mRNA of the pro-apoptotic gene BBC3 serves as a molecular marker for TNF-α-induced islet damage in humans

Abstract

Aims/hypothesis TNF-α plays important roles in the pathogenesis of type 1 and type 2 diabetes mellitus. In light of this, we examined the involvement of a pro-apoptotic gene, BBC3 (also known as PUMA), in TNF-α-mediated beta cell dysfunction and destruction in human islets. Methods Human islets were exposed in vitro to TNF-α alone or in combination with IFN-γ. Gene expression was assessed by RT-PCR using a set of single islets. Protein abundance and cellular localisation of BBC3 were assessed by immunoblot and immunohistochemistry. A marginal number of islets were transplanted into diabetic NODscid mice to correlate in vivo islet function with BBC3 expression. Results BBC3 and IL8 mRNA were upregulated in TNF-α-stimulated islets in a dose-dependent manner and enhanced through addition of IFN-γ, but not upregulated by IFN-γ alone. Immunohistochemistry revealed that TNF-α in combination with IFN-γ upregulated basal BBC3 abundance in the cytoplasm of beta cells along with the perinuclear clustering of mitochondria partially colocalised with BBC3. TNF-α alone did not induce beta cell death, but did abrogate preproinsulin precursor mRNA synthesis in response to high glucose stimulation, which was inversely associated with upregulation of BBC3 mRNA expression by TNF-α. Higher BBC3 mRNA expression in islets correlated with decreased graft function in vivo. Conclusions/interpretation These results suggest that BBC3 mRNA can serve as a molecular marker to detect early TNF-α-induced beta cell stress and may help identify isletprotective compounds for the treatment of diabetes.

Microglia-derived TNFα induces apoptosis in neural precursor cells via transcriptional activation of the Bcl-2 family member Puma

“…Similar to our findings, it has recently been demonstrated that pro-inflammatory cytokines including TNF α can induce Puma expression via an NF- κ B-mediated pathway in colorectal cancer cells and islet β -cells. 46, 47, 48 NF- κ B has been reported to promote survival or cell death depending on the context. 27, 28 The protective effects of NF- κ B have been attributed to its ability to induce the expression of anti-apoptotic factors such as Bcl-2, Bcl-X L , and cIAP2.…”

Blood concentration of proinflammatory cytokines TNF-α and IL-6 in the Chornobyl NPP accident consequences clean-up workers of iodine period suffering type 2 diabetes mellitus

“…Він визнаний одним із головних цитокінів, які індукують апоптоз інсулінпродукувальних клітин шляхом впливу на проапоптичний ген BBC3 (РИМА), і таким чином відіграє суттєву роль у патогенезі ЦД [5,19,21].…”

Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming

“…Three days prior to transfection, INS-1 cells were cultured in a 24-well plate (Corning Life Sciences, Tewksbury, MA) (70 ϫ 10 3 cells/well) with INS-1 culture media not containing antibiotic-antimycotic. INS-1 cells and islets isolated from the pancreata of Firefly rats were transfected with 50 nM of Bbc3 siRNA or 50 nM of a nontargeting scrambled control siRNA (SMARTpool siRNA, GE Dharmacon), using the DharmaFECT transfection reagent (GE Dharmacon) as described previously (31). The transfection rate was confirmed to be Ͼ80% in the INS-1 cells and Ͼ25% in the islets.…”

“…We have shown previously that BBC3 expression is upregulated in human islets after exposure to recombinant human TNF␣ or in combination with IFN␥, leading to ␤-cell apoptosis through the mitochondrial pathway in vitro (31). High BBC3 mRNA levels in human islets are associated with poor graft function after transplantation in streptozotocin-induced diabetic NOD Scid mice.…”