Dominant negative mutant forms of the cAMP response element binding protein induce apoptosis and decrease the anti-apoptotic action of growth factors in human islets

Abstract

Aims/hypothesis Transplantation of islets is a viable option for the treatment of diabetes. A significant proportion of islets is lost during isolation, storage and after transplantation as a result of apoptosis. cAMP response element binding protein (CREB) is an important cell survival factor. The aim of the present study was to determine whether preservation of CREB function is needed for survival of human islets. Materials and methods To determine the effects of downregulation of CREB activity on beta cell apoptosis in a transplantation setting, adenoviral vectors were used to express two dominant negative mutant forms of CREB in human islets isolated from cadaveric donors. Markers of apoptosis were determined in these transduced islets under basal conditions and following treatment with growth factor. Results Expression of CREB mutants in human islets resulted in significant (p<0.001) activation of caspase-9, a key regulatory enzyme in the mitochondrial pathway of apoptosis, when compared with islets transduced with adenoviral beta galactosidase. Immunocytochemical analysis showed the activation of caspase-9 to be predominantly in beta cells. Other definitive markers of apoptosis such as parallel activation of caspase-3, accumulation of cleaved poly-(ADP-ribose) polymerase and nuclear condensation were also observed. Furthermore, the anti-apoptotic action of growth factors exendin-4 and betacellulin in human islets exposed to cytokines was partially lost when CREB function was impaired. Conclusions/interpretation Our findings suggest that impairment of CREB-mediated transcription could lead to loss of islets by apoptosis with potential implications in islet transplantation as well as in the mechanism of beta cell loss leading to diabetes.

Anti-inflammatory action of exendin-4 in human islets is enhanced by phosphodiesterase inhibitors: potential therapeutic benefits in diabetic patients

“…8). Previously, we have reported that CREB is needed for the anti-apoptotic effects of exendin-4 [19]. Thus the present study suggests that a combination of exendin-4 and phosphodiesterase 3 and 4 inhibitors could decrease islet inflammation in addition to improving beta cell mass in patients with diabetes.…”

“…8a). CREB, an anti-apoptotic transcription factor that mediates the effects of exendin-4, is phosphorylated by cyclic AMP-activated protein kinase A (PKA) [19]. Therefore we determined the levels of phosphorylated CREB as a measure of increase in cyclic Human islets (2,000 islet equivalents) were pre-incubated with 100 nmol/l of exendin-4 (Ex), 15 μmol/l rolipram (R), 10 μmol/l cilostamide (C), 1 mmol/l DBC, 15 μmol/l Bay 11-7085 (Bay) and 1 μmol/l JAK inhibitor (JI) as indicated for 30 min, followed by exposure to a combination of 2 ng/ml each of IL-1β, TNF-α and IFN-γ (Cyt) for 6 h. Total RNA was isolated and PCR-based expression profiling of genes specific to the inflammatory pathway was carried out *p<0.05, **p<0.01 and ***p<0.001…”

“…The objective of this study was to determine whether exendin-4-mediated generation of cyclic AMP in human islets leads to suppression of cytokineinduced CXCL10 expression. [18,19]. Islets were exposed to IL-1β (50 U/ng), IFN-γ (20 U/ng) and TNF-α (100 U/ng) (Roche Applied Science, Indianapolis, IN, USA), and rolipram and cilostamide (Enzo Life Sciences, Plymouth Meeting, PA, USA) or exendin-4 (Sigma Chemical, St Louis, MO, USA).…”

Exendin-4 protects pancreatic beta cells from palmitate-induced apoptosis by interfering with GPR40 and the MKK4/7 stress kinase signalling pathway

“…The cAMP/PKA/CREB cascade represents a central mechanism of GLP-1 action, specifically when this relates to promotion of beta cell survival [47], even though other signalling mediators may also be implicated [48,49]. Impairment of CREB-mediated transcription led to loss of islets by apoptosis and diminished the antiapoptotic action of exendin-4 in human islets [50]. In line with these results, our previous studies demonstrated that inhibition of TNF-α-induced JNK phosphorylation by exendin-4 was abrogated when cells were preincubated with H89, a specific inhibitor of PKA [15].…”

HER-2/neu Mediates Oncogenic Transformation via Altered CREB Expression and Function

“…So far, initial studies testing the potential to target CREB as a strategy for cancer therapy have focused on approaches that inhibit CREB function by shRNAs or by using dominant-negative CREB mutants, which target the CREB-CBP interaction, thereby preceding the CREB-dependent transcriptional activation of gene expression. Indeed, inhibition of CREB activity by dominant-negative mutants or by shRNAs was associated with a reduced in vitro and in vivo growth potential and increased radiosensitivity (39,43,44). In the HER-2/neu model used, shCREB-mediated inhibition also resulted in decreased proliferation of HER-2/neu þ cells, accompanied by cell-cycle arrest, increased apoptosis, and reduced cell migration and invasion, which was directly associated with an altered expression of proapoptotic and antiapoptotic genes as well as a transcriptional downregulation of MMP-2 and -9 expression.…”