Skeletal muscle endurance declines with impaired mitochondrial respiration and inadequate supply of acetyl-CoA during muscle fatigue in 5/6 nephrectomized rats.

Chronic kidney disease (CKD)-related cachexia increases the risks of reduced physical activity and mortality. However, the physiological phenotype of skeletal muscle fatigue and changes in intramuscular metabolites during muscle fatigue in CKD-related cachexia remain unclear. In the present study, we performed detailed muscle physiological evaluation, analysis of mitochondrial function, and comprehensive analysis of metabolic changes before and after muscle fatigue in a 5/6 nephrectomized rat model of CKD. Wistar rats were randomized to a sham-operation (Sham) group that served as a control group or a 5/6 nephrectomy (Nx) group. Eight weeks after the operation, in situ torque and force measurements in plantar flexor muscles in Nx rats using electrical stimulation revealed a significant decrease in muscle endurance during subacute phase related to mitochondrial function. Muscle mass was reduced without changes in the proportions of fiber type-specific myosin heavy chain isoforms in Nx rats. Pyruvate-malate-driven state 3 respiration in isolated mitochondria was impaired in Nx rats. Protein expression levels of mitochondrial respiratory chain complexes III and V were decreased in Nx rats. Metabolome analysis revealed that the increased supply of acetyl CoA in response to fatigue was blunted in Nx rats. These findings suggest that CKD deteriorates skeletal muscle endurance in association with mitochondrial dysfunction and inadequate supply of acetyl-CoA during muscle fatigue.NEW & NOTEWORTHY Mitochondrial dysfunction is associated with decreased skeletal muscle endurance in chronic kidney disease (CKD), but the muscle physiological phenotype and major changes in intramuscular metabolites during muscle fatigue in CKD-related cachexia remain unclear. By using a 5/6 nephrectomized CKD rat model, the present study revealed that CKD is associated with reduced tetanic force in response to repetitive stimuli in a subacute phase, impaired mitochondrial respiration, and inadequate supply of acetyl-CoA during muscle fatigue.

Dissociation of SH3 and cysteine-rich domain 3 and junctophilin 1 from dihydropyridine receptor in dystrophin-deficient muscles.

The disruption of excitation-contraction (EC) coupling and subsequent reduction in Ca2+ release from the sarcoplasmic reticulum (SR) have been shown to account for muscle weakness seen in patients with Duchenne muscular dystrophy (DMD). Here, we examined the mechanisms underlying EC uncoupling in skeletal muscles from mdx52 and DMD-null/NSG mice, animal models for DMD, focusing on the SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), which link the dihydropyridine receptor (DHPR) in the transverse tubule and the ryanodine receptor 1 in the SR. The isometric plantarflexion torque normalized to muscle weight of whole plantar flexor muscles was depressed in mdx52 and DMD-null/NSG mice compared with their control mice. This was accompanied by increased autolysis of calpain-1, decreased levels of STAC3 and JP1 content, and dissociation of STAC3 and JP1 from DHPR-α1s in gastrocnemius muscles. Moreover, in vitro mechanistic experiments demonstrated that STAC3 and JP1 underwent Ca2+-dependent proteolysis that was less pronounced in dystrophin-deficient muscles where calpastatin, the endogenous calpain inhibitor, was upregulated. Eccentric contractions further enhanced autolysis of calpain-1 and proteolysis of STAC3 and JP1 that were associated with severe torque depression in gastrocnemius muscles from DMD-null/NSG mice. These data suggest that Ca2+-dependent proteolysis of STAC3 and JP1 may be an essential factor causing muscle weakness due to EC coupling failure in dystrophin-deficient muscles.NEW & NOTEWORTHY The mechanisms underlying the disruption of excitation-contraction (EC) coupling in dystrophin-deficient muscles are not well understood. Here, using animal models for Duchenne muscular dystrophies (DMD), we show a Ca2+-dependent protease (calpain-1)-mediated proteolysis of SH3 and cysteine-rich domain 3 (STAC3) and junctophilin 1 (JP1), essential EC coupling proteins, in dystrophin-deficient muscle, and highlighting the dissociation of STAC3 and JP1 from dihydropyridine receptor as a causative factor in EC uncoupling of dystrophic muscles.

Preconditioning contractions prevent prolonged force depression and Ca2+-dependent proteolysis of STAC3 after damaging eccentric contractions.

Preconditioning contractions (PCs) have been shown to markedly improve recovery from eccentric contractions (ECCs)-induced force depression. We here examined the mechanism behind the effects of PCs with focusing on the SH3 and cysteine-rich domain 3 (STAC3) that is essential for coupling membrane depolarization to Ca2+ release from the sarcoplasmic reticulum. Rat medial gastrocnemius (MG) muscles were excised immediately (REC0), 1 day (REC1), and 4 days (REC4) after exposure to 100 repeated damaging ECCs in vivo. PCs with 10 repeated nondamaging ECCs were applied 2 days before the damaging ECCs. Damaging ECCs induced in vivo isometric torque depression at 50 and 100 Hz stimulation frequencies, which was accompanied by a significant decrease in the amount of full-length STAC3, an activation of calpain 1, and an increased number of Evans Blue dye-positive fibers in MG muscles at REC1 and REC4. Interestingly, PCs attenuated all these deleterious alterations induced by damaging ECCs. Moreover, mechanistic experiments performed on normal muscle samples exposed to various concentration of Ca2+ showed a Ca2+-dependent proteolysis of STAC3, which was prevented by calpain inhibitor MDL-28170. In conclusion, PCs may improve recovery from force depression after damaging ECCs, in part by inhibiting the loss of STAC3 due to the increased permeability of cell membrane and subsequent activation of calpain 1.NEW & NOTEWORTHY The SH3 and cysteine-rich domain 3 (STAC3) is a skeletal muscle-specific protein that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release. No studies, however, examined the role of STAC3 in protective effects of preconditioning contractions (PCs) against damaging eccentric contractions (ECCs). Here, we demonstrate that PCs may improve recovery from damaging ECCs-induced force depression, in part by an inhibition of Ca2+-dependent proteolysis of STAC3 due to increased membrane permeability and subsequent calpain 1 activation.

Eccentric Resistance Training Ameliorates Muscle Weakness in a Mouse Model of Idiopathic Inflammatory Myopathies.

OBJECTIVE: High-force eccentric contractions (ECCs) have traditionally been excluded from rehabilitation programs that include patients with idiopathic inflammatory myopathies (IIMs) due to unverified fear of causing muscle damage and inflammation. In an IIM animal model that used mice with experimental autoimmune myositis (EAM), we undertook this study to investigate whether ECC training can safely and effectively be used to counteract muscle weakness in IIM. METHODS: EAM was induced in BALB/c mice by immunization with 3 injections of myosin emulsified in Freund's complete adjuvant. Controls (n = 12) and mice with EAM (n = 12) were exposed to either an acute bout of 100 ECCs or 4 weeks of ECC training (20 ECCs every other day). To induce ECCs, plantar flexor muscles were electrically stimulated while the ankle was forcibly dorsiflexed. RESULTS: Less cell damage, as assessed by Evans blue dye uptake, was observed in the muscles of mice with EAM, compared to controls, after an acute bout of 100 ECCs (P < 0.05). Maximum Ca2+ -activated force was decreased in skinned gastrocnemius muscle fibers from mice with EAM, and this was accompanied by increased expression of endoplasmic reticulum (ER) stress proteins, including Gsp78 and Gsp94 (P < 0.05). ECC training prevented the decrease in force and the increase in ER stress proteins and also enhanced the expression and myofibrillar binding of small heat-shock proteins (HSPs) (P < 0.05), which can stabilize myofibrillar structure and function. CONCLUSION: ECC training protected against the reduction in myofibrillar force-generating capacity in an IIM mouse model, and this occurred via inhibition of ER stress responses and small HSP-mediated myofibrillar stabilization.

Cancer Cachexia Induces Preferential Skeletal Muscle Myosin Loss When Combined With Denervation.

Patients with cancer cachexia (CCX) suffer from muscle wasting, which is often but not always accompanied by selective loss of myosin. Here we examined the effects of CCX on muscle mass and myosin heavy chain (MyHC) expression in denervated (DEN) muscles, especially focusing on the protein synthesis and degradation pathways. Male CD2F1 mice were randomly divided into control (CNT) and CCX groups and their left sciatic nerve was transected. CCX was induced by an intraperitoneal injection of colon 26 cells. After 14 days, the serum concentration of IL-6 and corticosteroid was higher in CCX mice than in CNT mice. The combination of CCX with DEN (CCX + DEN) resulted in a marked reduction of the gastrocnemius muscle weight (-69%) that was significantly lower than DEN (-53%) or CCX (-36%) alone. CCX had no effect on MyHC content, but it elicited a preferential MyHC loss when combined with DEN. The expression levels of autophagy markers cathepsin D and LC3BII/I ratio were markedly higher in the CCX + DEN group than in the CNT + DEN and the CCX groups. Paradoxically, there was an increase in protein synthesis rate and phosphorylation levels of p70S6K and rpS6, markers of mTORC1 signaling, in the CNT + DEN group, and these molecular alterations were inhibited in the CCX + DEN group. Our data indicate that CCX aggravates muscle atrophy in DEN muscles by inducing seletive loss of myosin, which involves inactivity dependent mechanisms that is likely to be a consequence of increased autophagy-mediated protein breakdown coupled with impaired protein synthesis.

Myofibrillar function differs markedly between denervated and dexamethasone-treated rat skeletal muscles: Role of mechanical load.

Although there is good evidence to indicate a major role of intrinsic impairment of the contractile apparatus in muscle weakness seen in several pathophysiological conditions, the factors responsible for control of myofibrillar function are not fully understood. To investigate the role of mechanical load in myofibrillar function, we compared the skinned fiber force between denervated (DEN) and dexamethasone-treated (DEX) rat skeletal muscles with or without neuromuscular electrical stimulation (ES) training. DEN and DEX were induced by cutting the sciatic nerve and daily injection of dexamethasone (5 mg/kg/day) for 7 days, respectively. For ES training, plantarflexor muscles were electrically stimulated to produce four sets of five isometric contractions each day. In situ maximum torque was markedly depressed in the DEN muscles compared to the DEX muscles (-74% vs. -10%), whereas there was not much difference in the degree of atrophy in gastrocnemius muscles between DEN and DEX groups (-24% vs. -17%). Similar results were obtained in the skinned fiber preparation, with a greater reduction in maximum Ca2+-activated force in the DEN than in the DEX group (-53% vs. -16%). Moreover, there was a parallel decline in myosin heavy chain (MyHC) and actin content per muscle volume in DEN muscles, but not in DEX muscles, which was associated with upregulation of NADPH oxidase (NOX) 2, neuronal nitric oxide synthase (nNOS), and endothelial NOS expression, translocation of nNOS from the membrane to the cytosol, and augmentation of mRNA levels of muscle RING finger protein 1 (MuRF-1) and atrogin-1. Importantly, mechanical load evoked by ES protects against DEN- and DEX-induced myofibrillar dysfunction and these molecular alterations. Our findings provide novel insights regarding the difference in intrinsic contractile properties between DEN and DEX and suggest an important role of mechanical load in preserving myofibrillar function in skeletal muscle.

Eccentric training enhances the αB-crystallin binding to the myofibrils and prevents skeletal muscle weakness in adjuvant-induced arthritis rat.

Patients with rheumatoid arthritis (RA) frequently suffer from muscle weakness. We examined whether eccentric training prevents skeletal muscle weakness in adjuvant-induced arthritis (AIA) rat, a widely used animal model for RA. AIA was induced in the knees of Wistar rats by injection of complete Freund's adjuvant. To induce eccentric contractions (ECCs), neuromuscular electrical stimulation (45 V) was applied to the plantar flexor muscles simultaneously with forced dorsiflexion of the ankle joint (0-40°) and was given every 6 s. ECC exercise was applied every other day for a total of 11 sessions and consisted of 4 sets of 5 contractions. There was a significant reduction in in vitro maximum Ca2+-activated force in skinned fibers in gastrocnemius muscle from AIA rats. These changes were associated with reduced expression levels of contractile proteins (i.e., myosin and actin), increased levels of inflammation redox stress-related biomarkers (i.e., TNF-α, malondialdehyde-protein adducts, NADPH oxidase 2, and neuronal nitric oxide synthase), and autolyzed active calpain-1 in AIA muscles. ECC training markedly enhanced the steady-state levels of αB-crystallin, a small heat shock protein, and its binding to the myofibrils and prevented the AIA-induced myofibrillar dysfunction, reduction in contractile proteins, and inflammation-oxidative stress insults. Our findings demonstrate that ECC training preserves myofibrillar function without muscle damage in AIA rats, which is at least partially attributable to the protective effect of αB-crystallin on the myofibrils against oxidative stress-mediated protein degeneration. Thus ECC training can be a safe and effective intervention, counteracting the loss of muscle strength in RA patients. NEW & NOTEWORTHY Eccentric contractions (ECCs) are regarded as an effective way to increase muscle strength. No studies, however, assess safety and effectiveness of ECC training on muscle weakness associated with rheumatoid arthritis. Here, we used adjuvant-induced arthritis (AIA) rats to demonstrate that ECC training prevents intrinsic contractile dysfunction without muscle damage in AIA rats, which may be attributed to the protective effect of αB-crystallin on the myofibrils against inflammation-oxidative stress insults.

Preconditioning contractions prevent the delayed onset of myofibrillar dysfunction after damaging eccentric contractions.

KEY POINTS: We examined the mechanisms underlying the positive effect of preconditioning contractions (PCs) on the recovery of muscle force after damaging eccentric contractions (ECCs). The mechanisms underlying the immediate force decrease after damaging ECCs differ from those causing depressed force with a few days' delay, where reactive oxygen species (ROS) produced by invading immune cells play an important causative role. PCs counteracted the delayed onset force depression and this could be explained by prevention of immune cell invasion, which resulted in decreased myeloperoxidase-mediated ROS production, hence avoiding cell membrane disruption, calpain activation and degenerative changes in myosin and actin molecules. ABSTRACT: Preconditioning contractions (PCs) have been shown to result in markedly improved contractile function during the recovery periods after muscle damage from eccentric contractions (ECCs). Here, we examined the mechanisms underlying the beneficial effect of PCs with a special focus on the myofibrillar function. Rat medial gastrocnemius muscles were exposed to 100 repeated damaging ECCs in situ and excised immediately (recovery 0, REC0) or after 4 days (REC4). PCs with 10 repeated non-damaging ECCs were applied 2 days before the damaging ECCs. PCs improved in situ maximal isometric torque at REC4. Skinned muscle fibres were used to directly assess changes in myofibrillar function. PCs prevented the damaging ECC-induced depression in maximum Ca2+ -activated force at REC4. PCs also prevented the following damaging ECC-induced effects at REC4: (i) the reduction in myosin heavy chain and actin content; (ii) calpain activation; (iii) changes in redox homeostasis manifested as increased expression levels of malondialdehyde-protein adducts, NADPH oxidase 2, superoxide dismutase 2 and catalase, and activation of myeloperoxidase (MPO); (iv) infiltration of immune cells and loss of cell membrane integrity. Additionally, at REC0, PCs enhanced the expression levels of heat shock protein (HSP) 70, HSP25, and αB-crystallin in the myofibrils and prevented the increased mRNA levels of granulocyte-macrophage colony-stimulating factor and interleukin-6. In conclusion, PCs prevent the delayed force depression after damaging ECCs by an HSP-dependent inhibition of degenerative changes in myosin and actin molecules caused by myeloperoxidase-induced membrane lysis and subsequent calpain activation, which were triggered by an inflammatory reaction with immune cells invading damaged muscles.

High-intensity eccentric training ameliorates muscle wasting in colon 26 tumor-bearing mice.

Eccentric (ECC) contractions are used to maintain skeletal muscle mass and strength in healthy subjects and patients. Here we investigated the effects of ECC training induced by electrical stimulation (ES) on muscle wasting in colon 26 (C-26) tumor-bearing mice. Mice were divided into four groups: control (CNT), CNT + ECC, C-26, and C-26 + ECC. Cancer cachexia was induced by a subcutaneous injection of C-26 cells and developed for four weeks. In experiment 1, muscle protein synthesis rate and mammalian target of rapamycin complex (mTORC) 1 signaling were investigated six hours after one bout of ECC-ES (2 s contraction given every 6 s, 20°/s, 4 sets of 5 contractions). In experiment 2, ECC-ES training, a total of 14 sessions, was performed every other day starting one day after C-26 injection. Compared to the CNT mice, the gastrocnemius muscle weight was significantly decreased in the tumor-bearing mice. This change was accompanied by a reduction in protein synthesis rate and a marked increase in the expression levels of genes including regulated in development and DNA damage responses (REDD) 1, forkhead box protein O1 (FoxO1), muscle-specific E3 ubiquitin ligases atrogin-1, and muscle ring finger 1 (MuRF-1) mRNA. ECC-ES increased the protein synthesis rate and the phosphorylation levels of p70S6K (Thr389) and rpS6 (Ser240/244), markers for mTORC1 signaling, and reversed an upregulation of MuRF-1 mRNA in muscles from C-26 mice. Our findings suggest that ECC-ES training reduces skeletal muscle atrophy in C-26 tumor-bearing mice through activation of mTORC1 signaling and the inhibition of ubiquitin-proteasome pathway. Thus, ECC-ES training might be used to effectively ameliorate muscle wasting in patients with cancer cachexia.

Ability of Group IVB metallocene polyethers containing dienestrol to arrest the growth of selected cancer cell lines.

BACKGROUND: Monomeric Group IVB (Ti, Zr and Hf) metallocenes represent a new class of antitumor compounds. There is literature on the general biological activities of some organotin compounds. Unfortunately, there is little information with respect to the molecular level activity of these organotin compounds. We recently started focusing on the anti-cancer activity of organotin polymers that we had made for other purposes and as part of our platinum anti-cancer effort. METHODS: For this study, we synthesized a new series of metallocene-containing compounds coupling the metallocene unit with dienestrol, a synthetic, nonsteroidal estrogen. This is part of our effort to couple known moieties that offer antitumor activity with biologically active units hoping to increase the biological activity of the combination. The materials were confirmed to be polymeric using light scattering photometry and the structural repeat unit was verified employing matrix assisted laser desorption ionization mass spectrometry and infrared spectroscopy results. RESULTS: The polymers demonstrated the ability to suppress the growth of a series of tumor cell lines originating from breast, colon, prostrate, and lung cancers at concentrations generally lower than those required for inhibition of cell growth by the commonly used antitumor drug cisplatin. CONCLUSION: These drugs show great promise in vitro against a number of cancer cell lines and due to their polymeric nature will most likely be less toxic than currently used metal-containing drugs such as cisplatin. These drugs also offer several addition positive aspects. First, the reactants are commercially available so that additional synthetic steps are not needed. Second, synthesis of the polymer is rapid, occurring within about 15 seconds. Third, the interfacial synthetic system is already industrially employed in the synthesis of aromatic nylons and polycarbonates. Thus, the ability to synthesize large amounts of the drugs is straight forward.

Cepharanthin, a biscoclaurine alkaloid, prevents destruction of acinar tissues in murine Sjögren's syndrome.

OBJECTIVE: Our previous study suggested that suppression by cepharanthin of tumor necrosis factor-a (TNF-a)-induced matrix metalloproteinase-9 (MMP-9) could prevent destruction of the acinar structure in the salivary glands of patients with Sjögren's syndrome (SS). In this study, we observed that in vivo administration of cepharanthin prevented severe damage to acinar tissues in the murine model of human SS. METHODS: Cepharanthin was intraperitoneally administered to thymectomized female NFS/sld mice. Inflammatory lesions in the salivary and lacrimal glands were then examined histologically. Expression of phosphorylated IkB-a, MMP-9, and type IV collagen was analyzed immunohistochemically. The apoptotic cell death of acinar cells was determined. RESULTS: Although extensive mononuclear cell infiltration and destruction of acinar tissue in salivary and lacrimal glands were observed in control mice, significant improvement of these lesions was evident in mice treated with cepharanthin. Immunohistochemical analysis revealed that p65, phosphorylated IkB-a, and MMP-9 were more strongly stained in the acinar cells of control mice than in cepharanthin-treated mice. Although no staining for type IV collagen was observed in the acinar tissues of control mice, continuity of staining for type IV collagen was observed in acinar tissues of cepharanthin-treated mice. Destruction of acinar tissues was attributed to the induction of apoptosis, suggesting that cepharanthin inhibits apoptosis by suppressing phosphorylation of IkB-a, followed by prevention of MMP-9 activation. CONCLUSION: Our findings suggest that cepharanthin may be a promising agent for use in preventing destruction of acinar tissues in murine SS.

Anesthetic management of a neonate with vein of Galen aneurysmal malformations and severe pulmonary hypertension.

Vein of Galen aneurysmal malformations (VGAMs) are rare congenital vascular malformations and excessive arteriovenous shunt causes intractable congestive high-output heart failure in the neonate. We report a case of successful staged transcatheter embolizations for a neonate with congestive heart failure and pulmonary hypertension (PH). Heart failure was dramatically relieved as the staged procedure progressed. Although transcatheter embolizations is essential for the treatment, inhaled nitric oxide (iNO) was helpful as a bridge treatment to reduce right-to-left shunt before the initial emergency embolization. Endovascular embolization is a less invasive therapy than open cranial surgery and allows hemodynamic stability. Perioperative iNO can be used to manage PH in VGAMs.

Potentiation of induction of apoptosis by sequential treatment with cisplatin followed by 5-fluorouracil in human oral cancer cells.

We examined the mechanism involved in the induction of apoptosis in human oral cancer (B88) cells with 5-fluorouracil (5-FU) and cisplatin (CDDP) combination. Three different combination treatment sequences were evaluated: i) 5-FU administered simultaneously with CDDP for 72 h (sequence I); ii) CDDP administered for 24 h before 5-FU treatment for 48 h (sequence II); and iii) 5-FU administered for 24 h before CDDP treatment for 48 h (sequence III). When combining the two drugs at doses 40% of their respective cytotoxicity, the growth-suppressing ratios were 49, 55, and 40% in sequences I, II, and III, respectively. Caspase 3 was significantly activated in sequence II as compared to its level of activation in sequence I or III. The mitochondrial release of cytochrome c was much greater in sequence II than in sequence III. In addition, activation of caspase 8 was most strongly activated in sequence II as compared with sequences I and III. Activation of caspase-9 was also observed in sequence II, and, to a lesser extent, in sequence III. Finally, although Bcl-2 was reduced in sequence II, no significant change was observed in sequence III. Accordingly, the data presented here demonstrate that sequence II showing a significant increase in the induction of apoptosis of cancer cells, is superior to sequences I and III.

Enhanced radiosensitization and chemosensitization in NF-kappaB-suppressed human oral cancer cells via the inhibition of gamma-irradiation- and 5-FU-induced production of IL-6 and IL-8.

We examined the mechanisms underlying the enhancement of radiosensitivity and chemosensitivity to gamma-irradiation (IR) and 5-Fluorouracil (5-FU) in human oral carcinoma cells (B88) in which NF-kappaB activity was constitutively suppressed. Three super-repressor form of IkappaBalpha cDNA-transfected cell (B88mI) clones and 1 empty vector-transfected cell clone (B88neo) have been established. We found that the tumor-forming ability in nude mice of B88mI clones was significantly lower than that of B88 or B88neo. This suppressed ability in tumorigenicity was attributed to the down-regulation of the expression of interleukin (IL)-1alpha, IL-6, IL-8, vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9 in B88mI cell clones as compared to that in B88 or B88neo. IR and 5-FU induced a much greater degree of apoptosis, as evidenced by flow cytometry analysis and annexin V staining, in B88mI cell clones than in B88 or B88neo. When tumor-bearing nude mice were treated with IR or 5-FU, the suppression of tumor growth was significantly augmented in B88mI cell clones as compared to that in B88 or B88neo. ELISA analysis indicated that although a remarkable increase in production of IL-6 and IL-8 was observed in B88 and B88neo after in vitro exposure to IR or treatment with 5-FU, radiotherapy and chemotherapy-induced production of these cytokines was significantly suppressed in B88mI cell clones. These findings suggest that production of angiogenic factors and growth factors in response to radiotherapy and chemotherapy is a principal mechanism of inducible radioresistance and chemoresistance in human oral cancers, and establish the inhibition of NF-kappaB as a rational approach to improve conventional radiotherapy and chemotherapy outcomes.

Suppression of tumor necrosis factor alpha-induced matrix metalloproteinase 9 production in human salivary gland acinar cells by cepharanthine occurs via down-regulation of nuclear factor kappaB: a possible therapeutic agent for preventing the destruction of the acinar structure in the salivary glands of Sjögren's syndrome patients.

OBJECTIVE: Our previous results suggested that suppression of tumor necrosis factor alpha (TNFalpha)-induced matrix metalloproteinase 9 (MMP-9) could prevent the destruction of acinar tissue in the salivary glands of patients with Sjögren's syndrome (SS). The present study was undertaken to investigate the effect of cepharanthine on the suppression of TNFalpha-induced MMP-9 production in NS-SV-AC, an SV40-immortalized normal human acinar cell clone. METHODS: After pretreatment with or without cepharanthine, NS-SV-AC cells were treated with TNFalpha alone or with a combination of TNFalpha and cepharanthine. The expression of MMP-9 was then examined at the protein and messenger RNA levels. In addition, the effect of cepharanthine on the morphogenetic behavior of NS-SV-AC cells cultured on type IV collagen-coated dishes in the presence of TNFalpha was examined. RESULTS: Although TNFalpha induced the production of MMP-9 in NS-SV-AC cells, this production was greatly suppressed when cells were pretreated with cepharanthine, followed by treatment with both TNFalpha and cepharanthine. In addition, cepharanthine suppressed the TNFalpha-stimulated NF-kappaB activity by partly preventing the degradation of IkappaBalpha protein in NS-SV-AC cells. When NS-SV-AC cells were seeded on type IV collagen-coated dishes in the presence of both TNFalpha and plasmin, type IV collagen interaction with the cells was lost and the cells entered apoptosis. However, pretreatment with cepharanthine restored the aberrant in vitro morphogenesis of the NS-SV-AC cells. CONCLUSION: These results may indicate a molecular mechanism by which cepharanthine is able to protect against the destruction of the acinar structure in salivary glands from patients with SS.

Stable inhibition of NF-kappa B in salivary gland cells does not enhance sensitivity to TNF-alpha-induced apoptosis due to upregulation of TRAF-1 expression.

The transcription factor NF-kappa B inhibits the apoptotic response induced by TNF-alpha. However, in salivary gland cell clones (ACMT-6 and ACMT-7) in which NF-kappa B activation was suppressed by introduction of a super-repressor form of I kappa B-alpha cDNA, TNF-alpha did not cause apoptosis. Thus, to investigate the molecular mechanism involved in the unresponsiveness of these cell clones to TNF-alpha-induced apoptosis, we examined the effect of TNF-alpha on the expression of antiapoptotic proteins, including TNF receptor-associated factor (TRAF)-1, TRAF-2, cellular inhibitor of apoptosis protein (cIAP)-1, and cIAP-2. Here we show that expression of TRAF-1 was commonly detected by treatment with TNF-alpha in ACMT-6, ACMT-7, and an empty vector-transfected cell clone (ACpRc-1) and that downregulation of TRAF-1 protein by either treatment with an antisense oligonucleotide or introduction of an antisense plasmid resulted in the induction of apoptosis in these cell clones. Our results, therefore, suggest that one of the mechanisms by which cells acquire resistance to TNF-alpha-induced apoptosis is a TNF-alpha induction of TRAF-1.