Abstract
We have developed a generic model for in vitro high-throughput screening for agents regulating transcription of genes in the mouse genome here exemplified by Foxc2, a forkhead transcription factor involved in regulation of adipocyte metabolism. We made a Foxc2-LacZ reporter “knock-in” mouse in which one of the two Foxc2 alleles has been inactivated and replaced by a LacZ reporter gene. Mouse embryonic fibroblasts, derived from such mice, were differentiated in vitro to adipocytes and used in cell-based screens. Forskolin as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) increased levels of Foxc2nLacZ fusion protein. We could also demonstrate that this was paralleled by an increase in Foxc2 mRNA, transcribed from the wild type allele. This generic method offers a novel way of identifying both positive and negative upstream regulators of a gene, using high-throughput screening methodology. In a cell-based screen using such methodology we demonstrate efficacy by identifying NKH477 as a Foxc2 activating compound.
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References
Arner P (2006) Visfatin—a true or false trail to type 2 diabetes mellitus. J Clin Endocrinol Metab 91:28–30. doi:10.1210/jc.2005-2391
Capecchi MR (2005) Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 6:507–512. doi:10.1038/nrg1619
Carlsson E, Almgren P, Hoffstedt J et al (2004) The FOXC2 C-512T polymorphism is associated with obesity and dyslipidemia. Obes Res 12:1738–1743. doi:10.1038/oby.2004.215
Carlsson E, Groop L, Ridderstrale M (2005) Role of the FOXC2–512C>T polymorphism in type 2 diabetes: possible association with the dysmetabolic syndrome. Int J Obes Lond 29:268–274. doi:10.1038/sj.ijo.0802876
Cederberg A, Gronning LM, Ahren B et al (2001) FOXC2 is a winged helix gene that counteracts obesity, hypertriglyceridemia, and diet-induced insulin resistance. Cell 106:563–573. doi:10.1016/S0092-8674(01)00474-3
Dagenais SL, Hartsough RL, Erickson RP et al (2004) Foxc2 is expressed in developing lymphatic vessels and other tissues associated with lymphedema-distichiasis syndrome. Gene Expr Patterns 4:611–619. doi:10.1016/j.modgep.2004.07.004
Di Gregorio GB, Westergren R, Enerback S et al (2004) Expression of FOXC2 in adipose and muscle and its association with whole body insulin sensitivity. Am J Physiol Endocrinol Metab 287:E799–E803. doi:10.1152/ajpendo.00155.2004
Farmer SR (2003) The forkhead transcription factor Foxo1: a possible link between obesity and insulin resistance. Mol Cell 11:6–8. doi:10.1016/S1097-2765(03)00003-0
Friedrich G, Soriano P (1991) Promoter traps in embryonic stem cells: a genetic screen to identify and mutate developmental genes in mice. Genes Dev 5:1513–1523. doi:10.1101/gad.5.9.1513
Gronning LM, Cederberg A, Miura N et al (2002) Insulin and TNF alpha induce expression of the forkhead transcription factor gene Foxc2 in 3T3–L1 adipocytes via PI3K and ERK 1/2-dependent pathways. Mol Endocrinol 16:873–883. doi:10.1210/me.16.4.873
Iida K, Koseki H, Kakinuma H et al (1997) Essential roles of the winged helix transcription factor MFH-1 in aortic arch patterning and skeletogenesis. Development 124:4627–4638
Jat PS, Noble MD, Ataliotis P et al (1991) Direct derivation of conditionally immortal cell lines from an H-2Kb-tsA58 transgenic mouse. Proc Natl Acad Sci USA 88:5096–5100. doi:10.1073/pnas.88.12.5096
Kaestner KH, Bleckmann SC, Monaghan AP et al (1996) Clustered arrangement of winged helix genes fkh-6 and MFH-1: possible implications for mesoderm development. Development 122:1751–1758
Kovacs P, Lehn-Stefan A, Stumvoll M et al (2003) Genetic variation in the human winged helix/forkhead transcription factor gene FOXC2 in Pima Indians. Diabetes 52:1292–1295. doi:10.2337/diabetes.52.5.1292
Miura N, Wanaka A, Tohyama M et al (1993) MFH-1, a new member of the fork head domain family, is expressed in developing mesenchyme. FEBS Lett 326:171–176. doi:10.1016/0014-5793(93)81785-X
Osawa H, Onuma H, Murakami A et al (2003) Systematic search for single nucleotide polymorphisms in the FOXC2 gene: the absence of evidence for the association of three frequent single nucleotide polymorphisms and four common haplotypes with Japanese type 2 diabetes. Diabetes 52:562–567. doi:10.2337/diabetes.52.2.562
Pressman CL, Chen H, Johnson RL (2000) LMX1B, a LIM homeodomain class transcription factor, is necessary for normal development of multiple tissues in the anterior segment of the murine eye. Genesis 26:15–25. doi:10.1002/(SICI)1526-968X(200001)26:1<15::AID-GENE5>3.0.CO;2-V
Ridderstrale M, Carlsson E, Klannemark M et al (2002) FOXC2 mRNA Expression and a 5′ untranslated region polymorphism of the gene are associated with insulin resistance. Diabetes 51:3554–3560. doi:10.2337/diabetes.51.12.3554
Smith RS, Zabaleta A, Kume T et al (2000) Haploinsufficiency of the transcription factors FOXC1 and FOXC2 results in aberrant ocular development. Hum Mol Genet 9:1021–1032. doi:10.1093/hmg/9.7.1021
Winnier GE, Hargett L, Hogan BL (1997) The winged helix transcription factor MFH1 is required for proliferation and patterning of paraxial mesoderm in the mouse embryo. Genes Dev 11:926–940. doi:10.1101/gad.11.7.926
Wolfrum C, Shih DQ, Kuwajima S et al (2003) Role of Foxa-2 in adipocyte metabolism and differentiation. J Clin Invest 112:345–356
Yanagisawa K, Hingstrup Larsen L, Andersen G et al (2003) The FOXC2–512C>T variant is associated with hypertriglyceridaemia and increased serum C-peptide in Danish Caucasian glucose-tolerant subjects. Diabetologia 46:1576–1580. doi:10.1007/s00125-003-1213-6
Yang X, Enerback S, Smith U (2003) Reduced expression of FOXC2 and brown adipogenic genes in human subjects with insulin resistance. Obes Res 11:1182–1191. doi:10.1038/oby.2003.163
Zaman GJ (2004) Cell-based screening. Drug Discov Today 9:828–830. doi:10.1016/S1359-6446(04)03241-6
Acknowledgments
The following plasmids were kind gifts; pCH110-nls from Dr Jean-Jacques Panthier, pPGKneobpAlox2PGKDTA from Dr. Arianna Tocchetti, and PGK-cre from Dr. Per Lindblom. This work was supported by the Swedish Research Council (grant K2005-32BI-15324-01A) to S.E., EU grants (QLK3-CT-2002-02149) to S. E. and (LSHM-CT-2003-503041) to S. E., The Arne and IngaBritt Foundation and The Söderberg Foundation.
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Anna Cederberg and Mats Grände have contributed equally to this work.
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Cederberg, A., Grände, M., Rhedin, M. et al. In vitro differentiated adipocytes from a Foxc2 reporter knock-in mouse as screening tool. Transgenic Res 18, 889–897 (2009). https://doi.org/10.1007/s11248-009-9280-1
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DOI: https://doi.org/10.1007/s11248-009-9280-1