Selective inhibition of protein kinase C β2 attenuates the adaptor P66Shc-mediated intestinal ischemia–reperfusion injury

Apoptosis is a major mode of cell death occurring during ischemia–reperfusion (I/R) induced injury. The p66Shc adaptor protein, which is mediated by PKCβ, has an essential role in apoptosis under oxidative stress. This study aimed to investigate the role of PKCβ2/p66Shc pathway in intestinal I/R injury. In vivo, ischemia was induced by superior mesenteric artery occlusion in mice. Ruboxistaurin (PKCβ inhibitor) or normal saline was administered before ischemia. Then blood and gut tissues were collected after reperfusion for various measurements. In vitro, Caco-2 cells were challenged with hypoxia–reoxygenation (H/R) to simulate intestinal I/R. Translocation and activation of PKCβ2 were markedly induced in the I/R intestine. Ruboxistaurin significantly attenuated gut damage and decreased the serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Pharmacological blockade of PKCβ2 suppressed p66Shc overexpression and phosphorylation in the I/R intestine. Gene knockdown of PKCβ2 via small interfering RNA (siRNA) inhibited H/R-induced p66Shc overexpression and phosphorylation in Caco-2 cells. Phorbol 12-myristate 13-acetate (PMA), which stimulates PKCs, induced p66Shc phosphorylation and this was inhibited by ruboxistaurin and PKCβ2 siRNA. Ruboxistaurin attenuated gut oxidative stress after I/R by suppressing the decreased expression of manganese superoxide dismutase (MnSOD), the exhaustion of the glutathione (GSH) system, and the overproduction of malondialdehyde (MDA). As a consequence, ruboxistaurin inhibited intestinal mucosa apoptosis after I/R. Therefore, PKCβ2 inhibition protects mice from gut I/R injury by suppressing the adaptor p66Shc-mediated oxidative stress and subsequent apoptosis. This may represent a novel therapeutic approach for the prevention of intestinal I/R injury.


Subject Category: Experimental Medicine
Critical massive intestinal ischemia occurs in response to conditions such as acute mesenteric thrombotic or embolic occlusion, which are associated with high mortality. 1,2 Other gut ischemia cases followed by hemorrhagic shock, volvulus, sepsis, and abdominal aortic aneurysm surgery have a more subtle but no less damaging injury. Although restoration of the blood supply to the ischemic gut is critical to salvage, the reperfusion may paradoxically aggravate ischemic tissue damage and systemic inflammatory response. 3 During the reperfusion period, a vicious cascade occurs including massive reactive oxygen species (ROS) generation, the activation of pro-apoptotic factors, and systemic inflammatory responses such as cytokine/chemokine release and polymorphonuclear neutrophil infiltration. 4,5 It becomes recognized that oxidative stress-induced ischemia/reperfusion (I/R) damage involves multiple signaling pathways.
PKC, a family of serine/threonine protein kinases comprising at least 12 members, has a central role in signal transduction and intracellular crosstalk. 6 PKCb 1 and PKCb 2 isoforms are encoded by the same gene, PKCb, and are not expressed in homozygous PKCb À / À mice (NCBI Gene Database, identification number 18751). Multiple PKC isozymes are expressed in the intestine. 7 Gene deletion or pharmacological blockade of PKCb protects ischemic myocardium, decreases infarct size, and enhances recovery of ventricular function. 8 Homozygous PKCb-null mice and WT mice fed with ruboxistaurin (LY333531, selective PKCb inhibitor) and subjected to single-lung I/R display increased survival, indicating that PKCb has a pivotal role in the I/R-induced apoptosis. 9 Despite these observations, the underlying mechanism by which PKCb exerts deleterious effects in the intestinal I/R remains unclear.
The Shc adaptor protein family, consisting of the p66 Shc , p52 Shc , and p46 Shc isoforms, is encoded by the ShcA locus. 10 Due to the presence of a unique N-terminal domain (CH2), which is required for redox activity, p66 Shc is the only isoform that acts as a redox enzyme implicated in mitochondrial ROS generation and the translation of oxidative signals into apoptosis. 11 Phosphorylation at Ser36 of p66 Shc is required for conferring increased susceptibility to oxidative stress, and is critical for the cell apoptosis elicited by oxidative damage. 12 Migliaccio et al. 11 reported the p66 Shc À / À mouse increased resistance to oxidative stress and extended lifespan by 30%. Deletion of the p66 Shc gene in mice is shown to protect hind limb, 13 brain, 14 and ex vivo hearts 15 from I/R injury. It suggests that p66 Shc would be a target to decrease the injury caused by intestinal I/R.
Hydrogen peroxide (H 2 O 2 ) and hyperglycemic stress activate the PKCb 2 isoform to induce p66 Shc phosphorylation at Ser-36, allowing transfer of the adaptor protein from the cytosol to the inner mitochondrial membrane, where it amplifies oxidative stress and catalyzes ROS production via cytochrome c oxidation. [16][17][18] Therefore, we hypothesize that there may be a PKCb 2 /p66 Shc signaling pathway in the pathogenesis of intestinal I/R.

Results
Membrane translocation and phosphorylation of PKCb 2 in response to intestinal I/R. To test the hypothesis that PKC could be activated by I/R injury, we assessed cell membranous fraction of patterns for distinct PKC isoforms in the intestinal tissue subjected to 45 min ischemia followed by 45, 90, or 180 min reperfusion. A selective membrane translocation of PKCb 2 was detected, whereas PKCb 1 , PKCd, and PKCe showed no differences in membrane fraction after various reperfusion times (Figure 1a), indicating that PKCb 2 is specifically activated by I/R. To support this notion, we detected that a 90-min reperfusion significantly increased PKCb 2 phosphorylation at the thr-641 residue, leading to a markedly increased ratio of phosphorylated PKCb 2 /total PKCb 2 ( Figure 1b). These results demonstrated that both membrane translocation and activation of PKCb 2 occurred in the model of intestinal I/R. Ruboxistaurin attenuates gut damage and the systemic inflammatory response after intestinal I/R. Next, ruboxistaurin (oral PKCb inhibitor) and normal saline were given as a pretreatment before the superior mesenteric artery was occluded for 45 min followed by 90 min reperfusion. On examination of the histological changes, ruboxistaurin preserved the integrity of morphological structure well, and reduced both hemorrhage and neutrophil infiltration in the I/R intestine (Figure 2a). Similarly, the gut histological injury scores were significantly increased following I/R injury versus sham, and was reduced by ruboxistaurin ( Figure 2b). Additionally, intestinal I/R significantly increased the serum levels of tumor necrosis factor-a (TNF-a) and interleukin 6 (IL-6). Ruboxistaurin, however, almost abrogated the increase in TNF-a and IL-6 concentrations ( Figure 2c).
Ruboxistaurin suppresses intestinal I/R-induced activation of PKCb 2 and p66 Shc . Figure 3a showed that ruboxistaurin greatly suppressed the translocation of PKCb 2 in the I/R intestine over the same time period in which PKCb 1 was not impacted. Meanwhile, ruboxistaurin prevented the intestinal I/R-induced increase in the phosphorylation of PKCb 2 without affecting the expression of total PKCb 2, and suppressed the increased ratio of phosphorylated PKCb 2 /total PKCb 2 ( Figure 3b). Intestinal I/R moderately increased the expression of p66 Shc , and greatly induced p66 Shc phosphorylation. However, ruboxistaurin significantly reduced I/R-induced p66 Shc overexpression and phosphorylation at ser36 (Figure 3c). Therefore, our study indicated that ruboxistaurin inhibited both PKCb 2 activation and PKCb 2 -mediated p66 Shc activation in the I/R intestine.  (Figure 5a). ROS accumulation was increased in intestinal I/R tissue based on the assessment of MDA activity, which was reduced by ruboxistaurin ( Figure 5b). In parallel, ruboxistaurin preserved intestinal I/R-induced GSH exhaustion and GSH-PX activity reduction (Figures 5c and d). Taken together, these data indicated that blockade of PKCb 2 decreased gut oxidative stress after intestinal I/R.
Inhibition of PKCb 2 activation by ruboxistaurin inhibits gut apoptosis after intestinal I/R. To determine the apoptosis state of the gut after I/R, a terminal deoxynucleotidyl transferase mediated deoxyuridinetriphosphate nick end labeling (TUNEL) assay was conducted. The apoptotic cells in the gut were elevated from non-detectable to well observed after intestinal I/R, whereas ruboxistaurin significantly reduced the number of apoptotic cells (Figure 6a). In addition, ruboxistaurin significantly suppressed the increased levels of cleaved caspase-3, another marker of cell apoptosis, in the I/R intestinal tissue (Figure 6b).

Discussion
In the present study, we have demonstrated that I/R-induced intestinal dysfunction involved the PKCb 2 /p66 Shc signaling pathway. PKCb 2 activation played an essential role in the     8,19,20 activation of PKCb 2 associated with the response to single-lung I/R, 9 and activation of PKCd and PKCe related to cerebral I/R. 21 Our results demonstrated that the activated principal isoform of PKC in intestinal I/R was specifically PKCb 2 , not PKCb 1 , PKCd, or PKCe (Figures 1a and b). These data suggested that the activation of individual PKC isoforms in ischemia or I/R is tissue specific. Moreover, our results indicated that in intestinal I/R, ruboxistaurin did not change the translocation of PKCb 1 over the same time period in which PKCb 2 was greatly impacted (Figure 3a). Taken together, it is likely that the primary role of ruboxistaurin was to inhibit the activation of PKCb 2 in intestinal I/R. H/R significantly induces the activation of p66 Shc , and ablation of p66 Shc is cytoprotective against oxidative stress  22 This may be clinically relevant as the mRNA level of p66 Shc is increased in peripheral blood mononuclear cells of patients with acute myocardial infarction. 23 In human aortic endothelial cells, selective inhibitor of PKCb 2 prevented p66 Shc activation after exposed to hyperglycemic stress or oxidized low-density lipoprotein, respectively. 18,24 Our data showed that inhibition of PKCb 2 activation by ruboxistaurin attenuated p66 Shc overexpression and phosphorylation at ser36 in the I/R intestine (Figure 3c). In vitro studies, knocking down PKCb 2 via siRNA inhibited the activation of PKCb 2 , and further prevented p66 Shc overexpression and phosphorylation under H/R conditions (Figure 4). By using both pharmacological blockade and gene knockdown PKCb 2 in vivo and in vitro experiments, we tested the above hypothesis that there may be a PKCb 2 /p66 Shc signaling pathway in intestinal I/R. Gut I/R produces excessive amounts of ROS, which is responsible for the intestinal mucosa damage. 25 Given exposure to ROS, mitochondrial proteins, lipids, and DNA are believed to be primary targets of oxidative damage, leading to alteration or loss of cellular functions, and causing inhibition of proliferation and induction of apoptosis. 26 A growing body of evidence links p66 Shc to oxidative stress as the adaptor protein has a pivotal role in modulating the intracellular redox state, increasing susceptibility to oxidative stress, and resulting in apoptosis elicited by oxidative damage. [27][28][29] Our data demonstrated ruboxistaurin increased the intestinal I/R-induced downregulation of MnSOD, a primary ROS scavenging enzyme, but suppressed the accumulation of MDA, an indicator of lipid peroxidation (Figures 5a and b). Meanwhile, ruboxistaurin preserved intestinal I/R-induced GSH exhaustion and GSH-PX activity reduction (Figures 5c and d). Furthermore, the apoptosis execution enzyme caspase-3 has a crucial role in cell apoptosis by resulting in DNA fragmentation, degradation of cytoskeleton, and formation of apoptotic bodies. Arany et al. 30 showed that p66 Shc was associated with cytochrome c, which is responsible for the activation of caspase-3 in the kidneys of mice with I/R injury. Our data showed that ruboxistaurin significantly attenuated intestinal caspase-3 activity and inhibited the apoptosis of the intestine subjected to I/R (Figures 6a and b). Therefore, it is conceivable that the inhibition of PKCb 2 activation by ruboxistaurin attenuates p66 Shc -mediated oxidative stress and subsequent apoptosis in intestinal I/R.
During the reperfusion period, mucosal barrier integrity is destroyed and the systemic release of pro-inflammatory cytokines occurs, with concurrent leukocyte activation and bacterial translocation. 31 In this study, intestinal I/R injury significantly increased the serum levels of TNF-a and IL-6, suggesting that a severe systemic inflammation response was induced during the reperfusion period. Ruboxistaurin administration almost abrogated the increase in TNF-a and IL-6 serum concentration (Figure 2c).
Ruboxistaurin, an oral PKCb inhibitor, is currently undergoing phase 2 and phase 3 clinical testing for several cardiovascular diseases, such as diabetic retinopathy and diabetic kidney disease. 32,33 Due to be administrated orally, ruboxistaurin was gavaged for 3 days before I/R, which would be a potential limitation in acute clinical cases. However, the focus of this study was to investigate the role of PKCb 2 in regulating p66 Shc -mediated intestinal I/R injury.
In summary, our results demonstrate that the inhibition of PKCb 2 activation attenuated intestinal I/R injury and systemic inflammation response by inhibiting the adaptor p66 Shcmediated oxidative stress and subsequent apoptosis. Furthermore, the activated principal isoform of PKC in intestinal I/R was specifically PKCb 2 , not PKCb 1 , PKCd, or PKCe. These may represent a novel therapeutic avenue for intestinal I/R injury.

Materials and Methods
Murine model of intestinal I/R. Male ICR mice (aged 4 weeks) weighing 20±2 g were obtained from the Animal Center of Dalian Medical University (Dalian, China), and kept under standard laboratory conditions with standard laboratory chow and water. The mouse intestinal occlusion-and-reperfusion procedure was performed as described previously. 5 Briefly, the superior mesenteric artery was occluded by a microvascular clamp for 45 min and then 45, 90, or 180 min reperfusion was performed. Normal saline and ruboxistaurin (LY 333531; ENZO, Lausen, Switzerland) were given by oral gavage before sham and 45 min ischemia, followed by 90 min reperfusion at a dose of 10 mg/kg daily for 3 days (demonstrated to adequately inhibit PKCb activation in mice heart and vasculature). 9 All procedures were conducted according to the Institutional Animal Care Guidelines, and were approved by the Institutional Ethics Committee.
Histological and TUNEL staining. For histological and TUNEL analysis, formalin-fixed tissues were embedded in paraffin and sectioned. The 4-mm sections were stained by hematoxylin-eosin. Intestinal I/R-induced mucosal injury was evaluated according to Chiu's score. 34 TUNEL staining was performed using an apoptosis assay kit (Roche, Mannheim, Germany) according to the manufacturer's instructions.
Measurement of cytokines. The levels of serum TNF-a and IL-6 were measured using Enzyme-linked immunosorbent assay (ELISA) kits (ENGTON Bio-engineering Limited Company, Shanghai, China), according to the manufacturer's protocols.
Intestinal GSH, GSH-PX, and MDA activity assay. The GSH and GSH-PX activities were determined using an assay kit (Nanjing Jiancheng Corp., Nanjing, China), according to the manufacturer's recommendations. The level of MDA in the intestinal tissues was quantified by a lipid peroxidation MDA assay kit (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer's protocol.
Cell culture. Caco-2 cells were cultured at 37 1C in a humidified atmosphere of 5% CO 2 in DMEM, supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% glutamide (Gibco, Carlsbad, CA, USA). To simulate physiologic conditions, Caco-2 cells were grown as monolayers on platforms, providing both apical and basolateral areas, thereby allowing cells to become polarized. The culture medium was then replaced with serum-free DMEM before experimental treatment.
H/R incubation and PMA exposure. To simulate in vivo intestinal ischemia, unless otherwise noted, cellular hypoxic conditions were created. For the hypoxic conditions, cells were incubated in a microaerophilic system (Thermo Fisher Scientific 8000, Marietta, GA, USA) at 5% CO 2 and 1% O 2 , and balanced Selective inhibition of PKCb 2 attenuates the P66 Shc -mediated intestinal I/R injury Z Chen et al