Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback

Abstract

Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.

Figure 1

The kynurenine pathway of tryptophan metabolism with the KMO catalytic reaction indicated in blue

Figure 2

Expression of active mitochondrial localised KMO. (a) Cellular staining image indicating mitochondrial localisation of KMO in HEK-KMO(V5-6His) cells obtained using the Opera HCS system with a × 40 water immersion objective (NA 0.9). Antibody-labelled KMO was detected using the 640 nm laser (2000 μW, 40 ms exposure time, emission filter 690/70), nuclear staining was detected using the UV light source (365 nm excitation, 40 ms, emission filter 450/50) and the 488 nm laser (1250 μW, 280 ms, emission filter 520/35) was used to detect the cell membrane stain. The white scale bar corresponds to 10 μm. (b) 3D image of KMO-expressing cells obtained using the Leica SP5C spectral confocal laser scanning microscope. The argon (488 nm) laser was used for detection of mitochondria and the 633 nm laser for detection of KMO confirming co-localisation. The white scale bar corresponds to 10 μm. Steady-state kinetics are shown for KMO at 37 °C, pH 7.0. Starting concentrations of (c) NADPH and (d) l-kynurenine are plotted versus 3HK produced and data fitted to the Michaelis–Menten equation (Y=Bmax*X/(K_d+X) using GraphPad Prism4 software

Figure 3

KMO protects against 3HK-induced cell death. Graphs showing (a) JC-1 readout, n=6 for each cell type and conditions, red:green ratio is significantly decreased (P=0.0498) in wild-type cells incubated with 3HK compared with KMO cells, (b) LDH assay readout, n=6 for each cell type and conditions, LDH release is significantly increased (P=0.0016) in wild-type cells incubated with 3HK compared with KMO cells, (c) caspase 3/7 activity readout, n=6 for each cell type and conditions, caspase activity is increased in wild-type cells incubated with 3HK compared with KMO cells. The S.E.M. is indicated by error bars on all graphs. Data are representative of three independent experiments for each of these assays. (d) Heat maps indicating DiOC6 staining in KMO cells verses wild-type cells with a 3HK-dose and time response in the presence and absence of Z-VAD-FMK, data are mean values of n=2 for each cell type and conditions from one independent experiment

Figure 4

Time-lapse confocal microscopy images of HEK293 cells transiently transfected with 10His-E2-Crimson-KMO (red) incubated with 500 μM 3HK in the presence of Annexin-V-Fluos (green) showing low or non-KMO-expressing cells undergoing apoptosis more rapidly than high-expressing cells. The black scale bar corresponds to 10 μm

Figure 5

Quantification of exogenous 3HK and KMO mRNA levels. (a) Plot showing 3HK concentration remaining in each cell and control incubation plotted against incubation time, n=4, the S.E.M. is indicated by error bars on the plot, data are representative of two independent experiments. (b) Table showing relative quantification of mRNA levels corresponding to each kynurenine pathway enzyme in HEK-KMO and HEK293 cells as determined by RT-qPCR

Figure 6

siRNA knockdown of the kynunenine pathway. (a) Graph showing cell death response indicated by LDH assay, n=2 for each knockdown conditions, the S.E.M. is indicated by error bars on the graph, data are from one independent experiment, knockdown of QPRT causes a significant increase (P=0.0077) in cell death as does knockdown of all four genes (P=0.0194). (b) Table showing the averaged knockdown efficiency for each gene in the cells