Cloning and characterization of the 5′ flanking region of the sialomucin complex/rat Muc4 gene: promoter activity in cultured cells

2000 ◽  
Vol 349 (2) ◽  
pp. 641-649 ◽  
Author(s):  
Shari A. PRICE-SCHIAVI ◽  
Aymee PEREZ ◽  
Roy BARCO ◽  
Kermit L. CARRAWAY
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Tissue Specific ◽  
Normal Rat ◽  
Hct 116 ◽  
Flanking Region ◽  
Sialomucin Complex ◽  
Muc4 Gene

Sialomucin complex (SMC/Muc4) is a heterodimeric glycoprotein complex consisting of a mucin subunit ascites sialoglycoprotein-1 (ASGP-1) and a transmembrane subunit (ASGP-2), which is aberrantly expressed on the surfaces of a variety of tumour cells. SMC is transcribed from a single gene, translated into a large polypeptide precursor, and further processed to yield the mature ASGP-1/ASGP-2 complex. SMC has complex spatial and temporal expression patterns in the normal rat, suggesting that it has complex regulatory mechanisms. A crude exon/intron map of the 5ʹ regions of the SMC/Muc4 gene generated from clones isolated from a normal rat liver genomic DNA library reveals that this gene has a small first exon comprising the 5ʹ untranslated region and signal peptide, followed by a large intron. The second exon appears to be large, comprising the 5ʹ unique region and a large part (probably all) of the tandem repeat domain. This structure is strikingly similar to that reported for the human MUC4 gene. Using PCR-based DNA walking, 2.4 kb of the 5ʹ-flanking region of the SMC/Muc4 gene was cloned and characterized. Promoter-pattern searches yielded multiple motifs commonly found in tissue-specific promoters. Reporter constructs generated from this 2.4 kb fragment demonstrate promoter activity in primary rat mammary epithelial cells (MEC), the human colon tumour cell line HCT-116, and the human lung carcinoma cell line NCI-H292, but not in COS-7 cells, suggesting epithelial cell specificity. Deletion constructs of this sequence transfected into rat MEC or HCT-116 cells demonstrate greatly varying levels of activity, suggesting that there are positive and negative, as well as tissue-specific, regulatory elements in this sequence. Taken together, these data suggest that the rat SMC/Muc4 promoter has been identified, that it is tissue- (epithelial cell-) specific, and that there are both positive and negative, as well as tissue-specific, regulatory elements in the sequence.

Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo

2007 ◽  
Vol 292 (4) ◽  
pp. C1305-C1312 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Minimal Promoter ◽  
Intestinal Cell ◽  
Epithelial Cell Line ◽  
Hek 293

The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5′ regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.

scholarly journals Contributions of Specificity Protein-1 and Steroidogenic Factor 1 to Adcy4 Expression in Y1 Mouse Adrenal Cells

Endocrinology ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3668-3678 ◽  
Author(s):  
Xianliang Rui ◽  
Jennivine Tsao ◽  
Joshua O. Scheys ◽  
Gary D. Hammer ◽  
Bernard P. Schimmer
Keyword(s):  
Adenylyl Cyclase ◽  
Cell Line ◽  
Promoter Activity ◽  
Mammalian Species ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Deficient Mutant ◽  
Small Interfering Rnas ◽  
Steroidogenic Factor 1 ◽  
Specificity Protein

The type 4 adenylyl cyclase, Adcy4, is the least abundant of five different adenylyl cyclase isoforms expressed in the Y1 mouse adrenocortical cell line and is deficient in a Y1 mutant with impaired steroidogenic factor 1 (SF1) activity. This study examines the contributions of SF1 and other DNA promoter/regulatory elements to Adcy4 expression in the Y1 cell line and its derivative Adcy4-deficient mutant. Primer extension and in silico analyses indicate that Adcy4 transcription initiates from multiple sites just downstream of a GC-rich sequence. Luciferase reporter gene assays identify a 124-bp sequence, situated 19 bp upstream of the major transcription start site and highly conserved among several mammalian species, as the major determinant of Adcy4 expression in Y1 cells and as a site of compromised activity in the Adcy4-deficient mutant. EMSAs using competitor nucleotides and specific antibodies indicate that this conserved region contains three specificity protein (Sp)-1/Sp3-binding sites and one SF1-binding site. As determined by site-specific mutagenesis, the 5′-most Sp1/Sp3-site enhances promoter activity, whereas the middle Sp1/Sp3 and SF1 sites each repress Adcy4 promoter activity. In the Adcy4-deficient mutant, mutating the SF1 site restores Adcy4 promoter activity and knocking down SF1 with small interfering RNAs increases Adcy4 expression, confirming the contribution of SF1 to the mutant phenotype. These studies demonstrate roles for Sp1/Sp3 and SF1 in Adcy4 expression in Y1 cells and establish a repressor function for SF1 in certain promoter contexts.

scholarly journals The 6-Fluoro-8-Methoxy Quinolone Gatifloxacin Down-Regulates Interleukin-8 Production in Prostate Cell Line PC-3

2006 ◽  
Vol 51 (1) ◽  
pp. 162-168 ◽  
Author(s):  
Koh Takeyama ◽  
Hiroaki Mitsuzawa ◽  
Chiaki Nishitani ◽  
Takeyuki Shimizu ◽  
Hitomi Sano ◽  
...  
Keyword(s):  
Cell Line ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Chronic Pelvic Pain Syndrome ◽  
Interleukin 8 ◽  
Pelvic Pain Syndrome ◽  
Prostate Cell ◽  
Prostate Cell Line ◽  
Tnf Α ◽  
Flanking Region

ABSTRACT Fluoroquinolones exhibit immunomodulatory effects on monocytes and macrophages, in addition to their bactericidal activities. It remains unknown even whether the quinolones act directly on the prostate. This study was based on the understanding of the molecular mechanisms of the actions of the fluoroquinolones that can be used for the treatment of chronic prostatitis/chronic pelvic pain syndrome. We investigated whether the 6-fluroro-8-methoxy quinolone gatifloxacin (GFLX) affected the production and secretion of interleukin-8 (IL-8) in the prostate cell line PC-3. GFLX decreased the level of IL-8 release from unstimulated PC-3 cells. GFLX also attenuated IL-8 secretion from PC-3 cells stimulated with peptidoglycan, Mycoplasma hominis, phorbol ester, and tumor necrosis factor alpha (TNF-α), indicating that GFLX exhibits an anti-inflammatory effect on the prostate cell line. However, GFLX failed to alter activation of the NF-κB and AP-1 elicited by these stimulants. GFLX significantly attenuated the expression of IL-8 mRNA in TNF-α-stimulated PC-3 cells and down-regulated the transcriptional activity of the 5′-flanking region of the IL-8 gene from −1481 to +44 bp. The deletion construct without the 5′-flanking region from −1481 to −170 bp but not the construct without the region from −1481 to −188 bp reversed the suppressive effect of GFLX on IL-8 promoter activity. These results demonstrate that GFLX suppresses IL-8 expression in the prostate cell line by decreasing the promoter activity of the IL-8 gene.

Ksp-cadherin gene promoter. I. Characterization and renal epithelial cell-specific activity

1999 ◽  
Vol 277 (4) ◽  
pp. F587-F598 ◽  
Author(s):  
Dilys A. Whyte ◽  
Congyi Li ◽  
R. Brent Thomson ◽  
Stacey L. Nix ◽  
Reza Zanjani ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Binding Sites ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Nuclear Proteins ◽  
Luciferase Reporter ◽  
Rnase Protection ◽  
Renal Epithelial Cells ◽  
Flanking Region

Kidney-specific cadherin (Ksp-cadherin, cadherin 16) is a novel, kidney-specific member of the cadherin superfamily that is expressed exclusively in the basolateral membrane of renal tubular epithelial cells. To characterize the Ksp-cadherin gene promoter, a λ bacteriophage clone containing 3.7 kb of the proximal 5′ flanking region of the mouse Ksp-cadherin gene was isolated. The transcription initiation site was mapped by RNase protection assays and 5′ rapid amplification of cDNA ends, and a 709-bp intron was identified within the 5′ untranslated region. The proximal 5′ flanking region was “TATA-less” but contained other consensus promoter elements including an initiator (Inr), GC boxes, and a CAAT box. Potential binding sites were identified for transcription factors that are involved in tissue-specific gene expression including activator protein-2 (AP-2), hepatocyte nuclear factor-3 (HNF-3), basic helix-loop-helix (bHLH) proteins, CCAAT/enhancer-binding protein (C/EBP), and GATA factors. Transfection of luciferase reporter plasmids containing 2.6 kb of the 5′ flanking region markedly increased luciferase activity in renal epithelial cells (MDCK and mIMCD-3) but not in mesenchymal cells (NIH 3T3 and MMR1). Deletion analysis identified an 82-bp region from −31 to −113 that was essential for promoter activity in transfected renal epithelial cells. Electrophoretic mobility-shift assays showed that mIMCD-3 cells contain nuclear proteins that bind to this region of the promoter. Mutational analysis showed that sequences within the HNF-3 consensus site and CAAT box were involved in protein binding and promoter activity. We conclude that the proximal 5′ flanking region of the mouse Ksp-cadherin gene contains an orientation-dependent promoter that is kidney epithelial cell specific. The region of the promoter from −113 to −31 is required for transcriptional activity and contains binding sites for nuclear proteins that are specifically expressed in renal epithelial cells.

A new stable epithelial cell line (RK-L) from normal rat kidney

1988 ◽  
Vol 252 (2) ◽  
Author(s):  
W. Jelkmann ◽  
U. Schramm ◽  
S. Gie�elmann ◽  
P. Schneede ◽  
F.P. Seydel
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Rat Kidney ◽  
Epithelial Cell Line ◽  
Normal Rat

Molecular mechanisms of epithelial cell-specific expression and regulation of the human anion exchanger (pendrin) gene

2008 ◽  
Vol 294 (5) ◽  
pp. C1261-C1276 ◽  
Author(s):  
Lior Adler ◽  
Edna Efrati ◽  
Israel Zelikovic
Keyword(s):  
Epithelial Cell ◽  
Inner Ear ◽  
Cell Lines ◽  
Anion Exchanger ◽  
Promoter Activity ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Acidic Ph ◽  
Alkaline Ph ◽  
Specific Expression

Pendrin, a Cl−/anion exchanger encoded by the gene PDS, is highly expressed in the kidney, thyroid, and inner ear epithelia and is essential for bicarbonate secretion, iodide accumulation, and endolymph ion balance, respectively. This study aimed to define promoter regulatory elements essential for renal, thyroid, and inner ear epithelial cell-specific expression of human PDS (hPDS) and to explore the effect of ambient pH and aldosterone on hPDS promoter activity. Endogenous pendrin mRNA and protein were detected in renal HEK293, thyroid LA2, and inner ear VOT36 epithelial cell lines, but not in the fibroblast cell line, NIH3T3. A 4.2-kb hPDS 5′-flanking DNA sequence and consecutive 5′-deletion products were cloned into luciferase reporter vectors and transiently transfected into the above cell lines. Distinct differences in expression/activity of deduced positive/negative regulatory elements within the hPDS promoter between HEK293, LA2, and VOT36 cells were demonstrated, with only basal activity in NIH3T3 cells. Acidic pH (7.0–7.1) decreased and alkaline pH (7.6–7.7) increased hPDS promoter activity in transfected HEK293 and VOT36, but not in LA2 cells. Aldosterone (10−8 M) reduced hPDS promoter activity in HEK293 but had no effect in LA2 and VOT36 cells. These pH and aldosterone-induced effects on the hPDS promoter occurred within 96-bp and 89-bp regions, respectively, which likely contain distinct response elements to these modulators. Acidic pH and aldosterone decreased, and alkaline pH increased, endogenous pendrin mRNA level in HEK293 cells. In conclusion, pendrin-mediated HCO3− secretion in the renal tubule and anion transport in the endolymph may be regulated transcriptionally by systemic pH and aldosterone.

scholarly journals Sp1 binding plays a critical role in Erb-B2- and v-ras-mediated downregulation of alpha2-integrin expression in human mammary epithelial cells.

1996 ◽  
Vol 16 (11) ◽  
pp. 6178-6189 ◽  
Author(s):  
J Ye ◽  
R H Xu ◽  
J Taylor-Papadimitriou ◽  
P M Pitha
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Cell Line ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Mammary Epithelial ◽  
Breast Cancers ◽  
Ras Pathway ◽  
Human Mammary Epithelial ◽  
Integrin Gene

The human alpha2-integrin gene is transcriptionally downregulated in a nontumorigenic human mammary epithelial cell line, MTSV1-7, and its clonal variant HB2, overexpressing the Erb-B2 oncogene. In this study, we have used deletion mutations within the alpha2-integrin promoter inserted 5' of the chloramphenicol acetyltransferase or luciferase reporter genes to identify the element that is responsible for the Erb-B2-mediated downregulation. The results of the transient-transfection assay showed that the Sp1 binding element located in the core region (positions --64 to +1) of the alpha2-integrin promoter plays an essential role in the alpha2-integrin promoter activity and its downregulation by Erb-B2. By gel shift assay, we have demonstrated that this element binds with a high degree of affinity not only to Sp1, but also to Sp3. The downregulation of the alpha2-integrin promoter activity could also be achieved by overexpression of v-Hras (v-ras), suggesting that the signals generated by Erb-B2, which lead to downregulation of the alpha2-integrin gene expression, may proceed through the ras pathway. Both the Erb-B2- and the v-ras-overexpressing cells exhibited a Sp1 DNA binding activity lower than that of the parental line, while the relative levels of Sp1 protein in these cells were not altered. The Erb-B2- and v-ras-mediated downregulation could be reversed by the overexpression of Sp1 and by a dominant negative variant of ras (rasN17), confirming the importance of Sp1 and the ras pathway. The inhibitory effects of Erb-B2 on transcriptional activity of the alpha2-integrin promoter were observed in transient-cotransfection assays using alpha2-integrin reporter plasmids and plasmids expressing the Erb-B2 or v-ras oncogene. The same effects were seen when an alpha2-integrin reporter gene construct was transfected into MTSV1-7 or HB2 cells permanently overexpressing Erb-B2 or v-ras. The effects of Erb-B2 or v-ras on the transcriptional activity of the alpha2-integrin promoter were observed in nontumorigenic luminal epithelial cell lines (MTSV1-7 and HB2) as well as in the breast cancer cell line T47D. These data suggest that in luminal epithelial cells and the breast cancers which develop from them, the Erb-B2 proto-oncogene signaling leads to inhibition of (alpha)2(beta)1-integrin gene expression and could contribute to the disruption of tissue architecture seen in breast cancers.

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