Requirement of NF-kappaB signalling pathway for modulation of the cholinergic muscarinic M3 receptor expression by INGAP-PP in insulin-producing cells.
The pentadecapeptide comprising the 104-118 amino acid sequence of the ilotropin-derived Reg3-related islet neogenesis-associated protein (INGAP-PP) has been implicated in beta cell neogenesis and enhancement of insulin secretion in pancreatic islets. The aim of this study was to investigate intracellular pathways by which INGAP-PP signals in insulin-producing cells. Treatment with INGAP-PP increased insulin secretion and intracellular calcium levels in MIN6 cells. INGAP-PP exposure activated c-Myc, serum and particularly nuclear factor-kappaB (NF-kappaB) response elements in insulin-producing cells (1.7+/-0.1, 1.8+/-0.1, 2.4+/-0.3 for RINm5F, and 1.3+/-0.1, 1.3+/-0.1 and 1.6+/-0.1 fold for MIN6 cells compared to controls, respectively). There was an increase in the proliferation rate of viable cells (162+/-17% for RINm5F and 155+/-13% for MIN6) that was accompanied by an increase in proliferating cell nuclear antigen (PCNA) protein expression (187+/-19% and 170+/-8% for RINm5F and MIN6 cells respectively) following INGAP-PP treatment. INGAP-PP increased the expression of the muscarinic M(3) receptor subtype (169+/-4% for RINm5F and 222+/-20% for MIN6 cells). Activation of multiple serum response elements by foetal calf serum also increased muscarinic M(3) receptor expression (173+/-9% for RINm5F and 140+/-7% for MIN6 cells). The blockade of NF-kappaB signalling pathway strongly decreased muscarinic M(3) receptor expression in response to both stimuli. In summary, a network of intracellular signals that includes activation of c-Myc signalling pathway and increased PCNA expression might be related to the increased proliferation rate of insulin-producing cells following incubation with INGAP-PP. NF-kappaB signalling plays an essential role in controlling the expression of the muscarinic M(3) receptor.
Paula
Flavia M M
FM
Department of Physiology and Biophysics, Institute of Biology, State University of Campinas, UNICAMP, Campinas 13083-970, Brazil.
Barbosa
Helena C L
HC
Carneiro
Everardo M
EM
Persaud
Shanta J
SJ
Gagliardino
Juan J
JJ
Boschero
Antonio C
AC
Souza
Kleber L A
KL
D000109:Acetylcholine / Q000378:metabolism*; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002217:Carbachol / Q000494:pharmacology; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D016207:Cytokines / Q000737:chemistry / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects*; D055785:Gene Knockdown Techniques; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D000073718:Pancreatitis-Associated Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D015203:Reproducibility of Results; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism; D027602:Serum Response Element / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects*; D051996:Transcription Factor RelA / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
D016207:Cytokines; C500438:INGAP peptide; D000073718:Pancreatitis-Associated Proteins; D010446:Peptide Fragments; D018809:Proliferating Cell Nuclear Antigen; D016271:Proto-Oncogene Proteins c-myc; C000612141:REG3A protein, human; D034741:RNA, Small Interfering; D043587:Receptor, Muscarinic M3; D051996:Transcription Factor RelA; D002217:Carbachol; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D000109:Acetylcholine