Manganese Superoxide Dismutase mRNA Expression Research Articles

Effect of Herbal Medicine Keishi-To (TJ-45) and Its Components on Rat Pancreatic Acinar Cell Injuries in vivo and in vitro

Background: In an attempt to clarify the mechanism of the effect of a herbal medicine, Saiko-keishi-to (TJ-10), which is a combination of Keishi-to (TJ-45) and Sho-saiko-to (TJ-9), we investigated the effects of these two herbal medicines and their components on pancreatic acinar cell injury models in vivo and in vitro. Methods: Four-week-old male WBN/Kob rats were fed an MB-3 pellet diet containing herbal medicine (TJ-9, TJ-10 and TJ-45). Expressions of pancreatitis-associated protein (PAP) and manganese superoxide dismutase (Mn-SOD) were analyzed with a reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. The herbal medicines and two of their components, Keihi (Cinnamomi cortex) and Shakuyaku (Paeoniae radix alba), were tested in vitro using an arginine-treated rat pancreatic acinar AR4-2J cell injury model. The inducible nitric oxide synthase (iNOS) was assayed in in vitro experiments. Results: TJ-45-treated WBN/Kob rats showed no evidence of pancreatitis whereas there were pathological changes of chronic pancreatitis in TJ-9-treated WBN/Kob rats. PAP was not expressed and Mn-SOD expression was increased in the TJ-10-, and TJ-45-treated rats. The herbal medicines and two components suppressed PAP mRNA expression and enhanced Mn-SOD and iNOS mRNA expression in arginine-treated AR4-2J cells. Conclusion: These results suggest that the herbal medicine TJ-45 is effective for chronic pancreatitis caused by pancreatic ischemia.

Reduced Expression of Manganese Superoxide Dismutase mRNA May Correlate with Invasiveness in Esophageal Carcinoma

Little is known about the expression and antioxidant function of manganese superoxide dismutase (Mn-SOD) in esophageal squamous cell carcinoma. To determine the significance of Mn-SOD in esophageal squamous cell carcinomas, Mn-SOD mRNA expression was examined in 45 esophageal squamous cell carcinomas and the corresponding normal mucosal tissues by reverse transcription-polymerase chain reaction. The tumor/normal (T/N) ratio of 45 patients with esophageal carcinoma was calculated, and the data were clinicopathologically analyzed. The T/N ratio of Mn-SOD mRNA expression was less than 0.5 in 11 (32.4%) of 34 esophageal carcinoma cases without any preoperative treatments, while none of 11 cases who underwent preoperative chemotherapy showed a T/N ratio of <0.5 (p < 0.05). There was an inverse correlation between the Mn-SOD expression level and the degree of venous invasion (p < 0.05) as well as lymphatic invasion (p < 0.05). Furthermore, poorly differentiated squamous cell carcinoma showed significantly lower Mn-SOD mRNA expression levels than well differentiated carcinoma (p < 0.05). Our results suggest that Mn-SOD mRNA was frequently reduced in esophageal carcinoma when compared to the normal mucosa and the reduced expression levels of Mn-SOD mRNA may lead to an accumulation of superoxide radicals in conjunction with the increased invasiveness of esophageal carcinoma.

Cytokines induce apoptosis in beta-cells isolated from mice lacking the inducible isoform of nitric oxide synthase (iNOS-/-).

Prolonged exposure of rodent beta-cells to combinations of cytokines induces the inducible form of nitric oxide synthase (iNOS) expression and Fas expression, nitric oxide (NO) production, and cell death. It also induces the expression of potential "defense" genes, such as manganese superoxide dismutase (MnSOD) and heat shock protein (hsp) 70. NO is a radical with multifaceted actions. Recent studies have shown that NO, in addition to having cytotoxic actions, may regulate gene transcription. It remains unclear whether NO mediates cytokine-induced gene expression and subsequent beta-cell death. Previous studies using NO synthase blockers yielded conflicting results, which may be due to nonspecific effects of these agents. In this study, we examined the effects of cytokines on gene expression, determined by reverse transcriptase-polymerase chain reaction (RT-PCR), and viability, determined by nuclear dyes, of pancreatic islets or fluorescence-activated cell sorter (FACS)-purified beta-cells isolated from iNOS knockout mice (iNOS-/-, background C57BL/6x129SvEv) or their respective controls (C57BL/6x129SvEv). The combination of cytokines used was interleukin-1beta (50 U/ml) plus gamma-interferon (1,000 U/ml) plus tumor necrosis factor-alpha (1,000 U/ml). The lack of cytokine-induced iNOS activity in the iNOS-/- islet cells was confirmed by RT-PCR and nitrite determination. Cytokines induced a >3-fold increase in Fas and MnSOD mRNA expression in wild-type (WT) and iNOS-/- islets. On the other hand, hsp 70 was induced in WT but not in iNOS-/- islets. Prolonged (6-9 days) exposure of WT islets to cytokines leads to an 80-90% decrease in islet cell viability, whereas viability decreased by only 10-30% in iNOS-/- islet cells. To determine the mode of cytokine-induced cell death, FACS-purified beta-cells were exposed to the same cytokines. After 9 days, the apoptosis index was similarly increased in WT (39 +/- 3%) and iNOS4-/- (33 +/- 4%) beta-cells. On the other hand, cytokines increased necrosis in WT (20 +/- 4%) but not in iNOS-/- (7 +/- 3%) beta-cells. From these data, we concluded that 1) NO is required for cytokine-induced hsp 70 mRNA expression but not for Fas and MnSOD expression, 2) cytokines induce both apoptosis and necrosis in mouse beta-cells, and 3) cytokine-induced apoptosis is mostly NO-independent, whereas necrosis requires NO formation.

Overexpression of manganese superoxide dismutase mRNA may correlate with aggressiveness in gastric and colorectal adenocarcinomas.

The expression or activity of manganese superoxide dismutase (Mn-SOD) is reduced in a variety of malignant tumors and Mn-SOD may act as a new type of tumor suppressor gene. On the other hand, increased expression of Mn-SOD can diminish the cytotoxic effects of several anticancer modalities, including tumor necrosis factor alpha, ionizing radiation, certain chemotherapeutic agents and hyperthermia. Although Mn-SOD expression and its role in various cancers are intensely studied, little is known about its function in gastrointestinal carcinomas. To examine the expression level and significance of Mn-SOD in gastrointestinal carcinomas, Mn-SOD mRNA expression was examined in 53 gastric carcinoma and 38 colorectal carcinoma by reverse transcription-polymerase chain reaction and was compared with those in the corresponding normal mucosal tissues. The tumor/normal (T/N) ratio was calculated and the data were clinicopathologically analyzed. The average T/N ratios of Mn-SOD mRNA expression in gastric and colorectal carcinomas were 2.19 and 3. 72, respectively. Clinicopathologic analyses revealed positive correlation between the Mn-SOD expression level and venous invasion in both gastric and colorectal carcinomas (p<0.05 and p<0.05, respectively). Furthermore, the colorectal carcinoma with lymph node metastasis showed significantly higher Mn-SOD expression than those without it (p<0.05). Our results suggest that Mn-SOD mRNA overexpression can occur in gastric and colorectal carcinomas and may be related to increased aggressiveness.

Expression of managanese superoxide dismutase in rat submandibular gland demonstrated by in situ hybridization and immunohistochemistry

Superoxide, an active oxygen species, plays an important role in protecting against bacterial infection. However, it also has adverse effects on health. Manganese superoxide dismutase (Mn-SOD) is a scavenger of superoxide. Antioxygen enzymes such as Mn-SOD exist in various tissues, and provide protections against oxidative injury. We investigated both the expression of Mn-SOD mRNA and the localization of Mn-SOD in adult rat submandibular glands using in situ hybridization and immunohistochemistry. Both Mn-SOD mRNA and Mn-SOD were detected in striated duct cells, and in some granular duct cells and excretory duct cells. With immunoelectron microscopy, many immunolabelings were observed on the mitochondria. These findings suggest that the expression of Mn-SOD mRNA and the localization of Mn-SOD in submandibular glands correlate with the number of mitochondria in cells.

INCREASE OF MnSOD EXPRESSION AND DECREASE OF JNK ACTIVITY DETERMINE THE TNF SENSITIVITY INbcl2-TRANSFECTED L929 CELLS

To investigate the protection mechanism of Bcl-2 against tumour necrosis factor (TNF)-mediated cell death, thebcl2gene was transfected into the L929 cells and stably expressed. Two clones having different sensitivity amongbcl2-transfected L929 clones had been isolated, and termed clone R1 and R2. It was observed that activation of manganese superoxide dismutase (MnSOD) and suppression of Jun kinase of clone R1 and R2 were correlated with protection from TNF cytotoxicity. Upon treatment with TNF, clone R1 and R2 were more resistant than control L929 cells against TNF cytotoxicity and the protective effect of clone R1 was stronger than clone R2. However, in case of TNF plus actinomycin D treatment, clone R1 was still resistant against TNF cytotoxicity, whereas clone R2 became more sensitive than control L929 cells. The JNK activities of clone R1 and R2 were suppressed upon TNF treatment and in case of TNF plus actinomycin D treatment, clone R2 showed a marked increase in JNK activities and had higher activity than control L929 cells. The specific activities of MnSOD of clone R1 and R2 upon TNF treatment were 70U/ml and 33U/ml, respectively, while the MnSOD activity was not detectable in control L929 cells. When TNF and actinomycin D were treated simultaneously, MnSOD activity was not detectable in control L929 cells andbcl2-transfected L929 cells (clone R1, R2). Consistent with these results, both clone R1 and R2 showed higher levels of MnSOD mRNA expression than control L929 cells after TNF treatment. These data suggest that suppression of Jun kinase and increase of MnSOD may be involved in inhibitory action of Bcl-2 against TNF, and the balance between MnSOD and JNK signalling pathway may be an important factor for the protection ofbcl2-transfected L929 cells from TNF cytotoxicity.

Effect of oxygen on lung superoxide dismutase activities in premature baboons with bronchopulmonary dysplasia.

We investigated the effects of gestational age and oxygen exposure on superoxide dismutase (SOD) activities in distal fetal lung tissue in primate models of bronchopulmonary dysplasia. During the final third of fetal life, lung coppper-zinc SOD (Cu,ZnSOD) specific activity decreased, whereas lung manganese SOD (MnSOD) specific activity tended to increase. In the premature newborn (140 days, 78% of term gestation), lung total SOD and Cu,ZnSOD specific activities decreased after 6-10 days of ventilation with as needed [pro re nada (PRN)] or 100% oxygen compared with fetal control animals. Neither Cu,ZnSOD mRNA nor protein expression changed after either oxygen exposure at this gestation (140 days) relative to fetal control animals. At this age (6-10 days), lung MnSOD specific activity did not change in oxygen-exposed relative to fetal control animals, even though lung expression of MnSOD mRNA and protein increased after PRN or 100% oxygen exposure. In the very premature 125-day newborn (69% of term), lung Cu,ZnSOD specific activity and protein decreased, whereas Cu,ZnSOD mRNA increased, after 6-10 days of ventilation with PRN oxygen compared with fetal control animals. In fetal lung explants, hyperoxia also decreased expression of SOD activity acutely (16-h exposure, 125 and 140 days gestation). To conclude, expression of SOD activity in the premature primate lung did not increase in response to elevated oxygen tension, apparently due to effects occurring subsequent to the expression of these mRNAs.

Neutrophil superoxide release is required for spontaneous and FMLP-mediated but not for TNF alpha-mediated apoptosis.

Polymorphonuclear leukocyte (PMN) lifespan is characterized by both rapid production and apoptotic cell death. The mechanisms triggering apoptosis in PMN are not completely understood. In this study, the relationship of neutrophil activation and apoptosis as related to released superoxide was investigated. PMN apoptosis was detected by DNA fragmentation, and ultraviolet and light microscopy, and was quantified by flow cytometry; superoxide release was measured by superoxide dismutase-inhibitable reduction of ferricytochrome C. Incubation of PMN with 20 ng/ml tumor necrosis factor (TNF)alpha induced superoxide release (8.8 +/- 7.5 nmol O2-/30 min, n = 7) in normal PMN and also resulted in apoptosis within 2 h, whereas a subactivating dose of 2 ng/ml TNF alpha, which did not trigger superoxide release (3.1 +/- 1.7 nmol O2-, n = 10), did facilitate apoptosis, although to a lesser degree. PMN cultured under nonstimulating conditions underwent apoptotic cell death after 8 h. Exogenous superoxide dismutase did not inhibit apoptosis induced by 20 ng/ml TNF alpha. No upregulation of endogenous manganese superoxide dismutase mRNA expression was observed in response to TNF alpha as measured by reverse transcription PCR. Formyl-methionyl-leucyl-phenylalanine (FMLP) stimulation (10(-7) M) resulting in superoxide release of 31.7 +/- 6.1 nmol O2-/30 min (n = 10) also significantly increased the percentage of apoptosis, but at 24 h (P < 0.05). Exogenous superoxide dismutase did inhibit FMLP-induced apoptosis, as well as apoptosis due to aging in culture. In conclusion, aging and FMLP-stimulated PMN undergo apoptosis by a superoxide release-dependent pathway, whereas TNF alpha-facilitated apoptosis appears to be unrelated to respiratory burst oxidase activity.

Enhanced Expression of Manganese Superoxide Dismutase mRNA and Increased TNFα mRNA Expression by Gastric Mucosa in Gastric Cancer

Manganese superoxide dismutase (Mn-SOD), a mitochondrial enzyme, is a cytokine-regulated acute-phase protein that protects cells from free radicals. The current investigations examined the in vivo regulation of the expression of Mn-SOD mRNA and tumor necrosis factor alpha (TNFalpha) mRNA in gastric carcinoma tissue. The expression of these transcripts in breast carcinoma tissue also was examined because breast cancer is a much more TNF-sensitive tumor than gastric cancer. TNFalpha mRNA was markedly increased in gastric carcinoma tissue (p < 0.005). There were significantly higher levels of Mn-SOD mRNA in gastric carcinoma tissue than in noncancerous tissue (p < 0.0001). The level of Mn-SOD mRNA in gastric carcinoma tissue was higher than that in breast carcinoma tissue (p < 0.005). Up-regulation of Mn-SOD mRNA in gastric carcinoma tissue most likely serves as a protective mechanism against superoxide radicals and TNF cytotoxicity.

Expression of manganese superoxide dismutase mRNA in reproductive organs during the ovulatory process and the estrous cycle of the rat.

Expression of manganese superoxide dismutase (Mn-SOD) mRNA during the pregnant mare serum gonadotrophin (PMSG)/human chorionic gonadotrophin (HCG)-induced ovulatory process, and during the estrous cycle was examined in rat female reproductive organs. Mn-SOD mRNA levels in theca interna cells markedly increased in PMSG-primed rats and high levels of the transcripts were maintained after HCG injection. The PMSG-enhanced expression of Mn-SOD mRNA in follicular epithelial cells increased concomitantly with luteinization of these cells. The levels of Mn-SOD mRNA remained high and became equivalent in both granulosa and theca lutein cells 24 h after HCG injection. Neither luteinization nor the expression of Mn-SOD mRNA was observed in the epithelial cells of unovulated follicles. Luteal bodies had formed 3 days after HCG injection, and the same level of Mn-SOD mRNA expression continued in lutein cells, but not in stromal cells. During the estrous cycle, Mn-SOD mRNA was localized to theca interna cells on proestrus, to the epithelial cells of luteinizing follicles on estrus, and to newly formed luteal bodies on diestrus. The epithelial cells in the oviduct did not express Mn-SOD mRNA throughout the ovulatory process or the estrous cycle. Expression of Mn-SOD mRNA in the luminal epithelial cells of the uterus increased after PMSG injection, reaching a maximum after 24 h, and became relatively negative 3 days after HCG injection when corpora lutea had formed in the ovary. During the estrous cycle, uterine epithelial cells and leukocytes showed marked increases in Mn-SOD mRNA expression on estrus and on proestrus, respectively. Expression in the vaginal epithelium became apparent 3 days after HCG injection and continued for at least 12 days after HCG injection. The expression was localized to the superficial layer of the epithelium. During the estrous cycle, expression occurs in the basal layer on proestrus and estrus, transferring to the superficial layer on diestrus day 1, and expression stops on diestrus day 2. The relationship between the expression of Mn-SOD mRNA and hormone-induced metabolic changes, including steroidogenesis, is discussed.

Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1β and TNF-α

Because reactive oxygen species have been implicated in the pathogenesis of various hyperproliferative and inflammatory diseases, the mRNA expression of the antioxidant enzyme superoxide dismutase was studied in psoriatic skin tissue. By using reverse transcription-PCR we found similar expression of copper, zinc superoxide dismutase (CuZnSOD) in the involved vs. uninvolved psoriatic skin. In contrast, the level of the manganese superoxide dismutase (MnSOD) mRNA message was consistently higher in lesional psoriatic skin as compared to adjacent uninvolved skin and healthy control skin. Parallel investigation of those cytokines that are thought to be direct or indirect inducers of the MnSOD activity revealed an increased mRNA expression of IL-1β, TNF-α, and GM-CSF in lesional psoriatic skin. To study if these cytokines exert a direct effect on dismutase expression in epidermal cells, human keratinocytes in culture were challenged with IL-1β, TNF-α, and GM-CSF. It was found that IL-1β and TNF-α, but not GM-CSF, induced the mRNA expression of MnSOD, and an additive effect was demonstrated for the two former cytokines. Further, the expression of both CuZnSOD and MnSOD transcripts was similar in cultured keratinocytes maintained at low differentiation (low Ca 2+ medium) and cells forced to terminal differentiation (by high Ca 2+ medium). Our results indicate that the abnormal expression of MnSOD mRNA in lesional psoriatic skin is not directly linked to the pathologic state of keratinocyte differentiation in the skin. It seems more likely that the cutaneous overexpression of MnSOD in psoriatic epidermis represents a protective cellular response evoked by cytokines released from inflammatory cells invading the diseased skin.

Expression of Manganese Superoxide Dismutase mRNA in the Rat Adrenal Cortex is Associated with the Occurrence of Mitochondrial Vesicular Cristae.

To investigate the possible role of manganese superoxide dismutase (Mn-SOD) in steroidogenesis, we studied the expression of Mn-SOD mRNA in the adrenal gland and ovaries of normal and hypophysectomized rats by in situ hybridization. Very strong expression of Mn-SOD mRNA was detected in the zona fasciculata and the zona reticularis of the rat adrenal cortex. The expression was very low in the zona glomerulosa, and was almost negative in the adrenal medulla. The mRNA levels in the adrenal cortex and the ovaries were markedly reduced 4 days after hypophysectomy. Marked adrenocortical atrophy occurred 14 to 24 days after the operation, while levels of Mn-SOD mRNA in the cortex remained low. Mitochondrial structure in the fasciculata cells of normal and hypophysectomized rats were compared by electron microscopy. Mitochondria in the fasciculata cells of normal rats were characterized by dense vesicular Cristae. Four days after hypophysectomy, the cristae became tubulo-vesicular in form and their density decreased significantly. Elongated mitochondria with tubular cristae appeared. Oval mitochondria with a few tubulo-vesicular cristae were present in these cells 14 days after the operation. Our results are consistent with the hypothesis that high Mn-SOD levels might play an important role in the regulation of steroidogenesis. The possible roles of superoxide radicals and hydrogen peroxide in the formation of vesicular cristae in the mitochondria are discussed with respect to steroidogenesis.

Dissociation between Interleukin-1β-Induced Expression of mRNA for Superoxide-Dismutase and Nitric Oxide Synthase in Insulin-Producing Cells

We presently investigated the induction of manganese superoxide dismutase (MnSOD) and nitric oxide synthase (iNOS) mRNA by interleukin-1β (IL-1β) in insulin-producing RINm5F cells. IL-1β induced both mRNAs in parallel, with increased levels detectable after 4 h and further increase at 6 h. Aminoguanidine, a blocker of NO production, did not prevent IL-1β-induced MnSOD mRNA expression, and SNP, a NO releasing agent, did not induce MnSOD mRNA. Actinomycin D, an inhibitor of gene transcription, prevented IL-1β induction of both MnSOD and iNOS mRNA. Cycloheximide, an inhibitor of protein synthesis, prevented IL-1β-induced expression of iNOS mRNA, but not MnSOD mRNA. These data suggest that induction of MnSOD mRNA by IL-1β is independent of iNOS expression and NO production. Moreover, while expression of iNOS mRNA depends on protein synthesis, MnSOD mRNA induction does not necessarily require this step. Thus, it seems that IL-1 induces genes potentially involved in cell damage (NOS) and defense (MnSOD) by different mechanisms.

Distribution of Manganese Superoxide Dismutase mRNA in Normal and Hyperoxic Rat Lung

Aerobic metabolism requires a continuous oxygen supply, which in turn can form partially reduced species (free radicals) that damage cellular components. To prevent this, organisms have elaborate free radical-scavenging defenses that include the superoxide dismutases. The lungs are unique in their role as an oxygen-gathering system, making these defenses critical to lung integrity. Manganese superoxide dismutase (Mn-SOD) levels increase in rats exposed to sublethal doses of hyperoxia and correlate with the development of tolerance to higher levels of hyperoxia. Although pulmonary Mn-SOD protein and mRNA levels both change with hyperoxia, the timing and levels differ dramatically. Lung heterogeneity makes extrapolation of data from whole tissue homogenates or cultures difficult. In this study, in situ hybridization of Mn-SOD in the lungs of adult rats exposed to air or to 85% O2 for 3 days was performed. In animals exposed to either air or 85% O2, Mn-SOD transcripts were present in arterioles, the septal tips of alveolar ducts, alveolar type II cells, and mesothelial cells. Hyperoxic lung had an intense, continuous labeling of the pleura that was distinctly greater than the intermittent labeling of the pleura found in control animals. The high level of expression of Mn-SOD mRNA in alveolar duct septal tips in both control and O2-exposed animals may be secondary to increased aerobic activity in these regions, which contain collagen and elastin and are important stress-bearing elements in the lung. Alveolar type II cells are metabolically active secretory cells and thus may experience increased endogenously generated oxidative stress. Pleural effusions are common after hyperoxic exposures, suggesting damage to the mesothelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of antioxidant enzymes in response to oxidants.

We have demonstrated the selective induction of manganese superoxide dismutase (MnSOD) or catalase mRNA after exposure of tracheobronchial epithelial cells in vitro to different oxidant stresses. Addition of H2O2 caused a dose-dependent increase in catalase mRNA in both exponentially growing and confluent cells. A 3-fold induction of catalase mRNA was seen at a nontoxic dose of 250 microM H2O2. Increase in the steady-state mRNA levels of glutathione peroxidase (GPX) and MnSOD were less striking. Expression of catalase, MnSOD, and GPX mRNA was highest in confluent cells. In contrast, constitutive expression of copper and zinc SOD (CuZnSOD) mRNA was greatest in dividing cells and was unaffected by H2O2 in both exponentially growing and confluent cells. MnSOD mRNA was selectively induced in confluent epithelial cells exposed to the reactive oxygen species-generating system, xanthine/xanthine oxidase, while steady-state levels of GPX, catalase, and CuZnSOD mRNA remained unchanged. The 3-fold induction of MnSOD mRNA was dose-dependent, reaching a peak at 0.2 unit/ml xanthine oxidase. MnSOD mRNA increases were seen as early as 2 h and reached maximal induction at 24 h. Immunoreactive MnSOD protein was produced in a corresponding dose- and time-dependent manner. Induction of MnSOD gene expression was prevented by addition of actinomycin D and cycloheximide. These data indicate that epithelial cells of the respiratory tract respond to different oxidant insults by selective induction of certain antioxidant enzymes. Hence, gene expression of antioxidant enzymes does not appear to be coordinately regulated in these cell types.

Phorbol ester induces manganese-superoxide dismutase in tumor necrosis factor-resistant cells.

The effects of phorbol ester (TPA) and other known stimulators such as tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide on induction of mRNA for manganese-superoxide dismutase (Mn-SOD) were investigated in various cell lines. TPA enhanced Mn-SOD mRNA expression in TNF-resistant cell lines including HeLa cells, in which the other reagents also induced expression of the gene, but did not affect TNF-sensitive cells, in which the other stimulators did not alter expression of the gene. HeLa cells which had been desensitized to TPA by pretreatment with TPA for 24 h expressed Mn-SOD mRNA at a slightly higher level than the cells without TPA treatment. TPA-pretreated cells stimulated with TNF, however, expressed Mn-SOD mRNA at about twice the level of TNF-stimulated, TPA-untreated cells. When protein synthesis was inhibited by cycloheximide during TPA pretreatment, TNF no more enhanced the Mn-SOD mRNA accumulation. These data suggest that at least two separate signal-transducing pathways are involved in expression of this gene. One is triggered by protein kinase C activation itself in the absence of new protein synthesis. The other can be activated by stimulation with TNF, interleukin-1, or lipopolysaccharide and in which a protein factor that can be induced by TPA treatment is involved.