TRANSFECTION AND OVEREXPRESSION OF THE CALCIUM-BINDING PROTEIN CALBINDIN-D-28K RESULTS IN A STIMULATORY EFFECT ON INSULIN SYNTHESIS IN A RAT BETA-CELL LINE (RIN-1046-38)

Calbindin-D-28k, 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-D-28k is also p resent in pancreatic beta cells, but its function in these cells is no t known. To determine a role for calbindin-D(28)k in the beta cell, ra t calbindin-D28k was overexpressed in the pancreatic beta cell line RI N 1046-38 by transfection of calbindin in expression vector, and chang es in insulin mRNA, were examined, Five transfected RIN cell clones we re found to overexpress calbindin 6- to 35-fold as determined by radio immunoassay, Northern blot analysis revealed increases in abundance in calbindin mRNA (>20-fold for most clones), Overexpressed calbindin wa s functional because it was capable of buffering calcium in response t o a rapid calcium influx induced by 1 and 5 mu M 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 vec tor transfected cells), Besides an increase in insulin mRNA, calbindin overexpression was also associated with an increase in insulin conten t and release (a 5.8-fold increase in insulin release was noted for cl one C10, and a 54-fold increase was noted for clone C2). To begin to a ddress the mechanism whereby overexpression of calbindin results in in creased 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 acet yltransferase coding sequence, Transient transfection with reporter pl asmids bearing the regulatory sequences of the rInsI promoter (-345/+1 ) or five copies of the Far-FLAT minienhancer (-247/-198) from the rIn sI promoter suggests that increased insulin mRNA in calbindin transfec ted cells is due, at least in part, to enhanced insulin gene transcrip tion. These studies provide the first direct evidence (to our knowledg e) for a role for calbindin in beta cell function.