Nitric oxide synthase-2 regulates mitochondrial Hsp60 chaperone function during bacterial peritonitis in mice

Abstract

Nitric oxide synthase-2 (NOS2) plays a critical role in reactive nitrogen species generation and cysteine modifications that influence mitochondrial function and signaling during inflammation. Here, we investigated the role of NOS2 in hepatic mitochondrial biogenesis during Escherichia coli peritonitis in mice. NOS2(-/-) mice displayed smaller mitochondrial biogenesis responses than Wt mice during E. coli infection according to differences in mRNA levels for the PGC-1 alpha coactivator, nuclear respiratory factor-1, mitochondrial transcription factor-A (Tfam), and mtDNA polymerase (Pol gamma). NOS2(-/-) mice did not significantly increase mitochondrial Tfam and Pol gamma protein levels during infection in conjunction with impaired mitochondrial DNA (mtDNA) transcription, loss of mtDNA copy number, and lower State 3 respiration rates. NOS2 blockade in mitochondrial-GFP reporter mice disrupted Hsp60 localization to mitochondria after E. coli exposure. Mechanistically, biotin-switch and immunoprecipitation studies demonstrated NOS2 binding to and S-nitros(yl)ation of Hsp60 and Hsp70. Specifically, NOS2 promoted Tfam accumulation in mitochondria by regulation of Hsp60-Tfam binding via S-nitros(yl)ation. In hepatocytes, site-directed mutagenesis identified (237)Cys as a critical residue for Hsp60 S-nitros(yl)ation. Thus, the role of NOS2 in inflammation-induced mitochondrial biogenesis involves both optimal gene expression for nuclear-encoded mtDNA-binding proteins and functional regulation of the Hsp60 chaperone that enables their importation for mtDNA transcription and replication.

Figure 1. Hepatic apoptosis and survival after live E. coli peritonitis in mice

E. coli inoculation is 1× 10⁷ CFU. Fig. 1A shows TUNEL staining of liver sections of Wt and NOS2^-/- mice pre and 3 days post fibrin clot implantation. Top row, Wt mice showed occasional scattered small clusters and independent TUNEL-positive cell nuclei (red), while NOS2^-/- mice show many stained nuclei throughout. Bottom images show TUNEL merger with DAPI nuclear staining. Fig. 1B: NOS2^-/- mice showed greater hepatic cleavage of caspase-3 than Wt mice. Fig. 1C: NOS2^-/- mice have a higher 7-day mortality than Wt mice (P<0.05).

Figure 2. NOS2 effect on transcriptional control of mitochondrial biogenesis

Fig. 2A-C: Real time RT-PCR analysis of nuclear-encoded transcriptional activators of mitochondrial biogenesis, NRF-1 and NRF-2, and the PGC-1α co-activator, indicated increases in all three mRNA levels after E. coli challenge in Wt and NOS2^-/- mice. NRF-1 and PGC-1α transcript responses were attenuated and PGC-1α response was late-shifted in NOS2^-/- mice. Fig. 2D&E: Transient elevations of nuclear-encoded transcript levels for Polγ and Tfam, downstream genes involved in mtDNA transcription and replication. In NOS2^-/- mice, transcript levels for these mitochondrial proteins were attenuated compared with the Wt responses (n=4 mice for all time points; * P<0.05 vs. baseline; † P<0.05 Wt vs. NOS2^-/-).

Figure 3. Nuclear Western analysis of NRF-1 and PGC-1α proteins

Nuclear protein levels for upstream regulators of Tfam and Polγ gene expression by Western analysis at 0, 1, 2, and 3 days after E. coli administration in Wt and NOS2^-/- mice (Fig. 3A). Densitometry confirms delayed nuclear enrichment of NRF-1 and PGC-1α in NOS2^-/- compared with Wt mice (Fig. 3B; n= 4 samples at each time point; * P<0.05 vs. baseline; † P<0.05 Wt vs. NOS2^-/- mice).

Figure 4. Molecular and functional responses in liver mitochondria of Wt and NOS2^-/- mice

Fig. 4A: Administration of E. coli decreased hepatic mtDNA copy number at day 1 in both lines with recovery by day 2 in Wt but not in NOS2^-/- mice. MtDNA copy number remained significantly reduced in NOS2^-/- mice at day 3 (P<0.05). Fig. 4B & C: Inter-genotype differences in mtDNA transcription measured by mRNA levels for cytochrome b (Cyt b) and NADH dehydrogenase subunit 1 (ND1) showed significant increases by day 2 for Cyt band ND1 in Wt mice but not in NOS2^-/- mice. Fig. 4D: Box plots of State 3 (peak) respiration rates for succinate (S) and malate + glutamate (M+G) in mitochondria isolated from mice with E. coli peritonitis showed significant expansion of State 3 respiration by day 3 in Wt but not NOS^-/- mice (n=3-4 mice for all time points; * P<0.05 vs. baseline; † P<0.05 Wt vs. NOS2^-/-).

Figure 5. NOS2-dependent association of Hsp60 with mitochondrial Tfam and Polγ

Fig. 5A: Western blots of NOS2 in liver at days 0, 1, 2, and 3 after E. coli challenge. NOS2 increased similarly in total homogenate and in the mitochondrial fraction with a peak response at day 1 (Fig. 5B). Comparable samples from NOS2^-/- mice are provided as negative controls (*P<0.05 vs. baseline).

Figure 6. Mitochondrial Hsp Tfam and Polγ proteins

Tfam and Polγ protein in Wt and NOS2^-/- mouse liver mitochondrial extract by Western blot relative to porin (Fig. 6A). These mitochondrial proteins increased significantly in Wt, but not in NOS2^-/- mice. Mitochondrial Hsp60 and Hsp70 content showed a small increase in Hsp60 relative to porin after E. coli in Wt, but a minimal difference compared with NOS2^-/- mice. Hsp70 levels were stable in both types of mice. Fig. 6B: SOD2 mRNA increased on day 1 after E. coli by 50%, but then returned to baseline in both lines. Mitochondrial SOD2 protein responded with less than a twofold increase in NOS2^-/- and Wt mice. Fig. 6C: Total hepatic mRNA and mitochondrial protein levels for Hsp60 increased comparably experiment-wide in both types of mice. Fig. 6D: Total hepatic mRNA and mitochondrial protein levels for Hsp 70 as in 6C (Densitometry for 6A-D is n= 4 samples per time point; * P<0.05 vs. baseline; † P<0.05 vs. baseline and vs. NOS2^-/-).

Figure 7. Mitochondrial Hsp60 interactions with Tfam and Polγ protein

Fig. 7A: Hepatic Hsp60 total/mitochondrial ratio at day 0, and day 1 and 3 after E. coli peritonitis for both lines of mice. Hsp60 was stable in mitochondria relative to porin and total Hsp60 in the extracts. Fig. 7B: Mitochondrial antioxidant, anti-apoptotic proteins, thioredoxin-2 (Trx2), thioredoxin reductase-2 (TrxR2), and peroxiredoxin-3 (Prx3) increased by Western analysis in Wt mice but failed to respond in NOS2^-/- mice (* P<0.05 vs. baseline; † P<0.05 vs. baseline and vs. NOS2^-/-). In Fig. 7C, NOS2 protein was not detectable in mitochondria pre-challenge, but co-precipitated with mitochondrial Hsp60 at day 1 post E.coli (top gel). Hsp60 co-precipitated with Tfam and Polγ before, but more strongly at 1 day post challenge in Wt mice. In NOS2^-/- mice, the Hsp60 association was unchanged or declined after challenge (second and third gels). Fig. 7D: In contrast, in both lines of mice, mitochondrial Hsp60 co-precipitated weakly with SOD2 (left 4 lanes), while SOD2 co-precipitated strongly with Tfam before and after challenge (right 4 lanes).

Figure 8. Biotin switch assays of mitochondrial Hsp60 and Hsp70 and site-directed mutagenesis of Hsp60

Constitutive S-nitros(yl)ation of both chaperones in Wt and NOS2^-/- mice. SNO protein levels increased in Wt mice after E. coli challenge (Fig. 8A; left panel) but fell in NOS2^-/- mice. In liver mitochondria, the low molecular weight NO donor, CSNO, fully nitros(yl)ated Hsp60 at 15μM and Hsp70 at 50μM CSNO (Fig. 8A middle panel). Equivalent ascorbate-dependent Hsp60- and Hsp70-SNO formation was demonstrated in control Wt and NOS2^-/- hepatic mitochondria (Fig. 8A right panel). Fig. 8B shows studies of flagged wild-type and mutant Hsp60 protein in H4IIE rat hepatocytes. Hsp60 in mitochondria and association with Tfam was demonstrated in cells transfected with flagged native Hsp60 and treated for 24h with LPS+TNF-α to induce NOS2, but neither occurs when ²³⁷Cys is replaced by Ala in mut-Hsp60. Mitochondrial NOS2 and protein loading are shown for comparison.