Evidence of calcium-dependent pathway in the regulation of human beta1,3-glucuronosyltransferase-1 (GlcAT-I) gene expression: a key enzyme in proteoglycan synthesis.
The importance of heparan- and chondroitin-sulfate proteoglycans in physiological and pathological processes led to the investigation of the regulation of beta1,3-glucuronosyltransferase I (GlcAT-I), responsible for the completion of glycosaminoglycan-protein linkage tetrasaccharide, a key step prior to polymerization of chondroitin- and heparan-sulfate chains. We have cloned and functionally characterized GlcAT-I 5'-flanking regulatory region. Mutation analysis and electrophoretic mobility shift assays demonstrated the importance of Sp1 motif located at -65/-56 position in promoter activity. Furthermore, we found that elevation of intracellular calcium concentration by the calcium ionophore ionomycin stimulated GlcAT-I gene expression as well as glycosaminoglycan chain synthesis in HeLa cells. Bisanthracycline, an anti-Sp1 compound, inhibited GlcAT-I basal promoter activity and suppressed ionomycin induction, suggesting the importance of Sp1 in calcium induction of GlcAT-I gene expression. Nuclear protein extracts from ionomycin-induced cells exhibited an increased DNA binding of Sp1 factor to the consensus sequence at position -65/-56. Signaling pathway analysis and MEK inhibition studies revealed the important role of p42/p44 MAPK in the stimulation of GlcAT-I promoter activity by ionomycin. The present study identifies, for the first time, GlcAT-I as a target of calcium-dependent signaling pathway and evidences the critical role of Sp1 transcription factor in the activation of GlcAT-I expression.
Barré
Lydia
L
UMR CNRS 7561-Université Henri Poincaré Nancy 1, Faculté de Médecine, BP 184, Vandoeuvre-lès-Nancy 54505, France.
Venkatesan
Narayanan
N
Magdalou
Jacques
J
Netter
Patrick
P
Fournel-Gigleux
Sylvie
S
Ouzzine
Mohamed
M
D001665:Binding Sites; D002118:Calcium / Q000502:physiology*; D002809:Chondroitin Sulfates / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D005786:Gene Expression Regulation; D014453:Glucuronosyltransferase / Q000235:genetics*; D006367:HeLa Cells; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011509:Proteoglycans / Q000096:biosynthesis; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000378:metabolism*
D002118:Calcium; D002809:Chondroitin Sulfates; D004247:DNA; D014453:Glucuronosyltransferase; D006497:Heparan Sulfate; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D011509:Proteoglycans; D016329:Sp1 Transcription Factor; C067806:glucuronyltransferase GlcAT-1