Thyroid hormone (T3) is an important regulator of gut mucosal development and differentiation, inducing intestinal alkaline phosphatase (IAP) and repressing lactase gene transcription. In contrast, cyclin D1 (CD1) appears to be a growth promoter in the gut, functioning to maintain the undifferentiated state. The present studies were designed to examine the effects of CD1 on T3 action within intestinal epithelia. Caco-2 cells were maintained in hypothyroid medium and transiently transfected with either rat lactase (3.0 kb) or human IAP (2.4 kb) luciferase (Luc) reporter plasmids. Cotransfections were carried out using two T3 receptor (TR) isoforms, TR"-1 and TR$-1, as well as plasmids expressing CD1, CD3, CA, or CB1. Cells were then treated +/- 10 nmol/L T3 for 24 hours and luciferase activity was determined. With T3 treatment, IAP-Luc activity was induced (TR"-1 = eightfold, TR$-1 = ninefold), but these effects were dramatically inhibited (> 50%) by CD1 and CD3. In contrast, CA and CB1 did not alter T3-mediated IAP gene activation. The ability of CD1 and CD3 to inhibit T3 action was also tested in the context of the lactase gene, which is negatively regulated by T3. As expected, lactase reporter gene activity was repressed by T3 treatment in the case of both receptor isoforms, TR"-1 = 30% and TR$-1 = 40%. In contrast to its effects on the IAP gene, CD1 did not inhibit T3-mediated changes in lactase reporter gene activity. The D-type cyclins (CD1 and CD3), but not CA or CB1, specifically inhibit T3-mediated activation of the IAP gene. In contrast, the D-type cyclins do not inhibit T3-mediated repression of the lactase gene. These studies have identified a novel molecular interaction that exists between the pathways of growth and differentiation within intestinal epithelia.

Alkaline Phosphatase↗

PubMedDate not supplied

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.

Animals↗