Mutation at position -132 in the islet amyloid polypeptide ( IAPP) gene promoter enhances basal transcriptional activity through a new CRE-like binding site.
OBJECTIVE
Mutations in the islet amyloid polypeptide ( IAPP) gene may play a potential role in the abnormal regulation or expression of the peptide. The aim of this study was to determine the functional role of the -132 G/A mutation reported in the promoter region of the IAPP gene in a population of Spanish Type 2 diabetic patients.


METHODS
We investigated the transcriptional activity using MIN6 cells and luciferase reporter plasmids in several culture conditions. Key regulatory elements of the IAPP promoter region were also analysed by electrophoretic mobility shift assays (EMSA).


RESULTS
The mutant construct doubled IAPP transcriptional activity ( p<0.001). Both constructs showed severely reduced promoter activity (four-fold decrease) in the presence of verapamil and diazoxide. In contrast, IAPP promoter activity was doubled after incubation with forskolin or dexamethasone, regardless of the glucose concentrations in the culture media. EMSA revealed that the -132 G/A mutation increased the binding affinity through two DNA-protein complexes. In addition, a cAMP-responsive element binding protein (CREB) was identified by super-shift EMSA.


CONCLUSIONS
Our studies show that the wild-type and the mutant constructs are regulated in a similar pattern under all conditions, strongly indicating that the -132 G/A mutation increases basal but not inducible transcription. These results may be explained by new binding to the mutant region through CREB and other transcription factors not yet identified.
Novials
A
A
Diabetes Institute Sarda Farriol Foundation, Barcelona, Spain.
Mato
E
E
Diabetes Institute Sarda Farriol Foundation, Barcelona, Spain.|Endocrinology and Diabetes Unit, Department of Medicine, Hospital Clinic, Biomedical Research Institute August Pi Sunyer (IDIBAPS), University of Barcelona, Villarroel 170, 08036, Barcelona, Spain.
Lucas
M
M
Diabetes Institute Sarda Farriol Foundation, Barcelona, Spain.|Endocrinology and Diabetes Unit, Department of Medicine, Hospital Clinic, Biomedical Research Institute August Pi Sunyer (IDIBAPS), University of Barcelona, Villarroel 170, 08036, Barcelona, Spain.
Franco
C
C
Endocrinology and Diabetes Unit, Department of Medicine, Hospital Clinic, Biomedical Research Institute August Pi Sunyer (IDIBAPS), University of Barcelona, Villarroel 170, 08036, Barcelona, Spain.
Rivas
M
M
Biomedical Research Institute Alberto Sols (CSIC), Autonomous University of Madrid, Madrid, Spain.
Santisteban
P
P
Biomedical Research Institute Alberto Sols (CSIC), Autonomous University of Madrid, Madrid, Spain.
Gomis
R
R
Endocrinology and Diabetes Unit, Department of Medicine, Hospital Clinic, Biomedical Research Institute August Pi Sunyer (IDIBAPS), University of Barcelona, Villarroel 170, 08036, Barcelona, Spain. rgomis@clinic.ub.es.
D019943:Amino Acid Substitution; D000682:Amyloid / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D007328:Insulin / Q000235:genetics; D058228:Islet Amyloid Polypeptide; D051379:Mice; D008822:Mice, Transgenic; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D018741:Replication Origin; D014158:Transcription, Genetic / Q000235:genetics*
D000682:Amyloid; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D007328:Insulin; D058228:Islet Amyloid Polypeptide