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.
OBJECTIVE
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.


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
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.
Sarkar
S A
SA
Barbara Davis Center for Childhood Diabetes and Rocky Mountain Islet Transplantation Program, Aurora, CO, USA.
Gunter
J
J
Bouchard
R
R
Reusch
J E-B
JE
Wiseman
A
A
Gill
R G
RG
Hutton
J C
JC
Pugazhenthi
S
S
D000256:Adenoviridae / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D066259:Betacellulin; D002102:Cadaver; D053453:Caspase 9 / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D016207:Cytokines / Q000494:pharmacology; D000077270:Exenatide; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000494:pharmacology; D014162:Transfection; D014688:Venoms / Q000494:pharmacology
C587851:BTC protein, human; D066259:Betacellulin; D017362:Cyclic AMP Response Element-Binding Protein; D016207:Cytokines; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014688:Venoms; D049452:Green Fluorescent Proteins; D000077270:Exenatide; D053453:Caspase 9; D020169:Caspases