Figure 1.
Inhibition of cathepsins B and L triggers apoptosis in INS-1 cells.
(A) INS-1 cells were treated with E64d (E, 20 μg/mL) and pepstatin A (P, 20 μg/mL) in 11 mM glucose medium for 48 hr, and caspase activation was monitored by western blotting using antibodies detecting cleaved, active forms of caspases. (B) INS-1 cells were treated with cathepsin B inhibitor (CBi, 20 μM), cathepsin L inhibitor (CLi, 20 μM), and cathepsin K inhibitor (CKi, 20 μM) for 48 hr in 11 mM glucose medium, and caspase activation was monitored by western blotting. (C) INS-1 cells stained with Annexin-V and Hoechst were observed using a fluorescence microscope for quantification of apoptotic cells. Cells were treated with E, CBi, CLi and staurosporine (STS, 0.5 μM) in 11 mM glucose medium. Lysosomal protease inhibitors were added daily, and STS was added 6 hr prior to staining (three independent counts). STS is a known prototypic inducer of apoptosis. *p <0.01, ** p <0.05 compared with 11 mM glucose. The scale bar represents 20 μm.
Figure 2.
Inhibition of cathepsins B and L increases caspase-dependent apoptosis under glucotoxicity.
(A) INS-1 cells were treated with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM), and cathepsin L inhibitor (CLi, 20 μM) in INS-1 medium containing 11 mM or 30 mM glucose. Cell viability was assessed using the CellTiter-BlueⓇ Cell Viability assay and indicated as a percentage of the values measured at each time point (n = 15). Statistical significance was represented as * p <0.01, and *** p <0.001 compared with 11 mM glucose and 30 mM glucose. (B) Protein levels of active caspase-9 (cleaved cas-9), caspase-3 (cleaved cas-3) and Bcl-2 were measured in INS-1 cells cultured in 11 or 30 mM glucose medium for 48 hr. Cells were treated daily with E, CBi, and CLi. (C) Immunoblot analysis of pancreatic islets of SD rats treated with CBi and CLi in 11 mM or 30 mM glucose medium for 48 hr. (D) JNK inhibitor (SP600125, 5 μM) was treated with CBi and CLi in INS-1 cells cultured in 30 mM glucose medium for 24 hr.
Figure 3.
Inhibition of cathepsins B and L leads to LC3 accumulation.
(A) Activity of cathepsins B and L in 30 mM glucose with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM) or cathepsin L inhibitor (CLi, 20 μM) was measured at 24 hr, 48 hr and 72 hr (n = 9). Statistical significance is represented as *** p <0.001 compared with 30 mM glucose. (B) INS-1 cells cultured in 30 mM glucose for 24 or 48 hr were treated daily with E, P, CBi, and CLi. LC3-II accumulation was assessed by immunoblot analysis. (C) Islets from SD rat cultured in 30 mM glucose and treated with CBi, and CLi. Level of LC3 was measured by immunoblot assay. (D) GFP-LC3/INS-1 stable cells were treated daily with E, P, CBi, and CLiin in 30 mM glucose for 48 hr. The graph indicates the number of GFP-LC3 puncta counted among GFP positive cells (n = 5). *** p <0.001 compared with 30 mM glucose. The scale bar represents 5 μm.
Figure 4.
Cathepsin B- and L-knockdown with siRNA enhances apoptosis and impairs autophagy.
INS-1 cells were transfected with scrambled siRNA (con) or with either the cathepsin B (A) or cathepsin L (B) siRNA and cultured in 30 mM glucose for 48 and 72 hr. Immunoblot analysis was performed at the indicated time points post-transfection. Red dashed arrows indicate immature cathepsins, while block solid arrows indicate the mature active form of cathepsins.
Figure 5.
Inhibition of cathepsins B and L results in incomplete processing of cathepsins in lysosome, not in ER.
(A) Immunoblot analysis with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM), and cathepsin L inhibitor (CLi, 20 μM)-treated INS-1 cells in 30 mM glucose for 24 or 48 hr. (B) Immunoblot analysis of pancreatic islets of SD rats treated daily with CBi and CLi in 30 mM glucose for 48 hr. (C) Cytoplasmic and nuclear fractions were prepared from INS-1 cells cultured in 30 mM glucose medium with cathepsin B and L inhibitors for 48 hr then immunoblotted. (D) Localization of pro-cathepsins and cathepsins after treatment with cathepsin B and L inhibitors in 30 mM glucose for 48 hr by cytosol and mito- / lyso- (mitochondria / lysosomal) fractionation. (E) Lysosome-enrichment extraction was prepared and separated by 10% SDS-PAGE. Fraction purity and loading were controlled by immunoblotting for LAMP2 (a lysosomal marker). Red dashed arrows indicate immature cathepsins, while block solid arrows indicate mature cathepsins. (F) Co-localization of cathepsin B (CatB) and L (CatL) with lysotracker was observed in INS-1 cells treated daily with CBi and CLi in 30 mM glucose for 48 hr, as detected by immunofluorescence analysis. (G) Co-localization of CatB / CatL and protein disulfide isomerase (PDI) observed in INS-1 cells treated daily with CBi and CLi in 30 mM glucose for 48 hr. Nuclei were stained with Hoechst 33342 dye. The scale bar represents 5 μm.
Figure 6.
Inhibition of cathepsins B and L causes severe lysosomal dysfunction.
(A) Lysotracker staining was performed in INS-1 cells cultured in 30 mM glucose for 24 or 48 hr, treated daily with E64d (E, 20 μg/mL), cathepsin B inhibitor (CBi, 20 μM), and cathepsin L inhibitor (CLi, 20 μM). Nuclei were stained with Hoechst 33342 dye. (B) INS-1 cells were transfected mRFP-GFP-LC3 construct and then treated with cathepsin B and L inhibitors in 30 mM glucose for 48 hr. 30 mM glucose medium alone increased red and yellow puncta (autolysosome, left panel), whereas treatment with cathepsin inhibitors increased only yellow puncta (autophagosome, middle and right panel). The scale bar represents 5 μm.
Figure 7.
Lysosomal dysfunction by inhibition of cathepsins B and L causes cell death.
In the macroautophagy pathway, impaired organelles are enclosed by a phagophore or isolation-membrane (IM), expansion of which gives rise to the autophagosome, a double-membrane vacuole that engulfs cellular components. Subsequently, the autophagosome fuses with lysosomes, in which lysosomal cathepsins, i.e. cathepsin B, D, L, etc. play a key role to allow normal function of the lysosome. Fusion of autophagosomes with lysosomes forms autolysosomes, playing a role in the degradation of cytoplasmic organelles. Inhibition of cathepsins B and L resulted in the accumulation of abnormal unprocessed cathepsins (pro-cathepsins) in the lysosomes. Abnormal accumulation of pro-cathepsins in the lysosomes leads to impaired autophagic process, especially fusion with autophagosomes, resulting in enlarged lysosomes. This lysosomal dysfunction indicates the phenomenon manifesting in lysosomal storage, finally inducing cell death by the activation of caspases.