymis. A candidate to regulate genes crucial for epididymal function is reproductive homeobox gene on X chromosome (RHOX)5, a homeobox transcription factor essential for optimal sperm motility that is expressed in the caput region of the epididymis. Here, we report the identification of factors that control Rhox5 gene expression in epididymal cells in a developmentally regulated and region-specific fashion. First, we identify GATA transcription factor-binding sites in the Rhox5 proximal promoter (Pp) necessary for Rhox5 expression in epididymal cells in vitro and in vivo. Adjacent to the GATA sites are androgen-response elements, which bind to the nuclear hormone receptor androgen receptor (AR), and are responsible for the AR-dependent expression of Rhox5 in epididymal cells. We provide evidence that AR is recruited to the Pp in a region-specific and developmentally regulated manner in the epididymis that is dictated not only by differential AR availability but differential methylation of the Pp. Site-specific methylation of the Pp cytosine and guanine separated by one phosphate, most of which overlap with androgen-response elements, inhibited both AR occupancy at the Pp and Pp-dependent transcription in caput epididymal cells. Together, our data support a model in which DNA methylation, AR, and GATA factors collaborate to dictate the unique developmental and region-specific expression pattern of the RHOX5 homeobox transcription factor in the caput epididymis, which in turn controls the expression of genes critical for promoting sperm motility and function.
Subject(s)
DNA Methylation , Epididymis/metabolism , GATA Transcription Factors/physiology , Gene Expression Regulation, Developmental , Homeodomain Proteins/genetics , Receptors, Androgen/metabolism , Transcription Factors/genetics , Androgens/physiology , Animals , Cell Line , Genes, Reporter , Homeodomain Proteins/metabolism , Luciferases/biosynthesis , Luciferases/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Organ Specificity , Receptors, Androgen/genetics , Response Elements , Transcription Factors/metabolism , Transcription, GeneticABSTRACT
Vitamin D and its analogs are potent inhibitors of colorectal cancer growth and metastasis. A number of recent studies have defined the intersections between the ß-catenin-TCF pathway (a known contributor to colorectal cancer progression) and the vitamin D receptor (VDR) pathway, shedding light on the underlying mechanisms. Vitamin D also regulates the innate immune response, and as such influences susceptibility to inflammatory bowel disease, a predisposing factor in colorectal cancer. Understanding the role of vitamin D in these different contexts will enable development of next generation vitamin D analogs that will serve as both chemopreventatives and cancer therapeutics, without the accompanying side effects of hypercalcemia usually associated with high vitamin D intake. This review summarizes the mechanisms of action of vitamin D and the VDR in the context of the gastrointestinal tract and colorectal carcinogenesis.
Subject(s)
Colorectal Neoplasms/metabolism , Colorectal Neoplasms/prevention & control , Receptors, Calcitriol/metabolism , Vitamin D/metabolism , Animals , Humans , beta Catenin/metabolismABSTRACT
TCF7L2 transcription factor is a downstream effector of the canonical Wnt/beta-catenin signaling, which controls cell fate and homeostasis. However, the complexity of TCF7L2 expression with numerous mRNA isoforms coding for proteins with distinct N- and C-termini allows variability in TCF7L2 functions and regulations. Here, we show that although TCF7L2 mRNA isoforms distinguish fetal, immortalized and adult differentiated endothelial cells (EC), they cannot explain the lack of significant beta-catenin/TCF7 activities in ECs. Lithium, a Wnt-signaling activator, increases TCF7L2 mRNA levels and induces an RNA isoform switch favoring the expression of TCF7L2-short forms lacking the C-termini domains. Although the latter occurs in different cell types, its extent depends on the overall increase of TCF7L2 transcription, which correlates with cell responsiveness to Wnt/beta-catenin signaling. While GSK3beta down-regulation increases TCF7L2 expression, there is no concomitant change in TCF7L2 mRNA isoforms, which demonstrate the dual effects of lithium on TCF7L2 expression via a GSK3beta-dependent up-regulation and a GSK3beta-independent modulation of RNA splicing. TCF7L2E-long forms display a repressor activity on TCF7L2-promoter reporters and lithium induces a decrease of the endogenous TCF7L2 forms bound to native TCF7L2-promoter chromatin at two novel distal TCF7-binding sites. Altogether our data reveal a lithium-induced RNA switch favoring the expression of TCF7L2-short forms, which results in a transcriptional de-repression of lithium target genes negatively regulated by TCF7L2-long forms, like TCF7L2, and thus to an amplification of Wnt-signaling in responsive cells.
Subject(s)
Alternative Splicing , Lithium/metabolism , TCF Transcription Factors/genetics , Wnt Proteins/metabolism , Animals , Cattle , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Lithium Chloride/pharmacology , Protein Isoforms/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , TCF Transcription Factors/metabolism , Transcription Factor 7-Like 2 Protein , beta Catenin/metabolismABSTRACT
Predicting the potential physiological outcome(s) of any given molecular pathway is complex because of cross-talk with other pathways. This is particularly evident in the case of the nuclear hormone receptor and canonical Wnt pathways, which regulate cell growth and proliferation, differentiation, apoptosis, and metastatic potential in numerous tissues. These pathways are known to intersect at many levels: in the intracellular space, at the membrane, in the cytoplasm, and within the nucleus. The outcomes of these interactions are important in the control of stem cell differentiation and maintenance, feedback loops, and regulating oncogenic potential. The aim of this review is to demonstrate the importance of considering pathway cross-talk when predicting functional outcomes of signaling, using nuclear hormone receptor/canonical Wnt pathway cross-talk as an example.
Subject(s)
Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction/physiology , Wnt Proteins/metabolism , Animals , Cell Membrane/metabolism , Cell Nucleus/metabolism , Extracellular Space/metabolism , Humans , In Vitro Techniques , Male , Models, Biological , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Receptor Cross-Talk , Receptors, Androgen/metabolism , beta Catenin/metabolismABSTRACT
BACKGROUND: The vitamin D receptor (VDR) pathway is important in the prevention and potentially in the treatment of many cancers. One important mechanism of VDR action is related to its interaction with the Wnt/beta-catenin pathway. Agonist-bound VDR inhibits the oncogenic Wnt/beta-catenin/TCF pathway by interacting directly with beta-catenin and in some cells by increasing cadherin expression which, in turn, recruits beta-catenin to the membrane. Here we identify TCF-4, a transcriptional regulator and beta-catenin binding partner as an indirect target of the VDR pathway. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we show that TCF-4 (gene name TCF7L2) is decreased in the mammary gland of the VDR knockout mouse as compared to the wild-type mouse. Furthermore, we show 1,25(OH)2D3 increases TCF-4 at the RNA and protein levels in several human colorectal cancer cell lines, the effect of which is completely dependent on the VDR. In silico analysis of the human and mouse TCF7L2 promoters identified several putative VDR binding elements. Although TCF7L2 promoter reporters responded to exogenous VDR, and 1,25(OH)2D3, mutation analysis and chromatin immunoprecipitation assays, showed that the increase in TCF7L2 did not require recruitment of the VDR to the identified elements and indicates that the regulation by VDR is indirect. This is further confirmed by the requirement of de novo protein synthesis for this up-regulation. CONCLUSIONS/SIGNIFICANCE: Although it is generally assumed that binding of beta-catenin to members of the TCF/LEF family is cancer-promoting, recent studies have indicated that TCF-4 functions instead as a transcriptional repressor that restricts breast and colorectal cancer cell growth. Consequently, we conclude that the 1,25(OH)2D3/VDR-mediated increase in TCF-4 may have a protective role in colon cancer as well as diabetes and Crohn's disease.
Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/biosynthesis , Colorectal Neoplasms/metabolism , Gene Expression Regulation , Mammary Glands, Animal/metabolism , Receptors, Calcitriol/metabolism , Transcription Factors/biosynthesis , Vitamin D/metabolism , Animals , Caco-2 Cells , Cadherins/metabolism , Cell Line, Tumor , Humans , Mice , Mice, Knockout , Promoter Regions, Genetic , Transcription Factor 4 , Wnt Proteins/metabolism , beta Catenin/metabolismABSTRACT
Mouse mammary tumors arising during medroxyprogesterone-DMBA-mediated mammary carcinogenesis comprised three distinct phenotypes: adenocarcinoma, squamous cell carcinoma, and myoepithelial carcinoma. The molecular signature for each of the three tumor subsets was characterized by gene microarray analysis, and three distinct sets of gene expression profiles were obtained that were corroborated in part by quantitative RT-PCR and immunohistochemistry. These results suggest that this carcinogenesis and gene expression model will be useful for rapidly assessing the histopathological differences arising in mammary carcinogenesis and the effects of tumor promoting or chemoprevention agents.
