Abstract
The intestinal fatty acid binding protein gene (Fabpi) provides a good model system for studying how gene transcription is regulated in enterocytes as a function of their differentiation program and location along the duodenal-to-colonic axis. We have compared and contrasted the transcriptional activity of four fusion genes composed of elements from the 5'-nontranscribed domain of rat Fabpi linked to the human growth hormone gene (I-FABP/hGH) in transgenic mice and in five primate epithelial cell lines derived from intestine, liver, kidney, and cervix. Nucleotides -103 to +28 of rat Fabpi are able to direct appropriate lineage-specific and geographic patterns of hGH expression in transgenic mice. I-FABP-103 to +28/hGH is preferentially expressed in Caco-2 cells, which emulate some of the features of differentiated small intestinal enterocytes after they reach confluence. However, other I-FABP/hGH fusion genes that exhibit differentiation-dependent changes in their expression along the crypt-to-villus axis do not manifest the same pattern of differentiation-dependent change in activity in this cell line. Correlation of their patterns of expression in vivo and ex vivo suggest that nonproliferating Caco-2 cells mimic some of the features of the transcriptional regulatory environment of enterocytes located in the upper crypt. Nucleotides -103 to +28 of rat Fabpi contain one copy of a repeated 14-base pair element that is conserved in the orthologous mouse and human genes and represented in several other homologous and nonhomologous genes, which are expressed in villus-associated enterocytes. This element binds to two members of the steroid hormone receptor superfamily of transcription factors produced in enterocytes and Caco-2 cells: hepatic nuclear factor-4 (HNF-4) and apolipoprotein regulatory protein-1 (ARP-1). Co-transfection studies performed in Caco-2 cells and in a monkey kidney cell line (CV-1) that lacks endogenous pools of ARP-1 and HNF-4 suggest that ARP-1 and HNF-4 can function to activate I-FABP-103 to +28/hGH+3 through their interactions with the 14-base pair element. This activation appears to be affected by elements located between nucleotides -277 and -104 and other transcription factors.
Highlights
Introduction of a HNF4 expressionvector into proliferat- modebstusitgnificanint creaseins expression of I-ing Caco-2 cells produced no significant change in thelevels FABp-277 to +28/hGH+3 and I-FABPE2"277to +2R/hGH+i3n proof hGpHroduction by I-FABP-103" +" /hGH+I3, liferating CV-1 cells, (levels of hGH production rise by 2-3-FABp-277 to +28/hGH+3, or I-FABPE2/-277to +28/hGH+3(Fig. fold, p < 0.005; Fig. 7B)
Epithelial Cell Type-specific Expression of I-FABP/hGW3 Constructs-Five different primate epithelial cell lines were included in our study
Caco-2 cells are derived from a human colon adenocarcinoma. After achieving confluence they are able to complete a differentiationprogram that results in the acquisition of some features of human small intestinal enterocytes (Pintoet al,1, 983;Rousset 1986).CV-1cells are derived from the kidney of an African Green monkey (Jensen et al, 1964)
Summary
Of DNAs, 401) were used to transfect Caco-2 cells using the protocol described above. 1987) linked tothe human growth hormone gene starting a t its ble, (2418-resistant populations were obtained, IO6pooled cells were nucleotide +3 (Seeburg, 1982) Five-ml aliquots of culture mediumwere nated I-FABP-1178”+2B/hGH+3I-.FABP-2T’”+28/hGH+w3 as obtained obtained every 3 days at thetime cells were re-fed. These samples of by digesting pIFhGHl with EcoRI yielding a 2.4-kb fragment, which medium were subjected to centrifugation at 17,000 x g to remove was cloned into theEcoRI site of pBluescript SKII (Promega, Madi- residual cells, and hGH concentrationswere measured in the resulting ” son, WI).
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