Regulation of expression of matrix metalloproteinase-9 by JNK in Raw 264.7 cells: presence of inhibitory factor(s) suppressing MMP-9 induction in serum and conditioned media

Matrix metalloproteinase-9 (MMP-9) secreted from macrophages plays an important role in tissue destruction and inflammation through degradation of matrix proteins and proteolytic activation of cytokines/chemokines. Whereas the MEK-ERK and PI3K-Akt pathways up-regulate MMP-9 expression, regulation of MMP-9 by JNK remains controversial. Presently, we aimed to determine the role of JNK in MMP-9 regulation in Raw 264.7 cells. Inhibition of JNK by the JNK inhibitor SP600125 induced MMP-9 in the absence of serum and suppressed the expression of TNF-alpha, IL-6 and cyclooxygenase-2 in LPS-treated Raw 264.7 cells. In a knockdown experiment with small interfering RNA, suppression of JNK1 induced MMP-9 expression. Interestingly, mouse serum suppressed SP600125-mediated MMP-9 induction, similar to IFN-gamma. However, the inhibitory activity of mouse serum was not affected by pyridone 6, which inhibits Janus kinase downstream to IFN-gamma. In addition to mouse serum, conditioned media of Raw 264.7 cells contained the inhibitory factor(s) larger than 10 kDa, which suppressed SP600125- or LPS-induced MMP-9 expression. Taken together, these data suggest that JNK1 suppresses MMP-9 expression in the absence of serum. In addition, the inhibitory factor(s) present in serum or secreted from macrophages may negatively control MMP-9 expression.

Simvastatin inhibits induction of matrix metalloproteinase-9 in rat alveolar macrophages exposed to cigarette smoke extract

Matrix metalloproteinase-9 (MMP-9) may play an important role in emphysematous change in chronic obstructive pulmonary disease (COPD), one of the leading causes of mortality and morbidity worldwide. We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, attenuates emphysematous change and MMP-9 induction in the lungs of rats exposed to cigarette smoke. However, it remained uncertain how cigarette smoke induced MMP-9 and how simvastatin inhibited cigarette smoke-induced MMP-9 expression in alveolar macrophages (AMs), a major source of MMP-9 in the lungs of COPD patients. Presently, we examined the related signaling for MMP-9 induction and the inhibitory mechanism of simvastatin on MMP-9 induction in AMs exposed to cigarette smoke extract (CSE). In isolated rat AMs, CSE induced MMP-9 expression and phosphorylation of ERK and Akt. A chemical inhibitor of MEK1/2 or PI3K reduced phosphorylation of ERK or Akt, respectively, and also inhibited CSE-mediated MMP-9 induction. Simvastatin reduced CSE-mediated MMP-9 induction, and simvastatin-mediated inhibition was reversed by farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP). Similar to simvastatin, inhibition of FPP transferase or GGPP transferase suppressed CSE-mediated MMP-9 induction. Simvastatin attenuated CSE-mediated activation of RAS and phosphorylation of ERK, Akt, p65, IkappaB, and nuclear AP-1 or NF-kappaB activity. Taken together, these results suggest that simvastatin may inhibit CSE-mediated MMP-9 induction, primarily by blocking prenylation of RAS in the signaling pathways, in which Raf-MEK-ERK, PI3K/Akt, AP-1, and IkappaB-NF-kappaB are involved.

SP600125, a selective JNK inhibitor, aggravates hepatic ischemia-reperfusion injury

c-Jun N-terminal kinase (JNK) is activated during hepatic reperfusion, and JNK inhibitors are known to protect other major organs from ischemia-reperfusion (I/R) injury. We attempted to determine the effect of SP600125, a JNK inhibitor, on hepatic I/R injury using a partial ischemia model in mice. Compared to a vehicle-treated group, the SP600125-treated group showed a greater increase in serum ALT levels 24 h after reperfusion with more severe parenchymal destruction and leukocyte infiltration. Similarly, tissue myeloperoxidase and malondialdehyde levels were higher in the SP600125-treated group, and chemokine expression was also higher in the SP600125-treated group. These data, which are contradictory to previous results, indicate that JNK inhibition by SP600125 may be harmful in hepatic I/R injury. Therefore, care must be taken when investigating the therapeutic use of JNK inhibitors in hepatic I/R injury, especially in the context of the effects of JNK inhibition on inflammatory infiltration.

Overexpressed Mitochondrial Thioredoxin Protects PC12 Cells from Hydrogen Peroxide and Serum-deprivation

Oxidative damage to mitochondria is a critical mechanism in necrotic or apoptotic cell death induced by many kinds of toxic chemicals. Thioredoxin (Trx) family proteins are known to play protective roles in organisms under oxidative stress through redox reaction by using reducing equivalents of cysteines at a conserved active site, Cys-X-X-Cys. Whereas biological and physiological properties of Trx1 are well characterized, significance of mitochondrial thioredoxin (Trx2) is not well known. Therefore, we addressed physiological role of Trx2 in PC12 cells under oxidative stress. In PC12 cells, transiently overexpressed Trx2 significantly reduced cell death induced by hydrogen peroxide, whereas mutant Trx2, having serine residues instead of two cysteine residues at the active site did not. In addition, stably expressed Trx2 protected PC12 cells from serum deprivation. These results suggest that Trx2 may play defensive roles in PC12 cells by reducing oxidative stress to mitochondria.

Expression of hOGG1 protein during differentiation of HL-60 cells

Human 8-oxo-G-DNA glycosylase 1 (hOGG1) is a DNA glycosylase to cleave 8-oxo-7,8-dihydroguanine (8-oxo-G), a mutagenic DNA adduct formed by oxidant stresses. Here, we examined hOGG1 protein expression and repair activity to nick a DNA strand at the site of 8-oxo-G during differentiation of hematopoietic cells using HL-60 cells. Overall expression of hOGG1 protein was increased during granulocytic differentiation of HL-60 cells induced by DMSO and monocytic differentiation by vitamine D3. Greater level of hOGG1 protein was expressed in DMSO-treated cells. However, change in the DNA nicking activity was not in parallel with the change in hOGG1 protein expression, especially in PMA-treated cells. In PMA- treated cells, the level of hOGG1 protein was lowered, even though the DNA nicking activity was elevated, in a manner similar to the changes in serum- deprived HL-60 cells. These results indicate that hOGG1 expression change during differentiation of hematopoietic stem cells for adaptation to new environments. And the DNA cleaving activity may require additional factor(s) other than expressed hOGG1 protein, especially in apoptotic cell death.

Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.

Identification of Escherichia coli 8-oxoguanine endonuclease

7,8-Dihydro-8-oxoguanine (oh8Gua) endonuclease is a DNA repair enzyme in Escherichia coli to remove oh8Gua, a promutagenic DNA adduct. Due to the unique mode of enzyme action and substrate specificity, this DNA repair enzyme has been suggested to be identical to 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy)-DNA glycosylase (Fpg). However, oh8Gua endonuclease had not been definitely identified because it had not been homogeneously purified. In this study, we attempted to purify and identify the enzyme. Through several purification procedures, we obtained two proteins (32 kD and 29 kD). The larger protein co-migrated with Fpg in 12% SDS-PAGE gel. Sequences of N-terminal amino acids of these two proteins were identical to that of Fpg; the smaller one is a degraded product of oh8Gua endonuclease during purification steps. These results indicate that oh8Gua endonuclease is identical to Fpg, implying that oh8Gua in oxidatively damaged DNA rather than Fapy is more physiologically relevant substrate for Fpg.

Protective Effect of Thiore doxin on Bovine Corneal Endothelial Cells Damaged by Oxidative Stresses

Reactive oxygen species including hydrogen peroxide are known to induce morphological and functional derangement of corneal endothelium, thus causing loss of corneal deturgescence. Among the antioxidants, thioredoxin has been recently suggested to play an important role under oxidative stresses. The present study was undertaken to investigate the protective effect of thioredoxin, obtained from Escherichia coli, on cultured bovine endothelial cells against hydrogen peroxide and ultraviolet ray. Thioredoxin at concentrations of 1, 10 and 100 microgram/ml appeared to protect corneal endothelial cells from the reactive oxygen radical hydrogen peroxide as assessed by measuring the leakage of lactate dehydrogenase (LDH) and the reduction of 3-[4, 5-dimethyl-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT). In conclusion, thioredoxin may play an important physiological role (s) in protection of corneal endothelial cells which is continuously challenged by reactive oxygen species. Thus, It is natural that thioredoxin may have clinical implications in certain corneal disease such as corneal edema, which is known to be caused by reactive oxygen species.

Minimal amount of insulin can reverse diabetic heart function: Sarcoplasmic reticulum Ca2+ transport and phospholamban protein expression

In the present study, the underlying mechanisms for diabetic functional derangement and insulin effect on diabetic cardiomyopathy were investigated with respect to sarcoplasmic reticulum (SR) Ca2+-ATPase and phospholamban at the transcriptional and translational levels. The maximal Ca2+ uptake and the affinity of Ca2+-ATPase for Ca2+ were decreased in streptozotocin-induced diabetic rat cardiac SR, however, even minimal amount of insulin could reverse both parameters. Levels of both mRNA and protein of phospholamban were significantly increased in diabetic rat hearts, whereas the mRNA and protein levels of SR Ca2+-ATPase were significantly decreased. In case of phospholamban, insulin treatment reverses these parameters to normal levels. Minimal amount of insulin could reverse the protein levels; however, it could not reverse the mRNA level of SR Ca2+-ATPase at all. Thus, the decreased SR Ca2+ uptake appear to be largely attributed to the decreased SR Ca2+-ATPase level, which is further impaired due to the inhibition by the increased level of phospholamban. These results indicate that insulin is involved in the control of intracellular Ca2+ in the cardiomyocyte through multiple target proteins via multiple mechanisms for the decrease in the mRNA for both SR Ca2+-ATPase and phospholamban which are unknown and needs further study.

Effects of Reactive Oxygen Metabolite on the Calcium Transport of Cardiac Mitochondria

BACKGROUND: Intracellular calcium overload is a common final feature of the ischemic-reperfused heart and mediates the genesis of irreversible cell damage. Reactive oxygen medabolites have been known to play and important role as toxic mediators in myocardial injuries resulting from ischemia and reperfusion. In order to investigate the mechanism of intracellular calcium accumulation in the ischemic-reperfused myocardium, the present study observed the possible contribution of the reactive oxygen metabolite to the calcium transport of cardiac mitochondria. METHODS: Mitochondrial were isolated from rabbit hearts. The effects of a reactive oxygen metabolite, H2O2 on calcium uptake and release, redox states of endogenous pyridine nucleotides and glutathiones of mitochondria respiring with succinate were observed. Calcium uptake and release were monitored by dual-wave length spectrophotometer using a calcium indicator, arsenaze III. Contents and redox states of pyridine nucleotides and glutathiones were measured by enzymatic methods using spectrofluorometer and HPLC. RESULTS: Hydrogen peroxide(10-500microM) promoted calcium release dose-dependently from CA++-preloaded mitochondria, but did not affect the mitochondrial calcium uptake. The H2O2-induced calcium release was accompanied by simultaneous oxidation of the pyridine nucleotides and decrease in the content of the reduced form of glutathione(GSH). When mitochondria were treated with BCNU(N,N=bis(2-chloroethyl)-N-nitrosourea) to inhibit glutathione reductase and so as to reduce the GSH content, there were no increase in calcium release from the mitochondria. These results may indicate that H2O2 increases the permeability of cardiac mitochondrial membrane to calcium in association with the changes in redox state of endogenous pyridine nucleotides, but not with that of glutathiones. CONCLUSION: It is suggested that the reactive oxygen metabolites induce the release of calcium from mitochondria by altering the redox state of pyridine nucleotides, and it may partly be involved in the elevation of cytosolic calcium concentration in the ischemic-reperfused myocardial cells.

Production of Superoxide Anion and Damage of Mitochondria Isolated from Ischemic Repertused Heart

BACKGROUND: Oxygen free radicals have been implicated as a cause of deleterious effects in the setting of coronary reperfusion, and they are believed to be generated by the xanthine oxidase system, from activated neutrophiles and from mitochondria. We evaluate the contribution of mitochondria to the production of oxygen free radicals and clarify the mechanism of cellular damage in ischemic reperfused hearts. METHODS: Mitochondria isolated from the ischemic rabbit hearts were incubated in the reaction conditions with different oxygen tensions. Generation of superoxide anion and activities of defensive enzymes aginst oxidative stress were mesured. RESULTS: Superoxide anion genertion by mitochondria incubated in 21% oxygen condition were 0.54+/-0.09 and 0.27+/-0.04(O2./min/mg protein) in ischemic mitochondria and in control respectively(P<0.05). Activites of defensive enzymes against oxidative stress, superoxide dismutase and glutathione peroxidase, were significantly reduced in mitochondria isolated from either ischemic or reperfused hearts. With the lapse of respiration in 21% oxygen condition, ADP-stimulated state 3 oxygen consumption(306.4+/-31.5 vs 214.4+/-11.4n atoms O/min/mg protein) at 30 minutes, P : O ratio and phosphorylation rate were significantly decreased in ischemic mitochondria. CONCLUSION: Elevation of oxygen free radical generation as well as the reduction of defensive enzyme activities in ischemic reperfused mitochondria are injurious to mitochondrial respiratory function. It may contribute to the mechanism of cellular damage in ischemic reperfused hearts.