NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro.
OBJECTIVE
Using primary cultures of human pancreatic islets, purified human pancreatic beta cells and the mouse beta TC6-F7 cell line, we analysed the expression of nerve growth factor, (NGF/NGF) receptors in beta cells. To investigate whether NGF could sub-serve an autocrine antiapoptotic role in beta cells, we studied the effects of NGF withdrawal using a neutralizing monoclonal anti-NGF antibody.


METHODS
The expression of NGF and NGF receptors (gp140(Trk-A) and p75(NTR)) were analysed by RT-PCR and immunofluorescence. Pulse-chase experiments and beta cell/PC12 co-cultures were used to investigate NGF production and secretion from beta cells. Possible apoptosis induced by NGF withdrawal was monitored by phosphatidylserine translocation, nucleosomal formation, DNA laddering and FACS analysis. Involvement of transcription/translation mechanisms were investigated as well as the gp140(Trk-A) required. Finally, signal transduction pathways typically involved in apoptotic mechanisms were analysed by western blot analysis.


RESULTS
We show that NGF and both NGF receptors, gp140(Trk-A) and p75(NTR) are expressed in beta cells where NGF is produced and secreted in a biologically active form. NGF-withdrawal induces beta-cell transcription/translation independent apoptosis but mediated by gp140(Trk-A). Analysis of signal transduction pathways revealed that NGF withdrawal inhibits the PI3-K, protein kinase B (AKT), Bad survival pathway and activates c-Jun kinase (JNK) whereas ERKs and p38 mitogen-activated protein kinase (MAPK) are not affected. Moreover, Bcl-XL, but not Bcl-2 protein expression are reduced.


CONCLUSIONS
We suggest that the integrity of the NGF/NGF receptor system and NGF bioavailability participate in controlling beta-cell survival in culture which represents a key issue for improving possibilities for transplantations in the treatment of diabetes.
Pierucci
D
D
Laboratory Molecular Medicine, Department of Internal Medicine, University of Rome Tor Vergata, Rome, Italy.
Cicconi
S
S
Bonini
P
P
Ferrelli
F
F
Pastore
D
D
Matteucci
C
C
Marselli
L
L
Marchetti
P
P
Ris
F
F
Halban
P
P
Oberholzer
J
J
Federici
M
M
Cozzolino
F
F
Lauro
R
R
Borboni
P
P
Marlier
L N
LN
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D017209:Apoptosis; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D018920:Coculture Techniques; D053938:DNA Fragmentation; D004789:Enzyme Activation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology* / Q000378:metabolism*; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008875:Middle Aged; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D020932:Nerve Growth Factor / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D009707:Nucleosomes / Q000648:ultrastructure; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D020800:Receptor, Nerve Growth Factor; D020917:Receptor, trkA / Q000032:analysis / Q000235:genetics; D017475:Receptors, Nerve Growth Factor / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; D020932:Nerve Growth Factor; D009707:Nucleosomes; D019869:Phosphatidylinositol 3-Kinases; D010718:Phosphatidylserines; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D020800:Receptor, Nerve Growth Factor; D020917:Receptor, trkA; D017475:Receptors, Nerve Growth Factor; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; C494819:BAD protein, human; C494811:BCL2L1 protein, human; C494820:Bad protein, mouse; C494821:Bad protein, rat; C494812:Bcl2l1 protein, mouse; C494813:Bcl2l1 protein, rat; C494918:AKT1 protein, human; C494920:Akt1 protein, rat