The Novel Small Molecule BTB Inhibits Pro-Fibrotic Fibroblast Behavior though Inhibition of RhoA Activity.

Idiopathic pulmonary fibrosis (IPF) is a progressive, chronic, interstitial lung disease with a poor prognosis. Although specific anti-fibrotic medications are now available, the median survival time following diagnosis remains very low, and new therapies are urgently needed. To uncover novel therapeutic targets, we examined how biochemical properties of the fibrotic lung are different from the healthy lung. Previous work identified lactate as a metabolite that is upregulated in IPF lung tissue. Importantly, inhibition of the enzyme responsible for lactate production prevents fibrosis in vivo. Further studies revealed that fibrotic lesions of the lung experience a significant decline in tissue pH, likely due to the overproduction of lactate. It is not entirely clear how cells in the lung respond to changes in extracellular pH, but a family of proton sensing G-protein coupled receptors has been shown to be activated by reductions in extracellular pH. This work examines the expression profiles of proton sensing GPCRs in non-fibrotic and IPF-derived primary human lung fibroblasts. We identify TDAG8 as a proton sensing GPCR that is upregulated in IPF fibroblasts and that knockdown of TDAG8 dampens myofibroblast differentiation. To our surprise, BTB, a proposed positive allosteric modulator of TDAG8, inhibits myofibroblast differentiation. Our data suggest that BTB does not require TDAG8 to inhibit myofibroblast differentiation, but rather inhibits myofibroblast differentiation through suppression of RhoA mediated signaling. Our work highlights the therapeutic potential of BTB as an anti-fibrotic treatment and expands upon the importance of RhoA-mediated signaling pathways in the context of myofibroblast differentiation. Furthermore, this works also suggests that TDAG8 inhibition may have therapeutic relevance in the treatment of IPF.

Noise as Diagnostic Tool for Quality and Reliability of MEMS.

This perspective explores future research approaches on the use of noise characteristics of microelectromechanical systems (MEMS) devices as a diagnostic tool to assess their quality and reliability. Such a technique has been applied to electronic devices. In comparison to these, however, MEMS have much more diverse materials, structures, and transduction mechanisms. Correspondingly, we must deal with various types of noise sources and a means to separate their contributions. In this paper, we first provide an overview of reliability and noise in MEMS and then suggest a framework to link noise data of specific devices to their quality or reliability. After this, we analyze 13 classes of MEMS and recommend four that are most amenable to this approach. Finally, we propose a noise measurement system to separate the contribution of electrical and mechanical noise sources. Through this perspective, our hope is for current and future designers of MEMS to see the potential benefits of noise in their devices.

The self-fulfilling prophecy of pulmonary fibrosis: a selective inspection of pathological signalling loops.

Pulmonary fibrosis is a devastating, progressive disease and carries a prognosis worse than most cancers. Despite ongoing research, the mechanisms that underlie disease pathogenesis remain only partially understood. However, the self-perpetuating nature of pulmonary fibrosis has led several researchers to propose the existence of pathological signalling loops. According to this hypothesis, the normal wound-healing process becomes corrupted and results in the progressive accumulation of scar tissue in the lung. In addition, several negative regulators of pulmonary fibrosis are downregulated and, therefore, are no longer capable of inhibiting these feed-forward loops. The combination of pathological signalling loops and loss of a checks and balances system ultimately culminates in a process of unregulated scar formation. This review details specific signalling pathways demonstrated to play a role in the pathogenesis of pulmonary fibrosis. The evidence of detrimental signalling loops is elucidated with regard to epithelial cell injury, cellular senescence and the activation of developmental and ageing pathways. We demonstrate where these loops intersect each other, as well as common mediators that may drive these responses and how the loss of pro-resolving mediators may contribute to the propagation of disease. By focusing on the overlapping signalling mediators among the many pro-fibrotic pathways, it is our hope that the pulmonary fibrosis community will be better equipped to design future trials that incorporate the redundant nature of these pathways as we move towards finding a cure for this unrelenting disease.

Role of cAMP-phosphodiesterase 1C signaling in regulating growth factor receptor stability, vascular smooth muscle cell growth, migration, and neointimal hyperplasia.

RATIONALE: Neointimal hyperplasia characterized by abnormal accumulation of vascular smooth muscle cells (SMCs) is a hallmark of occlusive disorders such as atherosclerosis, postangioplasty restenosis, vein graft stenosis, and allograft vasculopathy. Cyclic nucleotides are vital in SMC proliferation and migration, which are regulated by cyclic nucleotide phosphodiesterases (PDEs). OBJECTIVE: Our goal is to understand the regulation and function of PDEs in SMC pathogenesis of vascular diseases. METHODS AND RESULTS: We performed screening for genes differentially expressed in normal contractile versus proliferating synthetic SMCs. We observed that PDE1C expression was low in contractile SMCs but drastically elevated in synthetic SMCs in vitro and in various mouse vascular injury models in vivo. In addition, PDE1C was highly induced in neointimal SMCs of human coronary arteries. More importantly, injury-induced neointimal formation was significantly attenuated by PDE1C deficiency or PDE1 inhibition in vivo. PDE1 inhibition suppressed vascular remodeling of human saphenous vein explants ex vivo. In cultured SMCs, PDE1C deficiency or PDE1 inhibition attenuated SMC proliferation and migration. Mechanistic studies revealed that PDE1C plays a critical role in regulating the stability of growth factor receptors, such as PDGF receptor ß (PDGFRß) known to be important in pathological vascular remodeling. PDE1C interacts with low-density lipoprotein receptor-related protein-1 and PDGFRß, thus regulating PDGFRß endocytosis and lysosome-dependent degradation in an low-density lipoprotein receptor-related protein-1-dependent manner. A transmembrane adenylyl cyclase cAMP-dependent protein kinase cascade modulated by PDE1C is critical in regulating PDGFRß degradation. CONCLUSIONS: These findings demonstrated that PDE1C is an important regulator of SMC proliferation, migration, and neointimal hyperplasia, in part through modulating endosome/lysosome-dependent PDGFRß protein degradation via low-density lipoprotein receptor-related protein-1.

Cyclic nucleotide phosphodiesterase 1 regulates lysosome-dependent type I collagen protein degradation in vascular smooth muscle cells.

OBJECTIVE: The phenotypic modulation of vascular smooth muscle cells (VSMCs) to a synthetic phenotype is vital during pathological vascular remodeling and the development of various vascular diseases. An increase in type I collagen (collagen I) has been implicated in synthetic VSMCs, and cyclic nucleotide signaling is critical in collagen I regulation. Herein, we investigate the role and underlying mechanism of cyclic nucleotide phosphodiesterase 1 (PDE1) in regulating collagen I in synthetic VSMCs. METHODS AND RESULTS: The PDE1 inhibitor IC86340 significantly reduced collagen I in human saphenous vein explants undergoing spontaneous remodeling via ex vivo culture. In synthetic VSMCs, high basal levels of intracellular and extracellular collagen I protein were markedly decreased by IC86340. This attenuation was due to diminished protein but not mRNA. Inhibition of lysosome function abolished the effect of IC86340 on collagen I protein expression. PDE1C but not PDE1A is the major isoform responsible for mediating the effects of IC86340. Bicarbonate-sensitive soluble adenylyl cyclase/cAMP signaling was modulated by PDE1C, which is critical in collagen I degradation in VSMCs. CONCLUSIONS: These data demonstrate that PDE1C regulates soluble adenylyl cyclase/cAMP signaling and lysosome-mediated collagen I protein degradation, and they suggest that PDE1C plays a critical role in regulating collagen homeostasis during pathological vascular remodeling.

Ogerin mediated inhibition of TGF-ß(1) induced myofibroblast differentiation is potentiated by acidic pH.

Transforming growth factor beta (TGF-ß) induced myofibroblast differentiation is central to the pathological scarring observed in Idiopathic Pulmonary Fibrosis (IPF) and other fibrotic diseases. Our lab has recently identified expression of GPR68 (Ovarian Cancer Gene Receptor 1, OGR1), a pH sensing G-protein coupled receptor, as a negative regulator of TGF-ß induced profibrotic effects in primary human lung fibroblasts (PHLFs). We therefore hypothesized that small molecule activators of GPR68 would inhibit myofibroblast differentiation. Ogerin is a positive allosteric modulator (PAM) of GPR68, inducing a leftward shift of the dose response curve to proton induced signaling. Using PHLFs derived from patients with both non-fibrotic and IPF diagnoses, we show that Ogerin inhibits, and partially reverses TGF-ß induced myofibroblast differentiation in a dose dependent manner. This occurs at the transcriptional level without inhibition of canonical TGF-ß induced SMAD signaling. Ogerin induces PKA dependent CREB phosphorylation, a marker of Gαs pathway activation. The ability of Ogerin to inhibit both basal and TGF-ß induced collagen gene transcription, and induction of Gαs signaling is enhanced at an acidic pH (pH 6.8). Similar findings were also found using fibroblasts derived from dermal, intestinal, and orbital tissue. The biological role of GPR68 in different tissues, cell types, and disease states is an evolving and emerging field. This work adds to the understanding of Gαs coupled GPCRs in fibrotic lung disease, the ability to harness the pH sensing properties of GPR68, and conserved mechanisms of fibrosis across different organ systems.

Cell-Type-Specific Effects of the Ovarian Cancer G-Protein Coupled Receptor (OGR1) on Inflammation and Fibrosis; Potential Implications for Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by irreversible lung scarring. The pathophysiology is not fully understood, but the working hypothesis postulates that a combination of epithelial injury and myofibroblast differentiation drives progressive pulmonary fibrosis. We previously demonstrated that a reduction in extracellular pH activates latent TGF-ß1, and that TGF-ß1 then drives its own activation, creating a feed-forward mechanism that propagates myofibroblast differentiation. Given the important roles of extracellular pH in the progression of pulmonary fibrosis, we sought to identify whether pH mediates other cellular phenotypes independent of TGF-ß1. Proton-sensing G-protein coupled receptors are activated by acidic environments, but their role in fibrosis has not been studied. Here, we report that the Ovarian Cancer G-Protein Coupled Receptor1 (OGR1 or GPR68) has dual roles in both promoting and mitigating pulmonary fibrosis. We demonstrate that OGR1 protein expression is significantly reduced in lung tissue from patients with IPF and that TGF-ß1 decreases OGR1 expression. In fibroblasts, OGR1 inhibits myofibroblast differentiation and does not contribute to inflammation. However, in epithelial cells, OGR1 promotes epithelial to mesenchymal transition (EMT) and inflammation. We then demonstrate that sub-cellular localization and alternative signaling pathways may be responsible for the differential effect of OGR1 in each cell type. Our results suggest that strategies to selectively target OGR1 expression may represent a novel therapeutic strategy for pulmonary fibrosis.

Role of Ca2+/calmodulin-stimulated cyclic nucleotide phosphodiesterase 1 in mediating cardiomyocyte hypertrophy.

RATIONALE: Cyclic nucleotide phosphodiesterases (PDEs) through the degradation of cGMP play critical roles in maintaining cardiomyocyte homeostasis. Ca(2+)/calmodulin (CaM)-activated cGMP-hydrolyzing PDE1 family may play a pivotal role in balancing intracellular Ca(2+)/CaM and cGMP signaling; however, its function in cardiomyocytes is unknown. OBJECTIVE: Herein, we investigate the role of Ca(2+)/CaM-stimulated PDE1 in regulating pathological cardiomyocyte hypertrophy in neonatal and adult rat ventricular myocytes and in the heart in vivo. METHODS AND RESULTS: Inhibition of PDE1 activity using a PDE1-selective inhibitor, IC86340, or downregulation of PDE1A using siRNA prevented phenylephrine induced pathological myocyte hypertrophy and hypertrophic marker expression in neonatal and adult rat ventricular myocytes. Importantly, administration of the PDE1 inhibitor IC86340 attenuated cardiac hypertrophy induced by chronic isoproterenol infusion in vivo. Both PDE1A and PDE1C mRNA and protein were detected in human hearts; however, PDE1A expression was conserved in rodent hearts. Moreover, PDE1A expression was significantly upregulated in vivo in the heart and myocytes from various pathological hypertrophy animal models and in vitro in isolated neonatal and adult rat ventricular myocytes treated with neurohumoral stimuli such as angiotensin II (Ang II) and isoproterenol. Furthermore, PDE1A plays a critical role in phenylephrine-induced reduction of intracellular cGMP- and cGMP-dependent protein kinase (PKG) activity and thereby cardiomyocyte hypertrophy in vitro. CONCLUSIONS: These results elucidate a novel role for Ca(2+)/CaM-stimulated PDE1, particularly PDE1A, in regulating pathological cardiomyocyte hypertrophy via a cGMP/PKG-dependent mechanism, thereby demonstrating Ca(2+) and cGMP signaling cross-talk during cardiac hypertrophy.

The effect of air pollution on the transcriptomics of the immune response to respiratory infection.

Combustion related particulate matter air pollution (PM) is associated with an increased risk of respiratory infections in adults. The exact mechanism underlying this association has not been determined. We hypothesized that increased concentrations of combustion related PM would result in dysregulation of the innate immune system. This epidemiological study includes 111 adult patients hospitalized with respiratory infections who underwent transcriptional analysis of their peripheral blood. We examined the association between gene expression at the time of hospitalization and ambient measurements of particulate air pollutants in the 28 days prior to hospitalization. For each pollutant and time lag, gene-specific linear models adjusting for infection type were fit using LIMMA (Linear Models For Microarray Data), and pathway/gene set analyses were performed using the CAMERA (Correlation Adjusted Mean Rank) program. Comparing patients with viral and/or bacterial infection, the expression patterns associated with air pollution exposure differed. Adjusting for the type of infection, increased concentrations of Delta-C (a marker of biomass smoke) and other PM were associated with upregulation of iron homeostasis and protein folding. Increased concentrations of black carbon (BC) were associated with upregulation of viral related gene pathways and downregulation of pathways related to antigen presentation. The pollutant/pathway associations differed by lag time and by type of infection. This study suggests that the effect of air pollution on the pathogenesis of respiratory infection may be pollutant, timing, and infection specific.

Computational methodology for absolute calibration curves for microfluidic optical analyses.

Optical fluorescence and absorption are two of the primary techniques used for analytical microfluidics. We provide a thorough yet tractable method for computing the performance of diverse optical micro-analytical systems. Sample sizes range from nano- to many micro-liters and concentrations from nano- to milli-molar. Equations are provided to trace quantitatively the flow of the fundamental entities, namely photons and electrons, and the conversion of energy from the source, through optical components, samples and spectral-selective components, to the detectors and beyond. The equations permit facile computations of calibration curves that relate the concentrations or numbers of molecules measured to the absolute signals from the system. This methodology provides the basis for both detailed understanding and improved design of microfluidic optical analytical systems. It saves prototype turn-around time, and is much simpler and faster to use than ray tracing programs. Over two thousand spreadsheet computations were performed during this study. We found that some design variations produce higher signal levels and, for constant noise levels, lower minimum detection limits. Improvements of more than a factor of 1,000 were realized.

GIT1 is a novel MEK1-ERK1/2 scaffold that localizes to focal adhesions.

Cell polarity is critical for cell migration and requires localized signal transduction in subcellular domains. Recent evidence demonstrates that activation of ERK1/2 (extracellular-signal-regulated kinase 1/2) in focal adhesions is essential for cell migration. GIT1 (G-protein-coupled receptor kinase-interacting protein 1) has been shown to bind paxillin and regulate focal-adhesion disassembly. We have previously reported that GIT1 binds to MEK1 [MAPK (mitogen-activated protein kinase)/ERK kinase 1] and acts as a scaffold to enhance ERK1/2 activation in response to EGF (epidermal growth factor). In the present study we show that GIT1 associates with ERK1/2 in focal adhesions and this association increases after EGF stimulation. The CC (coiled-coil) domain of ERK1/2 is required for association with GIT1, translocation to focal adhesions, and cell spreading and migration. Immunofluorescent staining showed that, after EGF stimulation, GIT1 co-localized with pERK1/2 (phosphorylated ERK1/2) in focal adhesions. The binding of GIT1 and ERK1/2 was functionally important, since transfecting an ERK2 mutant lacking the CC domain [ERK2(del CC)] significantly decreased pERK1/2 translocation to focal adhesions, cell spreading and migration induced by EGF. In summary, the CC domain of ERK1/2 is necessary for binding to GIT1, for ERK1/2 activation in focal adhesions, and for cell spreading and migration.

Role of nuclear Ca2+/calmodulin-stimulated phosphodiesterase 1A in vascular smooth muscle cell growth and survival.

In response to biological and mechanical injury, or in vitro culturing, vascular smooth muscle cells (VSMCs) undergo phenotypic modulation from a differentiated "contractile" phenotype to a dedifferentiated "synthetic" one. This results in the capacity to proliferate, migrate, and produce extracellular matrix proteins, thus contributing to neointimal formation. Cyclic nucleotide phosphodiesterases (PDEs), by hydrolyzing cAMP or cGMP, are critical in the homeostasis of cyclic nucleotides that regulate VSMC growth. Here, we demonstrate that PDE1A, a Ca2+-calmodulin-stimulated PDE preferentially hydrolyzing cGMP, is predominantly cytoplasmic in medial "contractile" VSMCs but is nuclear in neointimal "synthetic" VSMCs. Using primary VSMCs, we show that cytoplasmic and nuclear PDE1A were associated with a contractile marker (SM-calponin) and a growth marker (Ki-67), respectively. This suggests that cytoplasmic PDE1A is associated with the "contractile" phenotype, whereas nuclear PDE1A is with the "synthetic" phenotype. To determine the role of nuclear PDE1A, we examined the effects loss-of-PDE1A function on subcultured VSMC growth and survival using PDE1A RNA interference and pharmacological inhibition. Reducing PDE1A function significantly attenuated VSMC growth by decreasing proliferation via G1 arrest and inducing apoptosis. Inhibiting PDE1A also led to intracellular cGMP elevation, p27Kip1 upregulation, cyclin D1 downregulation, and p53 activation. We further demonstrated that in subcultured VSMCs redifferentiated by growth on collagen gels, cytoplasmic PDE1A regulates myosin light chain phosphorylation with little effect on apoptosis, whereas inhibiting nuclear PDE1A has the opposite effects. These suggest that nuclear PDE1A is important in VSMC growth and survival and may contribute to the neointima formation in atherosclerosis and restenosis.

Ischemic preconditioning suppresses apoptosis of rabbit spinal neurocytes by inhibiting ASK1-14-3-3 dissociation.

The mechanism by which a brief episode of sublethal ischemia followed by reperfusion (ischemic preconditioning, IPC) prevents the lethal effects of subsequent periods of prolonged ischemia, are poorly understood. A completely randomized, controlled study was designed to study the effect of IPC using a rabbit model of ischemic spinal cord injury. Twenty-four white adult New England rabbits were randomly assigned to one of 3 groups (n=8 per group); the groups were assigned as follows: Group I: sham-operation group, Group II: ischemic reperfusion (I/R) group, and Group III: ischemic preconditioning group. Spinal cord ischemia was induced by introducing an infra renal aortic cross-clamp for 30min. Following injury, rabbits were subjected to 30min, 2h, or 8h of reperfusion in Group II. In Group III, subjects underwent three cycles, 5min each, of ischemia followed by 5min of reperfusion, before receiving 30min of ischemia. We previously reported that the association between ASK1 (apoptosis signal-regulating kinase 1) and 14-3-3 played an important role in regulating ischemia/reperfusion spinal cord injuries. To evaluate the effect of ischemic preconditioning in injured spinal cords, we examined alterations in spinal tissue morphology, activation of key members of the ASK1-mediated signaling pathway, and the association between ASK1 and 14-3-3. Changes in spinal cord morphology were observed with hematoxylin and eosin (H&E) staining and electron microscopy. The phosphorylation levels of ASK1, JNK, and p38 were assessed by immunoblot analysis. The association between ASK1 and 14-3-3 was analyzed by co-immunoprecipitation experiments. We observed that swelling of the neurocyte bodies and hemorrhage of the spinal cord were dramatically decreased in Group III compared to Group II. In addition, the degree of apoptosis among neurocytes was reduced in Group III compared to Group II. Finally, the phosphorylation of ASK1, JNK, p38 and the dissociation of ASK1 from 14-3-3 were dramatically decreased in Group III compared with Group II. These results indicate that ischemic preconditioning may have a protective affect against ASK1/14-3-3 dissociation-induced spinal cord injuries.

Prevention and treatment of bleomycin-induced pulmonary fibrosis with the lactate dehydrogenase inhibitor gossypol.

Pulmonary fibrosis is a chronic and irreversible scarring disease in the lung with poor prognosis. Few therapies are available; therefore it is critical to identify new therapeutic targets. Our lab has previously identified the enzyme lactate dehydrogenase-A (LDHA) as a potential therapeutic target in pulmonary fibrosis. We found increases in LDHA protein and its metabolic product, lactate, in patients with idiopathic pulmonary fibrosis (IPF). Importantly, we described lactate as a novel pro-fibrotic mediator by acidifying the extracellular space, and activating latent transforming growth factor beta (TGF-ß1) in a pH-dependent manner. We propose a pro-fibrotic feed-forward loop by which LDHA produces lactate, lactate decreases pH in the extracellular space and activates TGF-ß1 which can further perpetuate fibrotic signaling. Our previous work also demonstrates that the LDHA inhibitor gossypol inhibits TGF-ß1-induced myofibroblast differentiation and collagen production in vitro. Here, we employed a mouse model of bleomycin-induced pulmonary fibrosis to test whether gossypol inhibits pulmonary fibrosis in vivo. We found that gossypol dose-dependently inhibits bleomycin-induced collagen accumulation and TGF-ß1 activation in mouse lungs when treatment is started on the same day as bleomycin administration. Importantly, gossypol was also effective at treating collagen accumulation when delayed 7 days following bleomycin. Our results demonstrate that inhibition of LDHA with the inhibitor gossypol is effective at both preventing and treating bleomycin-induced pulmonary fibrosis, and suggests that LDHA may be a potential therapeutic target for pulmonary fibrosis.

Activation of ASK1 during reperfusion of ischemic spinal cord.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK), which plays a pivotal role in cell apoptosis. To determine the mechanism of ASK1 induction during reperfusion of ischemic spinal tissue, we used a model of rabbit spinal cord ischemia and reperfusion. To assess the role of ASK1 in spinal cord ischemia-reperfusion injuries, we examined alterations in spinal tissue morphology, protein-protein interactions, and activation of key members of the ASK1-mediated signaling pathway. Changes in spinal cord morphology were observed with hematoxylin and eosin (H&E) staining and electron microscopy. The phosphorylation levels of ASK1, JNK, and p38 were assessed by immunoblotting proteins from animals that received 30 min of ischemia followed by 1 or 24h of reperfusion. We observed increased phosphorylation of ASK1, JNK, and p38 after reperfusing ischemic spinal cords. Immunohistochemical studies were performed to determine the cellular localization of phosphorylated ASK1 (pASK1) and 14-3-3. Following reperfusion for 24h, we observed increased cytoplasmic localization of pASK1 and decreased cytoplasmic localization of 14-3-3. Immunoprecipitation analyses suggested that 14-3-3 dissociates from ASK1 during reperfusion of ischemic spinal cords. These results indicate that activation of ASK1 may play an important role in the apoptotic signaling mechanisms that occur in reperfused spinal cord injuries.

Compact optical microfluidic uric acid analysis system.

We designed, fabricated and tested a novel compact fluorescence analysis system for quantification of uric acid (UA) in clinical samples at the point-of-care. To perform an analysis, diluted saliva, urine or blood samples are simply placed in a disposable thin-film sample holder using a dropper. A new enzyme immobilization technique was developed to retain within the sample holder two enzymes and a molecule, which transforms into a fluorescer in amounts depending on the UA concentration. The small instrument (7.5 cm × 5 cm × 5 cm) into which the sample holder is placed for analysis contains an LED, a narrow-band filter and an amplified photodiode. The analysis time is 30s, and the dynamic range of the system is 4-400 µM of UA. The calibration curve for transparent saliva and urine was made using solutions of UA. The calibration curve for opaque blood was obtained with spiked samples of blood. The three different types of clinical samples were collected from three subjects and simply diluted before their measurements. Analysis with our instrument yielded UA concentrations within the expected concentration ranges. Development of instruments based on the current laboratory prototype is expected to result in products for clinical trials and point-of-care.

A model of ischemia and reperfusion increases JNK activity, inhibits the association of BAD and 14-3-3, and induces apoptosis of rabbit spinal neurocytes.

It is now well established that the protein BAD (a pro-apoptotic Bcl-2 family protein) plays a pivotal role in determining cell death and survival. The c-Jun N-terminal kinase (JNK) pathway has been hypothesized to be involved in regulation of BAD. To clarify the role of BAD within the JNK pathway, a randomized, controlled study was designed using a rabbit model of ischemic spinal cord injury [5,8]. Forty-five white adult New England rabbits were randomly assigned to one of the three groups: sham-operation group (n=5), vehicle group (n=20), and JNK inhibitor group (n=20). We examined alterations in spinal tissue morphology, local concentration and cellular locations of key regulatory proteins, and protein-protein interactions. Changes in spinal cord morphology were observed with hematoxylin and eosin (H&E) staining and electron microscopy. In the vehicle group, the amount of JNK phosphorylation, cytochrome c release, and the interaction between BAD and Bcl-XL or Bcl-2 were increased compared with the JNK inhibitor group. Similarly, the phosphorylation of BAD (Ser136) and the interaction between BAD and 14-3-3 were decreased in the vehicle group. Immunohistochemical studies showed that cytoplasmic location of 14-3-3 and p-BAD (Ser136) were decreased in the vehicle group compared with the JNK inhibitor group. In addition, mitochondrial morphology was better preserved and the percentage of apoptosis was lower when JNK was inhibited. These results indicate that the JNK pathway has a critical role in the survival of neurocytes by regulating the interaction between BAD and 14-3-3.