PKCβ regulates ischemia/reperfusion injury in the lung (original) (raw)
Effect of PKC β on the consequences of lung I/R: enhanced survival and maintenance of vascular homeostatic mechanisms. PKC_β_–/– mice in the C57BL/6 background were subjected to single-lung ischemia for 1 hour followed by reperfusion for 3 hours. Following this treatment was a test period of 1 hour during which time reperfusion to the uninstrumented lung was blocked, rendering the animal dependent on the I/R lung. Compared with PKC_β+/+_ controls, deletion of the PKC_β gene conferred enhanced survival of about threefold (survival was 67% in PKC_β_–/– mice and 22% in PKC_β+/+ mice; P = 0.03; Figure 1A). In parallel, leukocyte accumulation in I/R tissue, an important indication of impending tissue damage, was decreased based on assessment of myeloperoxidase activity (Figure 1B). Pulmonary fibrin deposition, reflecting the balance of procoagulant and anticoagulant/fibrinolytic mechanisms in the injured lung, was significantly decreased in PKC_β_–/– mice compared with PKC_β+/+_ mice, based on immunoblotting of lung extracts with an antibody directed to a fibrin neoepitope (P = 0.003, Figure 1C). Consistent with the apparently protective phenotype in PKC_β_–/– mice subjected to lung I/R, WT mice fed the PKCβ inhibitor ruboxistaurin (LY333531) that were subjected to lung I/R also displayed increased survival (about threefold; P = 0.035; Figure 1D). Taken together, these data indicate that deletion or blockade of PKCβ resulted in maintenance of vascular homeostatic mechanisms.
Murine model of lung ischemia/reperfusion (I/R): effect of PKCβ. (A and D) Survival analysis. PKC_β+/+_ and PKC_β_–/– (A) and PKC_β+/+_ mice fed vehicle chow or ruboxistaurin chow (D), respectively, were subjected to left-lung ischemia for 1 hour and reperfusion for 3 hours. Blood flow to the uninstrumented right lung was then blocked, and mortality was determined after 1 hour with only the left lung in the circulation. (B) Myeloperoxidase activity. After I/R, lung samples were harvested from PKC_β+/+_ and PKC_β_–/– mice and subjected to myeloperoxidase activity assay (n = 5). (C) After left-lung I/R as described above, animals received systemic heparin and were sacrificed. Lung protein extract was digested with plasmin and subjected to SDS-PAGE (7.5%; 0.2 ∝g of total protein/lane). Immunoblotting with anti-fibrin antibody was performed. Data are shown as mean ± SEM of five experiments.
Activation of PKC β II in response to lung I/R. When WT C57BL/6 mice were subjected to I/R, rapid translocation of PKCβII to the cell membrane was observed, consistent with its activation. An increase of PKCβII associated with the membrane fraction reached an apparent maximum after 1 hour of ischemia and 10–15 minutes of reperfusion (Figure 2A). These studies were performed using an antibody selective to the PKCβII isoform. In contrast, immunoblotting with an antibody specific to the PKCβI isoform showed no change (not shown). Next, we examined the patterns of two other isoforms of PKC linked to I/R, PKCδ and PKCε. PKCδ and PKCε antigens in the membranous fraction from PKC_β+/+_ and PKC_β_–/– mice showed no changes resulting from 15 minutes of reperfusion after 1 hour of ischemia (Figure 2, B and C), thus confirming that the activated principal isoform of PKC relevant to I/R injury in the lung was the PKCβII isoform.
I/R-mediated activation of PKCβII in the lung. PKC_β+/+_ and PKC_β_–/– mice were subjected to left-lung ischemia for 1 hour and reperfusion for 10–15 minutes. Membranous fractions were prepared from I/R and uninstrumented lung, and were subjected to SDS-PAGE (7.5%, 5 ∝g of total protein/lane) and immunoblotting with antibody to PKCβII (A), PKCδ (B), and PKCε (C).
Activation of PKC β II upregulates expression of downstream MAPKs. In view of the observed close relationship between environmental stress and PKCβII translocation, we considered what impact deletion of PKCβII in I/R might have on MAPKs ERK1 and ERK2 (p44 and p42), which are indirect downstream targets of this PKC isoform (9, 10, 22). Immunoblotting of extracts from I/R lung harvested from PKC_β+/+_ mice displayed an approximately eightfold increase in intensity of phospho-p44/42 in samples from I/R lung compared with samples from uninstrumented lung (Figure 3A). In comparison, PKC_β_–/– mice displayed an increase of only about threefold in phosphorylation of p44/42 in I/R lung compared with tissue from uninstrumented lung (Figure 3A). Levels of total p44/42 antigen did not change in any of these experiments (Figure 3A). Immunostaining with antibody to phospho-p44/42 demonstrated increase of this epitope in the lung from PKC_β+/+_ mice subjected to I/R (Figure 3C), compared with the uninstrumented lung, in which it was undetectable. However, in PKC_β_–/– mice, there was a slight increase in immunoreactive phospho-p44/42 in I/R lung (Figure 3E) that was similar to that of immunoreactive phospho-p44/42 in the lungs of uninstrumented PKC_β_–/– mice (Figure 3D). In addition, immunofluorescence of PKC_β+/+_ I/R lung sections stained with anti–phospho-p44/42 and F4/80 IgG demonstrated that phospho-p44/42 antigen was localized predominantly in MPs. Figure 3F demonstrates staining with anti–phospho-p44/42 kinase (red), and Figure 3G demonstrates staining using anti-F4/80 IgG (green) to identify MPs. Figure 3H reveals the merge and indicates that phospho-p44/42 MAPK is, at least in part, expressed in MPs in the I/R lung.
Ischemia/reperfusion-mediated activation of MAPKs in the lung. PKC_β+/+_ and PKC_β_–/– mice underwent the indicated period of left-lung I/R. Animals were sacrificed and protein extracts from the I/R and uninstrumented lung were prepared and subjected to SDS-PAGE (12%, 50 ∝g of protein/lane). Immunoblotting with phospho-p44/42 MAPK antibody or total p44/42 MAPK antibody (B), phospho-p38 MAPK antibody, or total p38 MAPK antibody (I), and phospho-SAPK/JNK antibody or total SAPK/JNK antibody (J) was performed. Data are shown as mean ± SEM of five experiments. Immunohistochemical analysis of phospho-p44/42 expression in murine lung from uninstrumented (B) or I/R (C) PKC_β+/+_ mice, and from uninstrumented (D) or I/R (E) PKC_β_–/– mice was performed. Scale bar: 50 ∝m. I/R lungs from PKC_β+/+_ mice were subjected to immunofluorescence microscopy and double stained with an anti–phospho-p44/42 antibody (red) (F) and an anti-macrophage antibody (F4/80, green) (G). The merging of F (pERK1/2) and G (F4/80) is shown in H. Arrows in F and arrowheads in G indicate dually stained cells Original magnification in F–H, ∞1,000. *P < 0.001; **P = 0.0041.
These data led us to consider the effect of deleting PKCβ on other MAPKs, each of which has been implicated in inflammatory responses to cell stress. Immunoblotting of lung extracts was performed with an antibody selective for phospho-p38 (Figure 3I). An about 13-fold increase in intensity of the phospho-p38 band was observed in I/R lung from PKC_β+/+_ mice compared with uninstrumented lung from PKC_β+/+_ mice (Figure 3I). Immunohistochemical studies demonstrated that increased phospho-p38 was present predominantly in MPs following I/R of PKC_β+/+_ mouse lung, compared with uninstrumented controls, in which it was undetectable (not shown). However, in PKC_β_–/– mice, phosphorylation of p38 after I/R was suppressed (about fourfold) compared with PKC_β+/+_ mice undergoing I/R (Figure 3I). Similarly, there was an increase in intensity of about 13-fold of the phospho-SAPK/JNK band in extracts from I/R lung compared with the uninstrumented lung from PKC_β+/+_ mice (Figure 3J). In contrast, the increase in phospho-SAPK/JNK band was considerably blunted in I/R lung from PKC_β_–/– animals (∼2.5-fold, compared with uninstrumented control PKC_β_–/– mice). In addition, immunohistochemical studies demonstrated that increased phospho-SAPK/JNK were present predominantly in MPs (not shown). Taken together, these data show decreased activation of phospho-p44/42, phospho-p38, and phospho-SAPK/JNK of about 70% (P < 0.001), 77% (P = 0.004), and 75% (P < 0.001), respectively, in I/R lung from PKC_β_–/– compared with I/R lung from PKC_β+/+_ mice.
Effect of PKC β gene deletion on targets of MAPKs. Since expression of Egr-1 has been linked to PKCβ-dependent MAPK activity in global hypoxia (9, 10), we tested the role of PKCβ in modulating expression of Egr-1 specifically in I/R. Transcripts for Egr-1 were increased by 18- to 20-fold in I/R lung from PKC_β+/+_ mice compared with Egr-1 transcripts from uninstrumented PKC_β+/+_ animals (Figure 4, A and B). In contrast, I/R in PKC_β_–/– mice (Figure 4A) or WT mice fed PKCβ inhibitor ruboxistaurin (Figure 4B) caused only a two- to threefold increase in Egr-1 mRNA transcripts. At the protein level, immunohistochemical analysis for Egr-1 antigen demonstrated a strong increase of antigen in lungs from PKC_β+/+_ mice subjected to I/R (Figure 4D) compared with uninstrumented controls (Figure 4C). Immunoreactivity for Egr-1 antigen was largely expressed in MPs, as demonstrated by using immunofluorescence to colocalize Egr-1 in MPs using antibodies to Egr-1 (Figure 4G, red) and anti-F4/80 IgG (Figure 4H, green). Figure 4I represents the merge, indicating that Egr-1 was principally expressed in MPs in PKC_β+/+_ I/R lung. In contrast to these findings in PKC_β+/+_ mice, in PKC_β_–/– mice there was only a slight increase in immunoreactive Egr-1 in I/R lung (Figure 4F), which was not significantly different from that observed in uninstrumented lung retrieved from PKC_β_–/– mice (Figure 4E).
I/R induces Egr-1 and procoagulant and proinflammatory molecules in the lung: effect of PKCβ. Mice underwent left lung I/R or no instrumentation. Mice were sacrificed and total RNA was isolated from the lung and subjected to Northern analysis (20 ∝g/lane) with 32P-labeled cDNA probes for Egr-1 (A), tissue factor (TF) (J), PAI-1 (K), IL-1β (L), MIP-2 (M), ICAM-1 (N), or β-actin (as internal control). Real-time PCR analysis of total RNA for Egr-1 expression was performed on I/R and uninstrumented lungs from WT mice fed vehicle or ruboxistaurin chow (B). Immunohistochemical analysis of Egr-1 expression in murine lung from uninstrumented (C) or I/R (D) PKC_β+/+_ mice and from uninstrumented (E) or I/R (F) PKC_β_–/– mice. Ctrl, control. Scale bar in F: 50 ∝M. I/R lungs from PKC_β+/+_ mice were immunofluorescence double stained with an anti–Egr-1 antibody (red) (G) and an anti-MP antibody (F4/80, green) (H). The merging of G (Egr-1) and H (F4/80) is shown in I. Arrows in G and arrowheads in H indicate dually stained cells. Original magnification in G–I, ∞1,000. The units for the y axes of A, B, and J–N are fold increase.
We next examined the impact of PKCβ on expression of proinflammatory/prothrombotic genes in IR. Previous studies revealed that Egr-1 regulated a broad array of such mediators in I/R (11). Compared with PKC_β+/+_ mice, factors regulating the vascular coagulant environment, such as tissue factor and plasminogen activator inhibitor-1 (PAI-1), displayed only a slight increase in transcripts in PKC_β_–/– mice. Levels of tissue factor and PAI-1 mRNA were increased by 15- and tenfold, respectively, in I/R lung from PKC_β+/+_ mice compared with uninstrumented PKC_β+/+_ controls (Figure 4, J and K). In PKC_β_–/– mice, tissue factor and PAI-1 mRNA were enhanced only by about fourfold and twofold, respectively, in the I/R lung. The same pattern of gene expression was observed comparing PKC_β_–/– and PKC_β+/+_ mice with respect to I/R-induced upregulation of proinflammatory cytokines (IL–1β), chemokines (macrophage inflammatory protein-2, MIP-2), and ICAM-1 (Figure 4, L, M, and N). Whereas IL-1β, MIP-2, and ICAM-1 displayed robust induction of transcripts in I/R lung from PKC_β+/+_ mice (increases of 20-, 16-, and 18-fold, respectively), these increases were much less prominent in I/R lungs from PKC_β_–/– mice (increases of approximately two-, three-, and twofold, respectively). In each case, the differences between enhanced levels of transcripts for these genes in I/R lungs from PKC_β+/+_ and PKC_β_–/– mice were statistically significant (P < 0.01 for tissue factor, PAI-1, IL-1β, MIP-2, and ICAM-1, respectively).
Mechanisms of I/R-induced PKC β-dependent signaling pathways. These considerations provide important insights into the potential downstream consequences of PKCβ-dependent activation of these multifaceted signaling cascades after I/R. The lack of orally administered inhibitors (or ready-made chow containing such inhibitors) of the three MAPKs (ERK1/2, JNK, or p38) for chronic administration to animals in vivo renders it difficult to directly assess the impact of these kinases on downstream targets. However, inhibitors of ERK1/2 (PD98059), JNK (SP600125), and p38 (SB203580) are available and suitable for in vitro studies. Therefore, to dissect the molecular mechanisms by which PKCβ activation upregulated Egr-1 in I/R, we used a rat alveolar MP cell line (NR8383), since alveolar MPs prominently displayed MAPKs such as phospho-ERK1/2 and Egr-1 in I/R lung. H/R was chosen as an in vitro model system to mimic key components of the ischemic/reperfused vascular milieu. Cultured MPs were serum-starved for 24 hours and exposed to hypoxia (oxygen pressure, 14 torr) for 30 minutes, then reoxygenation for 15 minutes. Real-time PCR analysis of total RNA from H/R MPs demonstrated an approximate eightfold increase in Egr-1 transcripts compared with normoxic MPs (P < 0.001) (Figure 5), in a manner strikingly suppressed by the inhibitor of PKCβ (LY379196, 200 nM; displays similar _K_i for the βI and βII isoforms [refs. 23, 24]). The increase in Egr-1 transcripts in H/R was also strongly suppressed by the inhibitors of ERK1/2 (PD98059, 50 ∝M) and JNK (SP600125, 20 ∝M) but not by an inhibitor of p38 (SB203580, 10 ∝M)] (Figure 5). These findings suggest that the PKCβ pathway modulates Egr-1 upregulation via phosphorylation of ERK1/2 and JNK, but not via phosphorylation of p38 in MPs in response to I/R injury.
H/R-mediated induction of Egr-1 in rat alveolar MPs (NR8383): effect of inhibitors of PKCβ and MAPKs. Rat MPs were incubated with LY379196 (200 nM), PD98059 (50 ∝M), SP600125 (20 ∝M), and SB203580 (10 ∝M), respectively, for 1 hour before being subjected to hypoxia for 30 minutes followed by reoxygenation for 15 minutes. Comparison is with H/R alone or normoxia (N). Real-time PCR analysis of total RNA from the above cells was performed. Data are presented as the fold induction of mRNA for Egr-1 normalized to β-actin. These experiments were repeated five times. *P < 0.001.
Activation of transcription factors activator protein-1 and NF-κB in I/R: effect of PKCβ. Lastly, we addressed the question of whether other key transcriptional regulatory pathways, such as activator protein-1 (AP-1) or NF-κB, were modulated by PKCβ in I/R beyond Egr-1. Compared with WT mice, Egr1–/– mice subjected to I/R did not reveal modulation of AP-1 or NF-κB DNA binding (data not shown). Thus, the possibility that PKCβ might modulate these pathways in I/R was important to consider, as activation of AP-1 (25) and NF-κB (26) have been shown in cells in response to hypoxia or hydrogen peroxide stress. Therefore, we investigated induction of AP-1 and NF-κB DNA binding activities in I/R lung from PKC_β+/+_ and PKC_β_–/– mice. Electrophoretic mobility shift analysis (EMSA) for AP-1 DNA binding activity, using a 32P-labeled consensus AP-1 oligonucleotide and nuclear extract from I/R lung derived from WT animals, showed induction of a prominent gel shift band (Figure 6A, compare lane 3 with lane 2, showing about an 11-fold increase in band intensity with I/R). This I/R-induced gel shift band represented specific binding to the AP-1 probe, as demonstrated by competition experiments; excess unlabeled AP-1 oligonucleotide blocked appearance of the band, whereas an unrelated Sp1 probe was without effect (lane 7 and lane 6 in Figure 6A). The observation that incubation with antibody to phospho-specific c-Jun (p-c-Jun) resulted in a supershift of the band corresponding to AP-1 DNA binding activity (Figure 6A, lane 8; lane 9 shows nonimmune IgG) indicated the involvement of this protein in the AP-1 complex. However, antibody to c-Fos had no effect in this supershift assay (data not shown). In contrast, when nuclear extracts were prepared from PKC_β_–/– mice subjected to I/R, intensity of the AP-1 gel shift band was significantly attenuated (Figure 6A, P < 0.0001, compare lane 5 with lane 3) compared with AP-1 after I/R in WT mice.
I/R-mediated induction of AP-1 and NF-κB in the lung: effect of PKCβ. Mice underwent left-lung I/R or no instrumentation. Mice were sacrificed, and nuclear extracts were prepared from the lung and subjected to EMSA with 32P-labeled consensus AP-1 (A) and NF-κB (B) probes. Where indicated, nuclear extracts from uninstrumented (lane 2 and lane 4) and I/R (lane 3 and lane 5) lung were incubated with 32P-labeled AP-1 (A) or NF-κB (B) probe alone. Nuclear extracts from I/R lungs of PKC_β+/+_ mice were incubated with 32P-labeled AP-1 (A) or NF-κB (B) probe in the presence of either a 100-fold molar excess of Sp1 (cold Sp1; lane 6 in A and B), AP-1 (cold AP-1; lane 7 in A), or NF-κB (cold NF-κB; lane 7 in B), and either anti–p-c-Jun IgG (lane 8 in A), anti-p50 IgG (lane 8 in B), and anti-p65 IgG (lane 9 in B) or nonimmune (NI) IgG (lane 9 in A and lane 10 in B, respectively). F.P., free probe.
In addition, EMSA with a 32P-labeled NF-κB probe and nuclear extracts from I/R lung harvested from WT mice demonstrated a 7.3-fold increase in band intensity compared with uninstrumented lung (Figure 6B, compare lane 3 with lane 2). The issue of specificity was addressed by competition studies in which excess unlabeled NF-κB probe blocked appearance of the gel shift band, whereas excess of another unrelated probe, Sp1, was without effect (Figure 6B, lane 7 and lane 6, respectively). Supershift analysis showed the presence of NF-κB p50 and p65 in the gel shift complex (Figure 6B, lane 8 and lane 9; lane 10 shows nonimmune IgG). When similar experiments were performed with nuclear extracts from I/R lung harvested from PKC_β_–/– mice, the intensity of the gel shift band was strikingly reduced compared with that produced using I/R PKC_β+/+_ mice (Figure 6B, compare lane 5 with lane 3; P < 0.0001). These data indicated that modulation of AP-1 or NF-κB activation was dependent on PKCβ, and not Egr-1, in the I/R lung.