Shufen Meng

Butyrate inhibits colon carcinoma cell growth through two distinct pathways

BACKGROUND Dietary fiber and the resultant increase in colonic butyrate levels protect against colon carcinogenesis. Previous studies have shown that p21 and histone hyperacetylation are important in basal growth inhibition by butyrate. This study was designed to elucidate other mechanism underlying the butyrate effects on cell growth. METHODS HT-29 colon carcinoma cells (standard medium or medium lacking serum) were treated with sodium butyrate (NaBu), epidermal growth factor (EGF), or both. Northern blot analyses were performed with cDNA probes specific for c-fos, c-jun, and actin. Cell growth was measured by 3H-thymidine incorporation. Enzyme-linked immunosorbent assay (ELISA) was used to quantify EGF receptor levels. RESULTS Butyrate and serum starvation (SS) both induced a cell cycle withdrawal by 24 hours. In response to EGF treatment, SS cells exhibited a growth spurt and induced c-fos and c-jun proto-oncogene expression, whereas butyrate-treated cells exhibited minimal growth response to EGF. This relative unresponsiveness to EGF in butyrate-treated cells corresponded to a dramatic decline in EGF receptor levels when compared to untreated controls. CONCLUSIONS Butyrate appears to inhibit colon cancer cell growth by two mechanisms, one involving histone hyperacetylation and p21 induction and the other related to impaired EGF-responsiveness.

Na-K-2Cl cotransporter gene expression and function during enterocyte differentiation. Modulation of Cl- secretory capacity by butyrate.

The basolateral Na-K-2Cl cotransporter (NKCC1) is a key component of the intestinal crypt cell secretory apparatus. Its fate during the transition to absorptive enterocyte and the potential impact of its altered expression on secretory output have not been addressed. In this report, NKCC1 mRNA was found to be expressed in rat jejunal crypt but not villus cells. Butyrate treatment of intestinal epithelial HT29 cells induced a differentiation pattern that recapitulated the rat intestinal crypt-villus axis, with NKCC1 mRNA levels decreasing in a time- and dose-dependent fashion in parallel with upregulation of apical brush-border markers. Butyrate but not acetate or proprionate decreased basal and cAMP-stimulated bumetanide-sensitive K+ (86Rb) uptake in both HT29 cells and the Cl--secreting T84 line. Butyrate markedly decreased transepithelial Cl- secretion in confluent T84 monolayers without effect on cAMP-regulated apical Cl- efflux. We conclude that NKCC1 regulation during enterocyte differentiation occurs at the level of gene expression, and that selective downregulation of NKCC1 gene expression and function by butyrate leads to a profound decrease in transepithelial Cl- secretion. These data emphasize the importance of NKCC1 in determining epithelial secretory capacity and suggest the possibility of modulation of the enterocytic transport phenotype as therapy for diarrheal disorders.

Thyroid hormone and the gut: Selective transcriptional activation of a villus-enterocyte marker

BACKGROUND Thyroid hormone (T3) is an important regulator of gut mucosal growth, differentiation, and barrier function, but its mechanism of action in the gastrointestinal tract is largely unknown. The present studies were carried out to define the molecular mechanisms by which T3 alters gut gene expression. METHODS In vivo: Adult, male, Sprague-Dawley rats were given three daily injections (intraperitoneal) of either saline solution or 30 micrograms/kg triiodothyronine. Small intestinal tissues were harvested, and Northern blot analyses were performed by using specific radiolabeled cDNA probes. In vitro: HT-29 cells were transfected with reporter plasmids and treated with or without T3, and chloramphenicol acetyltransferase activity was measured. RESULTS The T3-induced changes in enterocyte gene expression occurred in villus enterocytes and not in crypt cells and were independent of food intake. Northern analyses with an intron-specific probe revealed that the T3 induction in intestinal alkaline phosphatase (IAP) expression occurs at the level of transcription. Transient transfection assays revealed no T3-induced changes under basal conditions but marked increases (sixfold, p < 0.001) when a T3-receptor (TR beta-1) plasmid was cotransfected. Furthermore, T3 was found to induce greater IAP reporter gene activity in differentiated (+ sodium butyrate) compared with undifferentiated HT-29 cells. CONCLUSIONS T3 induces IAP expression at the level of gene transcription. Both in vivo and in vitro, IAP transcriptional activation occurs to a greater extent in differentiated enterocytes than in undifferentiated crypt cells. Transactivation of the IAP gene by T3 is mediated via a DNA cis-element(s) located within the 2.4 kb segment present in the reporter gene.

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.

Small bowel adaptation: counterregulatory effects of epidermal growth factor and somatostatin on the program of early gene expression.

BACKGROUND Small intestinal crypt cell proliferation is essential to the normal renewal of the epithelium, as well as the adaptive responses that follow resection or injury. The present studies were designed to elucidate the molecular mechanisms by which epidermal growth factor (EGF) and somatostatin interact to regulate crypt cell proliferation. METHODS Rat crypt (IEC-6) cells were maintained in Dulbeccos modified Eagles medium plus 10% fetal calf serum and treated with EGF (10 or 100 ng/ml) or somatostatin (0.5 microgram/ml). Cell counts were done to examine the effects on cellular growth, and Northern blot analyses were carried out by using complementary DNA probes corresponding to various protooncogenes. RESULTS EGF caused a 41% increase in cellular growth, an effect that was almost completely blocked by pretreatment (30 minutes) with somatostatin. EGF led to dramatic increases in c-fos (greater than 20-fold), c-jun (2-fold), and jun B (3-fold) gene expression at 30 minutes, consistent with the previously characterized immediate-early gene response in IEC-6 cells. Somatostatin alone had no effects on protooncogene levels, but pretreatment with somatostatin resulted in a marked inhibition (80%, p < 0.001 in all cases) of the EGF-induced increases in protooncogene expression. CONCLUSIONS Somatostatin inhibits the EGF-induced protooncogene expression in IEC-6 cells. The somatostatin inhibition of immediate-early gene expression lends support to its role as a negative growth regulator in intestinal epithelia and indicates that its effect occur at an upstream site in the cellular growth response.

Thyroid hormone and the d-type cyclins interact in regulating enterocyte gene transcription.

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. Coaansfections were carried out using two T3 receptor (TR) isoforms, TRW1 and TRβ-1, as well as plasmids expressing CDl, 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 (TRα-1 = eightfold, TRβ-1 = ninefold), but these effects were dramatically inhibited (>50%) by CD1 and CD3. In contrast, CA and CBl 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, TRW1 = 30% and TRP-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.

Thyroid hormone and the D-type cyclins interactin regulating enterocyte gene transcription

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. Coaansfections were carried out using two T3 receptor (TR) isoforms, TRW1 and TRβ-1, as well as plasmids expressing CDl, 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 (TRα-1 = eightfold, TRβ-1 = ninefold), but these effects were dramatically inhibited (>50%) by CD1 and CD3. In contrast, CA and CBl 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, TRW1 = 30% and TRP-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.