title: Transfection and overexpression of the calcium binding protein calbindin-D28k results in a stimulatory effect on insulin synthesis in a rat beta cell line (RIN 1046-38).
abstract: Calbindin-D28k, a calcium binding protein that is thought to act as a facilitator of calcium diffusion in intestine and kidney, is known to be regulated by vitamin D in these tissues. Calbindin-D28k is also present in pancreatic beta cells, but its function in these cells is not known. To determine a role for calbindin-D28k in the beta cell, rat calbindin-D28k was overexpressed in the pancreatic beta cell line RIN 1046-38 by transfection of calbindin in expression vector, and changes in insulin mRNA were examined. Five transfected RIN cell clones were found to overexpress calbindin 6- to 35-fold as determined by radioimmunoassay. Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones). Overexpressed calbindin was functional because it was capable of buffering calcium in response to a rapid calcium influx induced by 1 and 5 microM calcium ionophore. In cells transfected with calbindin, there was a marked increase in the expression of insulin mRNA (>20-fold for most clones compared with vector transfected cells). Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin content and release (a 5.8-fold increase in insulin release was noted for clone C10, and a 54-fold increase was noted for clone C2). To begin to address the mechanism whereby overexpression of calbindin results in increased insulin gene expression, calbindin-overexpressing clones were transiently transfected with plasmids incorporating various regions of the rat insulin I (rInsI) promoter linked to the chloramphenicol acetyltransferase coding sequence. Transient transfection with reporter plasmids bearing the regulatory sequences of the rInsI promoter (-345/+1) or five copies of the Far-FLAT minienhancer (-247/-198) from the rInsI promoter suggests that increased insulin mRNA in calbindin transfected cells is due, at least in part, to enhanced insulin gene transcription. These studies provide the first direct evidence (to our knowledge) for a role for calbindin in beta cell function.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103, USA.
authors.forename: D
authors.initials: D
authors.lastname: Reddy
authors.forename: A S
authors.initials: AS
authors.lastname: Pollock
authors.forename: S A
authors.initials: SA
authors.lastname: Clark
authors.forename: K
authors.initials: K
authors.lastname: Sooy
authors.forename: R C
authors.initials: RC
authors.lastname: Vasavada
authors.forename: A F
authors.initials: AF
authors.lastname: Stewart
authors.forename: T
authors.initials: T
authors.lastname: Honeyman
authors.forename: S
authors.initials: S
authors.lastname: Christakos
mesh_terms: D000818:Animals; D064092:Calbindin 1; D064026:Calbindins; D000001:Calcimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051272:Glucose Transporter Type 1; D007328:Insulin / Q000096:biosynthesis*; D007340:Insulinoma; D007476:Ionophores / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D011863:Radioimmunoassay; D051381:Rats; D064030:S100 Calcium Binding Protein G / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: C576500:Calb1 protein, rat; D064092:Calbindin 1; D064026:Calbindins; D051272:Glucose Transporter Type 1; D007328:Insulin; D007476:Ionophores; D009004:Monosaccharide Transport Proteins; D064030:S100 Calcium Binding Protein G; D000001:Calcimycin; D015500:Chloramphenicol O-Acetyltransferase