Cathepsin C Regulates Cytokine-Induced Apoptosis in β-Cell Model Systems

Abstract

Emerging evidence suggests that several of the lysosomal cathepsin proteases are genetically associated with type 1 diabetes (T1D) and participate in immune-mediated destruction of the pancreatic β cells. We previously reported that the T1D candidate gene cathepsin H is downregulated by pro-inflammatory cytokines in human pancreatic islets and regulates β-cell function, apoptosis, and disease progression in children with new-onset T1D. In the present study, the objective was to investigate the expression patterns of all 15 known cathepsins in β-cell model systems and examine their role in the regulation of cytokine-induced apoptosis. Real-time qPCR screening of the cathepsins in human islets, 1.1B4 and INS-1E β-cell models identified several cathepsins that were expressed and regulated by pro-inflammatory cytokines. Using small interfering RNAs to knock down (KD) the cytokine-regulated cathepsins, we identified an anti-apoptotic function of cathepsin C as KD increased cytokine-induced apoptosis. KD of cathepsin C correlated with increased phosphorylation of JNK and p38 mitogen-activated protein kinases, and elevated chemokine CXCL10/IP-10 expression. This study suggests that cathepsin C is a modulator of β-cell survival, and that immune modulation of cathepsin expression in islets may contribute to immune-mediated β-cell destruction in T1D.

Characterization of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC-βH5 beta cells

“…By TUNEL assay, we observed a basal cell death rate for EndoC-βH5 cells of around 20%, which may foremost represent the non-viable cell fraction observed following thawing of the cells. This rate is higher as compared to what is typically observed for other beta-cell models, which have basal death rates of 5-10% ( 17 , 44 , 45 ). This may explain the rather modest ~2-fold cytokine effect on cell death observed in EndoC-βH5 cells.…”

“…Preparation of protein lysates, measurements of protein concentrations, and immunoblotting were performed as previously described ( 17 ). Antibodies used were anti-cleaved caspase-7 (#9491S, Cell Signaling, 1:500), anti-major histocompatibility complex (MHC)-I (the human leukocyte antigen (HLA)-A/B/C molecules (#ALX-805-711-C100, Enzo, Farmingdale, NY, USA, 1:2000), anti-phosho-c-Jun N-terminal kinase (P-JNK) (#9252, Cell Signaling, 1:1000), anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) (#J1512, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), anti-phospho-signal transducer and activator of transcription 1 (P-STAT1) (7649S, Cell Signaling, 1:1000), anti-Tubulin (T8203, Sigma-Aldrich, St. Louis, MO, USA, 1:2000), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#9482, Abcam, Cambridge, UK, 1:5000) and secondary HRP-conjugated anti-mouse (#7076, Cell Signaling, 1:1000) or anti-rabbit (#7074, Cell Signaling, 1:2000) IgG antibodies.…”

Characterisation of the functional and transcriptomic effects of pro-inflammatory cytokines on human EndoC-βH5 beta cells

“…By TUNEL assay, we observed a basal cell death rate for EndoC-βH5 cells of around 20%, which may foremost represent the non-viable cell fraction observed following thawing of the cells. This rate is higher as compared to what is typically observed for other beta-cell models, which have basal death rates of 5-10% [15, 30, 31]. This may explain the rather modest ~2-fold cytokine effect on cell death observed in EndoC-βH5 cells.…”

“…Immunoblotting Preparation of protein lysates, measurements of protein concentrations, and immunoblotting were performed as previously described [15]. Antibodies used were anti-cleaved caspase-7 (#9491S, Cell Signaling, 1:500), anti-MHC-I (#ALX-805-711-C100, Enzo, Farmingdale, NY, USA, 1:2000), anti-phosho-c-Jun N-terminal kinase (P-JNK) (#9252, Cell Signaling, 1:1000), anti-nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor α (IκBα) (#J1512, Santa Cruz Biotechnology, Dallas, TX, USA, 1:500), anti-phospho-signal transducer and activator of transcription 1 (P-STAT1) (7649S, Cell Signaling, 1:1000), anti-Tubulin (T8203, Sigma-Aldrich, St. Louis, MO, USA, 1:2000), anti-GAPDH (#9482, Abcam, Cambridge, UK, 1:5000) and secondary HRP-conjugated anti-mouse (#7076) or anti-rabbit (#7074) (Cell Signaling) IgG antibodies.…”

β Cell and Autophagy: What Do We Know?

“…Some of the lysosomal cathepsin proteases are genetically associated with T1D, i.e., cathepsin B (CTSB) and H (CTSH) [130][131][132], and some are modulators of β cell survival. We previously reported that cathepsin C (CTSC) and CTSH have anti-apoptotic functions in β cells [128,133,134], whereas CTSB is pro-apoptotic [63]. Hyperglycemia can lead to excessive ROS levels, leading to oxidative stress and the increased burden of chronically high levels of insulin secretion on the ER, resulting in ER stress and oxidative stress, which autophagy can alleviate to protect the β cells against apoptosis.…”