PAX3/forkhead homolog in rhabdomyosarcoma oncoprotein activates glucose transporter 4 gene expression in vivo and in vitro.
Increased levels of glucose uptake and increased expression of the glucose transporter (GLUT) genes are characteristic features of tumors. In the muscle-derived tumor alveolar rhabdomyosarcoma (ARMS), a chromosomal translocation t(2:13) generates the PAX3/forkhead homolog in rhabdomyosarcoma (FKHR) oncoprotein. In muscle tissues, glucose transport is primarily mediated by GLUT4. However, the mechanisms that regulate GLUT4 gene expression in tumor tissues are largely unknown. Therefore, we evaluated the role of PAX3/FKHR in the regulation of GLUT4 gene expression in muscle tumorigenesis. GLUT4 mRNA and protein were detected in ARMS-derived human biopsies and in ARMS-derived RH30 myoblasts, which both express the PAX3/FKHR chimeric protein, but not in either C2C12 or embryonal rhabdomyosarcoma-derived myoblasts. GLUT4 was functionally active in RH30 cells, because insulin induced a 1.4-fold stimulation of basal 2-deoxyglucose uptake rates. Coexpression of PAX3/FKHR increased basal transcriptional activity from a GLUT4 promoter reporter (GLUT4-P) in C2C12, SaOS-2, and Chinese hamster ovary-K1 cells in a dose-dependent and tissue-specific manner. PAX3/FKHR mutants with deletions in either the homeodomain (DeltaHD) or the FKHR-derived activation domain (DeltaFKHR), or in which the PAX3-derived paired domain (PD) was point-mutated (PD-R56L), were unable to activate GLUT4-P. Progressive 5'-deletion analysis of GLUT4-P further identified a specific region of the promoter, -66/+163 bp, which retained about 65% of the full transactivation effect. EMSA studies established that the PAX3/FKHR protein directly and specifically binds to this region and to a shorter fragment, -4/+36 bp, that contains potential binding sites for HD and PD, but not to a -4/+36-bp fragment whose HD and PD sites have been mutated. Thus, the functional interaction of PAX3/FKHR with GLUT4-P appears to require all of the functional domains of PAX3/FKHR, as well as a -4/+36-bp region within the GLUT4 promoter. Taken together, the data suggest that the GLUT4 gene is a downstream target of PAX3/FKHR and that GLUT4 is aberrantly transactivated by this oncoprotein both in vivo and in vitro.
Armoni
Michal
M
Institute of Endocrinology, Diabetes and Metabolism, Rambam Medical Center, Technion-Israel Institute of Technology, Haifa 31096, Israel. amichal@tx.technion.ac.il
Quon
Michael J
MJ
Maor
Gila
G
Avigad
Smadar
S
Shapiro
David N
DN
Harel
Chava
C
Esposito
Diana
D
Goshen
Yaacov
Y
Yaniv
Isaac
I
Karnieli
Eddy
E
D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004268:DNA-Binding Proteins / Q000235:genetics / Q000502:physiology*; D005347:Fibroblasts / Q000378:metabolism; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4; D018398:Homeodomain Proteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000235:genetics*; D009124:Muscle Proteins; D009132:Muscles / Q000378:metabolism; D051761:Paired Box Transcription Factors; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D018232:Rhabdomyosarcoma, Alveolar / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; C085126:FOXO1 protein, human; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; C467200:Foxo1 protein, mouse; D051275:Glucose Transporter Type 4; D018398:Homeodomain Proteins; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C491709:PAX4 protein, human; D051761:Paired Box Transcription Factors; C110218:Pax4 protein, mouse; C495692:SLC2A4 protein, human; C495693:Slc2a4 protein, mouse; D014157:Transcription Factors; D008156:Luciferases; D005947:Glucose