Cis-regulatory elements involved in species-specific transcriptional regulation of the SVCT1 gene in rat and human hepatoma cells.
Ascorbic acid is transported into cells by the sodium-coupled vitamin C transporters (SVCTs). Recently, we obtained evidence of differential regulation of SVCT expression in response to acute oxidative stress in cells from species that differ in their capacity to synthesize vitamin C, with a marked decrease in SVCT1 mRNA and protein levels in rat hepatoma cells that was not observed in human hepatoma cells. To better understand the regulatory aspects involved, we performed a structural and functional analysis of the proximal promoter of the SVCT1 rat gene. We cloned a 1476-bp segment containing the proximal promoter of the rat SVCT1 gene and generated deletion-derived truncated promoters of decreasing sizes and mutant promoters by modification of consensus binding sites for transcription factors by site-directed mutagenesis. We next analyzed their capacity to direct the transcription of a reporter gene after transfection into rat H4IIE and human HepG2 hepatoma cells, in experiments involving the coexpression of transcription factors whose consensus binding sequences are present in the SVCT1 promoter. This analysis revealed the presence of two critical cis-regulatory elements of the transcriptional activity of the rat SVCT1 gene promoter, sites containing consensus sequences for the binding of the transcription factors Bach1 and HNF4 that are not present in equivalent locations in the human SVCT1 gene promoter. Moreover, a consensus site for HNF1 that is crucial for the regulation of the human SVCT1 promoter is present in the SVCT1 rat promoter but has no effect on its transcriptional activity. These findings imply that regulation of vitamin C metabolism in the rat, a species with the capacity to synthesize large amounts of ascorbic acid, may differ from that of humans, a species that must obtain ascorbic acid from the diet through a transport mechanism that depends on proper SVCT1 expression.
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Alejandra
A
Muñoz
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Marcelo
M
Villagrán
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Paula
P
Guzmán
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Carlos
C
Solíz
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Marcell
M
Gatica
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Carlos
C
Aylwin
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Karen
K
Sweet
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Mafalda
M
Maldonado
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Elizabeth
E
Escobar
Instituto de Bioquímica y Microbiología, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja, Valdivia, Chile.
Alejandro M
AM
Reyes
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Jorge R
JR
Toledo
Departamento de Farmacología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Oliberto
O
Sánchez
Departamento de Especialidades Médicas, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile.
Sergio A
SA
Oñate
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile. Electronic address: juvera@udec.cl.
Juan
J
Carlos Vera
Departamento de Fisiopatología, Facultad de Medicina, Universidad de Concepción, Concepción, Bio-Bio 4070386, Chile. Electronic address: corivas@udec.cl.
Coralia I
CI
Rivas
D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D060347:Sodium-Coupled Vitamin C Transporters / Q000235:genetics*; D013045:Species Specificity
C555162:SLC23A1 protein, human; C555166:Slc23a1 protein, rat; D060347:Sodium-Coupled Vitamin C Transporters
Antioxidants; Ascorbic acid transporter 1 promoter; Free radicals; SLC23A1 gene promoter; SVCT1 promoter; SVCT1 transcriptional regulation; Species-specific transcription