Transcriptional activation of the human villin gene during enterocyte differentiation.
Enterocyte differentiation occurs along the crypt-villus axis and is generally thought to involve the transcriptional activation of cell-specific genes, among which is the brush-border structural protein villin. We have examined the molecular mechanisms of villin induction using both in vivo and in vitro systems. Total RNA was purified from rat tissues or cultured cells by the guanidinium thiocyanate method and Northern blot analyses carried out using radiolabeled complementary DNA probes specific for villin or the actin control. Transient transfection (calcium/phosphate method) assays were performed using a luciferase reporter gene containing 2 kb of the human villin gene 5'-flanking region. We have found that the villin mRNA was expressed at high levels in the small intestine, to a lesser degree in the colon, and was not detected in the brain or liver. In HT-29 cells, villin mRNA levels increased 2.5-fold (P<0.001) after 24 hours of sodium butyrate treatment, consistent with the process of enterocyte differentiation. Similarly, villin gene expression was induced in Caco-2 cells during postconfluence differentiation. Transient transfection assays demonstrated marked reporter gene activation (fourfold, P<0.001) in response to sodium butyrate in HT-29 cells, but no activation in the liver cell line HepG2. The effects of sodium butyrate were dose dependent, reaching a maximum at a concentration of 5 mmol/L. We conclude that a 2 kb region of the human villin gene is able to mediate its transcriptional activation during HT-29 cell differentiation. This DNA regulatory region appears to function in a cell type-specific (gut) manner.
Hodin
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RA
Department of Surgery, Beth Israel Deaconess Medical Center, Beth Israel Hospital, Harvard Medical School, Boston, MA 02215, USA.
Shei
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A
Meng
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S
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D020895:Enterocytes / Q000166:cytology*; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic / Q000502:physiology*
D008840:Microfilament Proteins; D012333:RNA, Messenger; C026615:villin