Transfection and overexpression of the calcium binding protein calbindin-D _28k results in a stimulatory effect on insulin synthesis in a rat β cell line (RIN 1046-38)

Abstract

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 present in pancreatic ␤ cells, but its function in these cells is not known. To determine a role for calbindin-D 28k in the ␤ cell, rat calbindin-D 28k was overexpressed in the pancreatic ␤ 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 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 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 (؊345͞؉1) or five copies of the Far-FLAT minienhancer (؊247͞؊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 ␤ cell function.Calbindin-D 28k belongs to a family of high affinity calcium-binding proteins that includes calmodulin, parvalbumin, troponin C, and S100 protein (1). Calbindin-D 28k is present in the highest concentrations in avian intestine and in avian and mammalian kidney, brain, and pancreas (2, 3). In intestine and kidney, calbindin is regulated by 1,25-dihydroxyvitamin D 3 . However, in brain calbindin is not influenced by vitamin D status. Various functions have been proposed for calbindin based on its calcium-binding properties, i.e., facilitation of the diffusional flux of calcium in the intestinal enterocyte (4), protection of neurons against excitatory calcium toxicity (5), and action as a mobile calcium buffer restricting evoked calcium signals in nerve synapses and hair cells (6, 7). In the pancreas, early autoradiographic and immunocytochemical studies have localized 1,25-dihydroxyvitamin D 3 receptors (8) and calbindin (9), respectively, to the ␤ cells. It had been suggested that ...

Calbindin-D28k Controls [Ca2+] and Insulin Release

“…Under basal conditions changes in insulin mRNA were also not observed between vector transfected and calbindin-transfected cells (not shown). This differs from calbindin overexpressing high passage RIN 1046-38 cells, which showed a marked increase in insulin mRNA under basal condition when compared with vector transfected cells (19). High passage RIN cells differ from ␤TC cells because they are not glucose-responsive, and the insulin content of RIN cells (0.1% of normal islets) is much lower than the insulin content of ␤TC cells (20 -30% of that of normal islets; 20).…”

“…Although these studies and others (14 -17) established a link between the pancreatic ␤ cell and the vitamin D endocrine system and although the importance of calcium in insulin secretion is well known, there is still little information available concerning the exact mechanism whereby vitamin D may affect ␤ cell function. It has been suggested that the role of vitamin D in calcium metabolism of the ␤ cell may involve a genomic effect of 1,25-(OH) 2 D 3 , including the production of calbindin.Although isolated islets and perfused pancreas from vitamin D-deficient animals have previously been used to study the effects of 1,25-(OH) 2 D 3 on ␤ cell function (14 -17), recently we reported that the ␤ cell line R1N1046-38 contains both calbindin and receptors for 1,25-(OH) 2 D 3 and suggested that ␤ cell lines may provide a useful in vitro system for studying the effects of the vitamin D endocrine system on ␤ cell function (18,19). Although interesting data have been generated in numerous studies using RIN cells, the RIN cell line may not be the best model because these cells have little or no response to glucose and the insulin content of these cells is only approximately 0.1% of the insulin content found in the normal ␤ cell.…”

“…Although isolated islets and perfused pancreas from vitamin D-deficient animals have previously been used to study the effects of 1,25-(OH) 2 D 3 on ␤ cell function (14 -17), recently we reported that the ␤ cell line R1N1046-38 contains both calbindin and receptors for 1,25-(OH) 2 D 3 and suggested that ␤ cell lines may provide a useful in vitro system for studying the effects of the vitamin D endocrine system on ␤ cell function (18,19). Although interesting data have been generated in numerous studies using RIN cells, the RIN cell line may not be the best model because these cells have little or no response to glucose and the insulin content of these cells is only approximately 0.1% of the insulin content found in the normal ␤ cell.…”

Cytosolic Ca2+ Buffers

“…Reported functions for CB-D28k include a role in Ca 2þ resorption in the kidney (Boros et al 2009) and modulation of insulin production and secretion in pancreatic beta cells (Reddy et al 1997;Sooy et al 1999). Data on a putative neuroprotective role against excitotoxicity have not yet resulted in a consistent picture (for a review, see Schwaller et al 2002 andSchwaller 2009).…”

Interaction of estrogen therapy with calcium and vitamin D supplementation on colorectal cancer risk: Reanalysis of Women's Health Initiative randomized trial

“…26,27 However, increase in calbindin consequently decreases intracellular free calcium, 28 with such intracellular free calcium decrease demonstrated to be important for subsequent inhibition of calcium-mediated apoptosis in multiple experimental studies. [29][30][31] Furthermore, in addition to studies indicating calbindin gene overexpression in mice colorectal adenomas, 32 a body of other evidence also indicates calbindin overexpression may stimulate cell growth and proliferation 33 as well as increase insulin levels, 34 which may also have cell proliferative effects for colorectal cancer. [35][36][37] However, modulation of vitamin D's effects by estrogen may also be important.…”