Oxidative stress-induced c-Jun N-terminal kinase (JNK) activation in tendon cells upregulates MMP1 mRNA and protein expression.

To explore the potential mechanisms of tendon degeneration, we investigated the role of c-Jun N-terminal Kinase (JNK) activation and the regulation of matrix metalloproteinase 1 (MMP1) in tendon matrix degradation under oxidative stress. JNK and MMP1 activity in samples from normal and ruptured human supraspinatus tendons was evaluated by immunohistochemistry. Real-time quantitative PCR was utilized to evaluate MMP1 mRNA expression and Western blotting for MMP1 and JNK protein detection. JNK activation and increased MMP1 activity were found in the torn human supraspinatus tendon tissue, as well as in human tendon cells under in vitro oxidative stress. Inhibition of JNK prevented MMP1 overexpression in oxidative stressed human tendon cells. Results from the current study indicated that stress activated JNK plays an important role in tendon matrix degradation, possibly through upregulating of MMP1.     

Role of protein kinase C and oxidative stress in interleukin-1beta-induced human proximal tubule cell injury and fibrogenesis

BACKGROUND: Interleukin (IL)-1beta, a pro-inflammatory macrophage-derived cytokine, is implicated as a key mediator of interstitial fibrosis and tubular loss or injury in progressive renal insufficiency. This study investigates some of the mechanisms of action of IL-1beta on the proximal tubule. METHODS: Confluent cultures of primary human proximal tubule cells (PTC) were incubated in serum-free media supplemented with either IL-1beta (0-4 ng/mL), phorbol-12-myristate 13-acetate (PMA, protein kinase C activator) (6.25-100 nmol/L), or vehicle (control), together with a non-specific protein kinase C inhibitor (H7), a specific protein kinase C inhibitor (BIM-1), an anti-oxidant (NAC) or a NADPH oxidase inhibitor (AEBSF). RESULTS: Interleukin-1beta-treated PTC exhibited time-dependent increases in fibronectin secretion (ELISA), cell injury (LDH release) and reactive nitrogen species (RNS) release (Griess assay). Proximal tubule cell DNA synthesis (thymidine incorporation) was also significantly suppressed. The effects of IL-1beta, which were reproduced by incubation of PTC with PMA (6.25-100 nmol/L), were blocked by H7 but not by BIM-1. The anti-oxidant (4 mmol/L) partially blocked IL-1beta-induced fibronectin secretion by PTC, but did not affect IL-1beta-induced LDH release, RNS release or growth inhibition. The NADPH oxidase inhibitor (AEBSF) significantly attenuated all observed deleterious effects of IL-1beta on PTC. CONCLUSION: Interleukin-1beta directly induces proximal tubule injury, extracellular matrix production and impaired growth. The anti-oxidant, NAC, appears to ameliorate part of the fibrogenic effect of IL-1beta on PTC through mechanisms that do not significantly involve protein kinase C activation or nitric oxide release.     

Adenoviral gene transfer of the interleukin-1 receptor antagonist protein to human islets prevents IL-1beta-induced beta-cell impairment and activation of islet cell apoptosis in vitro.

The beta-cells in the pancreatic islets of Langerhans are the targets of autoreactive T-cells and are destroyed in type 1 diabetes. Macrophage-derived interleukin-1beta (IL-1beta) is important in eliciting beta-cell dysfunction and initiating beta-cell damage in response to microenvironmental changes within islets. In particular, IL-1beta can impair glucose-stimulated insulin production in beta-cells in vitro and can sensitize them to Fas (CD95)/FasL-triggered apoptosis. In this report, we have examined the ability to block the detrimental effects of IL-1beta by genetically modifying islets by adenoviral gene transfer to express the IL-1 receptor antagonist protein. We demonstrate that adenoviral gene delivery of the cDNA encoding the interleukin-1 receptor antagonist protein (IL-1Ra) to cultured islets results in protection of human islets in vitro against IL-1beta-induced nitric oxide formation, impairment in glucose-stimulated insulin production, and Fas-triggered apoptosis activation. Our results further support the hypothesis that IL-1beta antagonism in in situ may prevent intra-islet proinsulitic inflammatory events and may allow for an in vivo gene therapy strategy to prevent insulitis and the consequent pathogenesis of diabetes.     

Apoptotic insults to human chondrocytes induced by sodium nitroprusside are involved in sequential events,including cytoskeletal remodeling,phosphorylation of mitogen‐activated protein kinase kinase kinase‐1/c‐Jun N‐terminal kinase,and Bax‐Mitochondria‐Mediated caspase activation

Nitric oxide (NO) can regulate chondrocyte activities. This study was aimed to evaluate the molecular mechanisms of NO donor sodium nitroprusside (SNP)‐induced insults to human chondrocytes. Exposure of human chondrocytes to SNP increased cellular NO levels but decreased cell viability in concentration‐ and time‐dependent manners. SNP time dependently induced DNA fragmentation and cell apoptosis. Treatment with 2‐phenyl‐4,4,5,5‐tetramethyl‐imidazoline‐1‐oxyl 3‐oxide, an NO scavenger, significantly lowered SNP‐induced cell injuries. Administration of SNP interrupted F‐actin and microtubule cytoskeletons and stimulated phosphorylation of mitogen‐activated protein kinase kinase kinase‐1 (MEKK1) and c‐Jun N‐terminal kinase (JNK). Similar to SNP, cytochalasin D, an inhibitor of F‐actin formation, disturbed F‐actin polymerization and increased MEKK1 and JNK activations. Overexpression of a dominant negative mutant of MEKK1 (dnMEK1) in human chondrocytes significantly ameliorated SNP‐induced cell apoptosis. Exposure to SNP promoted Bax translocation from the cytoplasm to mitochondria, but application of dnMEKK1 lowered the translocation. SNP time dependently decreased the mitochondrial membrane potential, complex I NADH dehydrogenase activity, and cellular ATP levels, but increased the release of cytochrome c from mitochondria to the cytoplasm. Activities of caspase‐9, ‐3, and ‐6 were sequentially increased by SNP administration. This study shows that SNP can induce apoptosis of human chondrocytes through sequential events, including cytoskeletal remodeling, activation of MEKK1/JNK, Bax translocation, mitochondrial dysfunction, cytochrome c release, caspase activation, and DNA fragmentation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1018–1026, 2008