Enterocyte differentiation marker intestinal alkaline phosphatase is a target gene of the gut-enriched Krüppel-like factor

2004 ◽  
Vol 286 (1) ◽  
pp. G23-G30 ◽  
Author(s):  
Brian F. Hinnebusch ◽  
Aleem Siddique ◽  
J. Welles Henderson ◽  
Madhu S. Malo ◽  
Wenying Zhang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Interactions ◽  
Target Gene ◽  
Marker Gene ◽  
Luciferase Reporter ◽  
Intestinal Alkaline Phosphatase ◽  
Human Colon Cancer ◽  
Cis Element ◽  
Differentiation Marker ◽  
Enterocyte Differentiation

We have examined the role that the transcription factor gut-enriched Krüppel-like factor (KLF4 or GKLF) plays in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase (IAP). A yeast one-hybrid screen was used to identify proteins interacting with a previously identified cis-element (IF-III) located within the human IAP gene promoter. DNA-protein interactions were determined by using EMSA. Northern blot analysis was used to study RNA expression in human colon cancer RKO cells engineered to overexpress KLF4. Transient transfections with IAP-luciferase reporter constructs were used to characterize the mechanisms by which KLF4 activates IAP transcription. The yeast one-hybrid screen and EMSA identified KLF4 as binding to IF-III. RKO cells induced to overexpress KLF4 demonstrated a corresponding dose-dependent increase in IAP expression, and EMSA with nuclear extract from these cells confirmed that KLF4 binds to the IF-III element. Transient transfections revealed that KLF4 transactivated the IAP gene largely via a critical segment in the IAP promoter that includes the IF-III cis-element. Mutant KLF4 constructs failed to fully activate IAP. We have identified the enterocyte differentiation marker IAP as a KLF4 target gene. IAP transactivation by KLF4 is likely mediated through a critical region located within the proximal IAP promoter region.

Differential regulation of intestinal alkaline phosphatase gene expression by Cdx1 and Cdx2

2005 ◽  
Vol 289 (2) ◽  
pp. G285-G290 ◽  
Author(s):  
Fuad Alkhoury ◽  
Madhu S. Malo ◽  
Moushumi Mozumder ◽  
Golam Mostafa ◽  
Richard A. Hodin
Keyword(s):  
Alkaline Phosphatase ◽  
Transcription Factors ◽  
Mrna Expression ◽  
Marker Gene ◽  
Human Colon ◽  
Luciferase Reporter ◽  
Intestinal Alkaline Phosphatase ◽  
Human Colon Cancer ◽  
Response Element ◽  
Cis Element

We have examined the role that the caudal-related homeobox transcription factors Cdx1 and Cdx2 play in activating the enterocyte differentiation marker gene intestinal alkaline phosphatase ( IAP). Human colon cancer Caco-2 cells were transiently transfected with Cdx1 and/or Cdx2, and semiquantitative RT-PCR was used to study the effects on IAP mRNA expression. Transfections with a variety of IAP-luciferase reporter constructs were used to identify a Cdx response element located within the human IAP gene promoter. Protein-DNA interactions were examined by EMSA. Results showed that Cdx1 markedly induced IAP mRNA expression, whereas Cdx2 did not, and, in fact, inhibited the Cdx1 effects. Functional analysis revealed that Cdx1 transactivates (fourfold, P < 0.05) the IAP promoter through a novel Cdx response element (GTTTAGA) located between −2369 and −2375 upstream of the translational start site. EMSA showed that both Cdx1 and Cdx2 could bind to the cis element, but in cotransfection experiments, Cdx2 inhibited the Cdx1 effects by ∼50%. Thus we have identified a previously unrecognized interaction between two important gut transcription factors, Cdx1 and Cdx2, in the context of IAP gene regulation. Cdx1 activates the IAP gene via a novel cis element, whereas Cdx2 inhibits the Cdx1 effects.

scholarly journals Thyroid Hormone Positively Regulates the Enterocyte Differentiation Marker Intestinal Alkaline Phosphatase Gene via an Atypical Response Element

2004 ◽  
Vol 18 (8) ◽  
pp. 1941-1962 ◽  
Author(s):  
Madhu S. Malo ◽  
Wenying Zhang ◽  
Fuad Alkhoury ◽  
Premraj Pushpakaran ◽  
Mario A. Abedrapo ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Thyroid Hormone ◽  
Intestinal Alkaline Phosphatase ◽  
Response Element ◽  
Differentiation Marker ◽  
Enterocyte Differentiation

Intestinal alkaline phosphatase gene expression is activated by ZBP-89

2006 ◽  
Vol 290 (4) ◽  
pp. G737-G746 ◽  
Author(s):  
Madhu S. Malo ◽  
Moushumi Mozumder ◽  
Xiao Bo Zhang ◽  
Shaluk Biswas ◽  
Alexander Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alkaline Phosphatase ◽  
Transcription Factor ◽  
Fat Absorption ◽  
Rt Pcr ◽  
Intestinal Alkaline Phosphatase ◽  
Reporter Assays ◽  
Differentiation Marker ◽  
Ht 29 ◽  
Enterocyte Differentiation

Intestinal alkaline phosphatase (IAP) is an enterocyte differentiation marker that functions to limit fat absorption. Zinc finger binding protein-89 (ZBP-89) is a Kruppel-type transcription factor that appears to promote a differentiated phenotype in the intestinal epithelium. The purpose of this study was to investigate the regulation of IAP gene expression by ZBP-89. RT-PCR, quantitative real-time RT-PCR, Western blot analyses, and reporter assays were used to determine the regulation of IAP by ZBP-89 in HT-29 and Caco-2 colon cancer cells. ZBP-89 knockdown was achieved by specific short interfering (si)RNA. EMSA and chromatin immunoprecipitation (ChIP) were performed to examine the binding of ZBP-89 to the IAP promoter. The results of RT-PCR, quantitative real-time PCR, and Western blot analyses showed that ZBP-89 was expressed at low levels in Caco-2 and HT-29 cells, whereas IAP was minimally expressed and absent in these cells, respectively. Transfection with ZBP-89 expression plamid increased IAP mRNA and protein levels in both cell lines, whereas knockdown of endogenous ZBP-89 by siRNA reduced basal levels of IAP gene expression in Caco-2 cells. IAP-luciferase reporter assays, EMSA, and ChIP established that ZBP-89 activated the IAP gene through a response element (ZBP-89 response element: 5′-CCTCCTCCC-3′) located between −1018 and −1010 bp upstream of the AUG start codon. We conclude that ZBP-89 is a direct transcriptional activator of the enterocyte differentiation marker IAP. These findings are consistent with the role that this transcription factor is thought to play as a tumor suppressor and suggests its possible function in the physiology of fat absorption.

scholarly journals MiR-27a-3p Targets GLP1R to Regulate Differentiation, Autophagy, and Release of Inflammatory Factors in Pre-Osteoblasts via the AMPK Signaling Pathway

2022 ◽  
Vol 12 ◽  
Author(s):  
Zhi Zeng ◽  
Liangyu Fei ◽  
Juntao Yang ◽  
Jun Zuo ◽  
Zelin Huang ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Target Gene ◽  
Marker Gene ◽  
Luciferase Assay ◽  
Inflammatory Factors ◽  
Luciferase Reporter ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bone Homeostasis ◽  
Downstream Target ◽  
Downstream Target Gene

Objective: Osteoporosis is caused by the dysregulation of bone homeostasis which is synergistically mediated by osteoclasts and osteoblasts. MiR-27a-3p is a key inhibitor of bone formation. Hence, unearthing the downstream target gene of miR-27a-3p is of great significance to understand the molecular mechanism of osteoporosis.Methods: Bioinformatics analysis was utilized to find the downstream target gene of miR-27a-3p, and dual-luciferase reporter assay was conducted to validate the interplay of miR-27a-3p and GLP1R. Besides, qRT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) were employed to verify the impact of miR-27a-3p on GLP1R expression and the differentiation, autophagy, and inflammatory response of MC3T3-E1 pre-osteoblasts.Results: Dual-luciferase assay validated that miR-27a-3p directly targeted GLP1R. Additionally, posttreatment of MC3T3-E1 cells with miR-27a-3p mimics resulted in a remarkable decrease in expression levels of GLP1R, cell differentiation marker gene, autophagy marker gene, and AMPK. These results indicated that miR-27a-3p targeted GLP1R to inhibit AMPK signal activation and pre-osteoblast differentiation and autophagy, while promoting the release of inflammatory factors.Conclusion: The miR-27a-3p/GLP1R regulatory axis in pre-osteoblasts contributes to understanding the molecular mechanism of osteoporosis.

Differentiation-dependent activation of the human intestinal alkaline phosphatase promoter by HNF-4 in intestinal cells

2005 ◽  
Vol 289 (2) ◽  
pp. G220-G226 ◽  
Author(s):  
Line Olsen ◽  
Simon Bressendorff ◽  
Jesper T. Troelsen ◽  
Jorgen Olsen
Keyword(s):  
Alkaline Phosphatase ◽  
Binding Site ◽  
Promoter Sequence ◽  
Computer Assisted ◽  
Intestinal Epithelial ◽  
Intestinal Alkaline Phosphatase ◽  
Cis Element ◽  
Nuclear Extracts ◽  
Brush Border Enzyme ◽  
Translational Start

The intestinal alkaline phosphatase gene ( ALPI) encodes a digestive brush-border enzyme, which is highly upregulated during small intestinal epithelial cell differentiation. To identify new putative promoter motifs responsible for the regulation of ALPI expression during differentiation of the enterocytes, we have conducted a computer-assisted cis-element search of the proximal human ALPI promoter sequence. A putative recognition site for the transcription factor hepatocyte nuclear factor (HNF)-4 was predicted at the positions from −94 to −82 in relation to the translational start site. The ability of HNF-4α to stimulate the expression from the ALPI promoter was investigated in the nonintestinal Hela cell line. Cotransfection with an HNF-4α expression vector demonstrated a direct activation of the ALPI promoter through this −94 to −82 element. EMSA showed that HNF-4α from nuclear extracts of differentiated intestinal epithelial cells (Caco-2) bound with high affinity to the predicted HNF-4 binding site. A 521 bp promoter fragment containing the HNF-4 binding site demonstrated a differentiation-dependent increase in promoter activity in Caco-2 cells. The presence of the HNF-4 binding site was necessary for this increase to occur.

Thyroid hormone positively regulates an enterocyte differentiation marker gene through an atypical response element

2003 ◽  
Vol 124 (4) ◽  
pp. A87
Author(s):  
Madhu S. Malo ◽  
Wenying Zhang ◽  
Mario A. Abedrapo ◽  
Joseph W. Henderson ◽  
Aleem Siddique ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Marker Gene ◽  
Response Element ◽  
Differentiation Marker ◽  
Enterocyte Differentiation

Increased C/EBP in fetal rat small intestine precedes initiation of differentiation marker mRNA synthesis

1997 ◽  
Vol 272 (3) ◽  
pp. G534-G544 ◽  
Author(s):  
R. K. Montgomery ◽  
E. H. Rings ◽  
J. F. Thompson ◽  
C. C. Schuijt ◽  
K. M. Aras ◽  
...  
Keyword(s):  
Small Intestine ◽  
Marker Gene ◽  
Light And Electron Microscopy ◽  
Columnar Epithelium ◽  
Rat Small Intestine ◽  
Intestinal Alkaline Phosphatase ◽  
Carbamoyl Phosphate ◽  
Differentiation Markers ◽  
Fetal Rat ◽  
Differentiation Marker

Morphogenesis, initiation of differentiation marker gene expression, and their correlation with CCAT/enhancer binding protein (C/EBP) expression were analyzed in the developing fetal rat small intestine. Expressions of mRNAs for lactase-phlorizin hydrolase (LPH), intestinal alkaline phosphatase (IALP), carbamoyl-phosphate synthetase (CPS), and three isoforms of C/EBP were simultaneously determined by Northern blot analysis from 15 to 19 days of gestation. At 17 days of gestation, prior to villus formation as demonstrated by light and electron microscopy, only CPS and C/EBPalpha, -beta, and -delta expression could clearly be detected. Both LPH and IALP mRNA were definitely detectable in proximal and middle intestine on day 18, as soon as the stratified epithelium of the early intestine had been transformed into a single layer of columnar epithelium lining villi. This distribution was confirmed by in situ hybridization for LPH mRNA. During the period of transformation when the columnar epithelium and villi were forming, no LPH or IALP mRNA was detectable in the immature distal one-third of the fetal intestine. Preceding villus morphogenesis, immunostaining demonstrated nuclear localization of C/EBPalpha protein in intestinal epithelial cells, with continued expression in all enterocytes through 19 days of gestation. Enhanced expression of C/EBPalpha mRNA and protein began 24 h prior to the initiation of the differentiation markers, suggesting that it may play a role in regulation of fetal intestinal differentiation.

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