Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression.

Henthorn, Paula S.; Zervos, Paula; Raducha, Michael; Harris, Harry; Kadesch, Tom

doi:10.1073/pnas.85.17.6342

Cited by 145 publications

(88 citation statements)

References 18 publications

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“…BHK-21 and InR1G9 cells were transfected by the calcium phosphate precipitation and the DEAE-dextran method, respectively [29,30]. We added pSV2A pap to monitor transfection efficiency [31]. Expression vectors contained the rat HNF3a (R. Costa, University of Illinois, Chicago, Ill., USA), hamster cdx-2/3 (M. S. German, University of California, San Francisco, Calif., USA), quail pax-6 (S. Saule, Institut Curie, Orsay cedex, France), and mouse pax-4 cDNA cloned in pSG5 (Stratagene) as well as rat brn-4 (M. A. Hussain, Harvard Medical School, Boston, Mass, USA) and p300 (S. Saule, Institut Curie, Orsay Cedex, France) in pCMV.…”

Section: Methodsmentioning

confidence: 99%

The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6

et al. 2002

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The endocrine pancreas is organised as islets of Langerhans embedded in the exocrine tissue and is composed of four cell types: beta, a, d, and g cells producing insulin, glucagon, somatostatin and the pancreatic polypeptide (PP), respectively. Gene knockout studies have shown the involvement of a variety of transcription factors in pancreas development. Null mutations of the homeodomain proteins pdx-1 and isl-1 result in pancreatic agenesis and the absence of the islets of Langerhans, respectively [1,2]. The lack of Beta2, Pax-6, Pax-4, Nk 2.2, and Nk 6.1 affects the organisation of the endocrine pancreas and the number of endocrine cells or both [3±9]. The formation of specific islet cell types can be affected by the absence of any of these factors: no or few glucagon-producing a cells are observed in homozygous pax-6 mutants, whereas pax-4 knockout mice lack beta and d cells; nk 2.2 and nk 6.1 mu-The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6 Abstract Aims/hypothesis. The paired-homeobox genes pax-4 and pax-6 are crucial for islet development; whereas the null mutation of pax-6 results in the nearly absence of glucagon-producing a cells, pax-4 homozygous mutant mice lack insulin and somatostatin-producing beta and d cells but contain an increased number of a cells suggesting that a cells could develop by a default mechanism. Methods. To investigate whether beta-cell specific factors act negatively on glucagon gene transcription, we ectopically expressed pax-4 in glucagon producing InR1G9 cells; Pax-4 inhibited basal transcription of the glucagon gene promoter by 60 %. To assess the mechanism of this inhibition, we cotransfected the non-islet cell line BHK-21 with Pax-4 and various transcription factors present in a cells.Results. In addition to a general repressor activity on basal glucagon gene promoter activity of 30±50 %, a specific 90 % inhibition of Pax-6 mediated transactivation was observed. In contrast, Pax-4 had no effect on Cdx-2/3 or HNF3a mediated transcriptional activation. Pax-4 showed similar affinity to the Pax-6 binding sites on the glucagon gene promoter compared to Pax-6, but varying with KCl concentrations. Conclusion/interpretation. Pax-4 impairs glucagon gene transcription specifically through inhibition of Pax-6 mediated transactivation. Transcriptional inhibition seems to be mediated by direct DNA binding competition with Pax-6 and potentially additional mechanisms such as protein-protein interactions and a general repressor activity of Pax-4. Glucagon gene expression in a cells could thus result from both the presence of islet cell specific transcription factors and the absence of Pax-4. [Diabetologia (2002) 45: 97±107]

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Section: Methodsmentioning

confidence: 99%

The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6

et al. 2002

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“…BHK-21 cells were transfected by the calcium phosphate precipitation technique (31) and InR1G9 cells by the DEAE-dextran method (32). pSV2A pap encoding the placental alkaline phosphatase was added to monitor transfection efficiency in BHK-21 cells (33). Cell extracts were prepared 48 h after transfection and analyzed for CAT and alkaline phosphatase activities as described previously (25).…”

Section: Methodsmentioning

confidence: 99%

The Paired Homeodomain Transcription Factor Pax-2 Is Expressed in the Endocrine Pancreas and Transactivates the Glucagon Gene Promoter

Ritz-Laser¹,

Estreicher²,

Gauthier³

et al. 2000

Journal of Biological Chemistry

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Glucagon gene expression is controlled by at least four DNA elements within the promoter; G2, G3, and G4 confer islet-specific expression, while G1 restricts glucagon transcription to ␣ cells. Two islet-specific complexes are formed on G3, the insulin-responsive element of the glucagon gene; one of these corresponds to the paired homeodomain protein Pax-6, a major glucagon gene transactivator that plays a crucial role in ␣ cell development. We describe here the identification of the second complex as Pax-2, another member of the paired box family. Pax-2 is known to be crucial for the development of the urogenital tract and of the central nervous system, but its presence in the endocrine pancreas has not been reported. We detected Pax-2 gene expression by RT-PCR; in islets, Pax-2 is present as two alternative splicing isoforms, Pax-2A and Pax-2B, whereas in the glucagon-and insulin-producing cell lines ␣TC1 and Min6, a distinct isoform, Pax-2D2, is found in addition to Pax-2B. Both islet-specific isoforms bind to the enhancer element G3 and to the ␣-specific promoter element G1 that also interacts with Pax-6. Pax-2A and Pax-2B dose-dependently activate transcription from the G3 and the G1 elements both in heterologous and in glucagon-producing cells. Our data indicate that Pax-2 is the third paired domain protein present in the endocrine pancreas and that one of its roles may be the regulation of glucagon gene expression.

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“…The cells were harvested 48 hours after transfection, and lysed with Cell Lysis Buffer (Promega). For normalizing efficiency, we cotransfected the vector pSV2Apap, containing the human placental alkaline phosphatase gene (34,35).…”

Section: Methodsmentioning

confidence: 99%

Transcriptional regulation of the HOX4C gene by basic fibroblast growth factor on rheumatoid synovial fibroblasts

Xue

Hasunuma

Asahara

et al. 1997

Arthritis & Rheumatism

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Objective. To examine the expression of genes of the HOX D cluster in the synovial tissue of patients with rheumatoid arthritis (RA), and to determine whether basic fibroblast growth factor (bFGF) influences the expression and transcriptional regulation of the gene.Methods. The expression of genes of the HOX D cluster, including HOX4C, HOX4D, HOX4H, and HOX41, was determined in the synovium of 4 patients with RA and 4 with osteoarthritis (OA) by in situ reverse transcription (RT) and RT-polymerase chain reaction (RT-PCR). The induction of HOX4C messenger RNA (mRNA) by bFGF was determined by RT-PCR. The binding activity of a transcriptional regulator of the HOX4C gene, C2, was analyzed by the mobility shift assay. NIH-3T3 cells transfected with a construct containing C2 binding sequence were incubated with bFGF, and the activity of the reporter was measured by luciferase assay.Results. Using an in situ RT assay, specific expression of HOX4C mRNA was detected in 3 of 4 RA synovial samples, whereas none of the OA synovia expressed HOX4C. HOX4D, HOX4H, and HOX41 genes were expressed in all synovial samples from RA and OA

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Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression.

Abstract: The human placental alkaline phosphatase gene has been cloned and reintroduced into mammalian cells.

Cited by 145 publications

References 18 publications

The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6

The pancreatic beta-cell-specific transcription factor Pax-4 inhibits glucagon gene expression through Pax-6

The Paired Homeodomain Transcription Factor Pax-2 Is Expressed in the Endocrine Pancreas and Transactivates the Glucagon Gene Promoter

Transcriptional regulation of the HOX4C gene by basic fibroblast growth factor on rheumatoid synovial fibroblasts

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