Skeletal muscle atrophy and weakness are thought to be stimulated by tumor necrosis factor alpha (TNF-alpha) in a variety of chronic diseases. However, little is known about the direct effects of TNF-alpha on differentiated skeletal muscle cells or the signaling mechanisms involved. We have tested the effects of TNF-alpha on the mouse-derived C2C12 muscle cell line and on primary cultures from rat skeletal muscle. TNF-alpha treatment of differentiated myotubes stimulated time- and concentration-dependent reductions in total protein content and loss of adult myosin heavy chain (MHCf) content; these changes were evident at low TNF-alpha concentrations (1-3 ng/ml) that did not alter muscle DNA content and were not associated with a decrease in MHCf synthesis. TNF-alpha activated binding of nuclear factor kappaB (NF-kappaB) to its targeted DNA sequence and stimulated degradation of I-kappaBalpha, an NF-kappaB inhibitory protein. TNF-alpha stimulated total ubiquitin conjugation whereas a 26S proteasome inhibitor (MG132 10-40 microM) blocked TNF-alpha activation of NF-kappaB. Catalase 1 kU/ml inhibited NF-kappaB activation by TNF-alpha; exogenous hydrogen peroxide 200 microM activated NF-kappaB and stimulated I-kappaBalpha degradation. These data demonstrate that TNF-alpha directly induces skeletal muscle protein loss, that NF-kappaB is rapidly activated by TNF-alpha in differentiated skeletal muscle cells, and that TNF-alpha/NF-kappaB signaling in skeletal muscle is regulated by endogenous reactive oxygen species.
Glucokinase (GK) has a major role in the control of blood glucose homeostasis and is a strong potential target for the pharmacological treatment of type 2 diabetes. We report here the mechanism of action of two novel and potent direct activators of GK: 6-[(3-isobutoxy-5-isopropoxybenzoyl)amino]nicotinic acid (GKA1) and 5-({3-isopropoxy-5-[2-(3-thienyl)ethoxy] benzoyl}amino)-1,3,4-thiadiazole-2-carboxylic acid (GKA2), which increase the affinity of GK for glucose by 4-and 11-fold, respectively. GKA1 increased the affinity of GK for the competitive inhibitor mannoheptulose but did not affect the affinity for the inhibitors palmitoylCoA and the endogenous 68-kDa regulator (GK regulatory protein [GKRP]), which bind to allosteric sites or to N-acetylglucosamine, which binds to the catalytic site. In hepatocytes, GKA1 and GKA2 stimulated glucose phosphorylation, glycolysis, and glycogen synthesis to a similar extent as sorbitol, a precursor of fructose 1-phosphate, which indirectly activates GK through promoting its dissociation from GKRP. Consistent with their effects on isolated GK, these compounds also increased the affinity of hepatocyte metabolism for glucose. GKA1 and GKA2 caused translocation of GK from the nucleus to the cytoplasm. This effect was additive with the effect of sorbitol and is best explained by a "glucose-like" effect of the GK activators in translocating GK to the cytoplasm. In conclusion, GK activators are potential antihyperglycemic agents for the treatment of type 2 diabetes through the stimulation of hepatic glucose metabolism by a mechanism independent of GKRP. Diabetes 53:535-541, 2004
The enzyme poly(ADP-ribose) glycohydrolase (PARG) performs a critical role in the repair of DNA single strand breaks (SSBs). However, a detailed understanding of its mechanism of action has been hampered by a lack of credible, cell-active chemical probes. Herein, we demonstrate inhibition of PARG with a small molecule, leading to poly(ADP-ribose) (PAR) chain persistence in intact cells. Moreover, we describe two advanced, and chemically distinct, cell-active tool compounds with convincing on-target pharmacology and selectivity. Using one of these tool compounds, we demonstrate pharmacology consistent with PARG inhibition. Further, while the roles of PARG and poly(ADP-ribose) polymerase (PARP) are closely intertwined, we demonstrate that the pharmacology of a PARG inhibitor differs from that observed with the more thoroughly studied PARP inhibitor olaparib. We believe that these tools will facilitate a wider understanding of this important component of DNA repair and may enable the development of novel therapeutic agents exploiting the critical dependence of tumors on the DNA damage response (DDR).
In some inflammatory diseases, TNF-alpha is thought to stimulate muscle catabolism via an NF-kappaB-dependent process that increases ubiquitin conjugation to muscle proteins. The transcriptional mechanism of this response has not been determined. Here we studied the potential role of UbcH2, a ubiquitin carrier protein and homologue of murine E220k. We find that UbcH2 is constitutively expressed by human skeletal and cardiac muscles, murine limb muscle, and cultured myotubes. TNF-alpha stimulates UbcH2 expression in mouse limb muscles in vivo and in cultured myotubes. The UbcH2 promoter region contains a functional NF-kappaB binding site; NF-kappaB binding to this sequence is increased by TNF-alpha stimulation. A dominant negative inhibitor of NF-kappaB activation blocks both UbcH2 up-regulation and the increase in ubiquitin-conjugating activity stimulated by TNF-alpha. In extracts from TNF-alpha-treated myotubes, ubiquitin-conjugating activity is limited by UbcH2 availability; activity is inhibited by an antiserum to UbcH2 or a dominant negative mutant of UbcH2 and is enhanced by wild-type UbcH2. Thus, UbcH2 up-regulation is a novel response to TNF-alpha/NF-kappaB signaling in skeletal muscle that appears to be essential for the increased ubiquitin conjugation induced by this cytokine.
A novel protein, proteolysis-inducing factor (PIF), has been isolated from the urine of patients with pancreatic cancer and is capable of inducing muscle proteolysis in vitro. Only adult skeletal muscle and liver exhibit substantial binding of PIF. We have investigated the effect of PIF on hepatic gene expression. Primary cultures of human hepatocytes and the human cell line HepG2 were incubated in the presence of PIF to assess its effects on hepatic transcription factors, proinflammatory cytokine production, and acute phase proteins. PIF activates both the transcription factors NF-kB and STAT3, which result in the increased production of IL-8, IL-6, and C-reactive protein and the decreased production of transferrin. The function of PIF, beyond muscle degradation, is unknown but here we show that it is involved in hepatic gene expression, and is thus likely to be involved in the proinflammatory response observed in cachexia. These results may also suggest a potential role for PIF during embryonic development. The expression of PIF peaks during the embryonic period E8 to E9, a stage that is crucial in the development of skeletal muscle and liver and during which both NF-kB and STAT3 activation can also be observed.
The recently discovered enzyme tyrosyl-DNA phosphodiesterase 2 (TDP2) has been implicated in the topoisomerase-mediated repair of DNA damage. In the clinical setting, it has been hypothesized that TDP2 may mediate drug resistance to topoisomerase II (topo II) inhibition by etoposide. Therefore, selective pharmacological inhibition of TDP2 is proposed as a novel approach to overcome intrinsic or acquired resistance to topo II-targeted drug therapy. Following a high-throughput screening (HTS) campaign, toxoflavins and deazaflavins were identified as the first reported sub-micromolar and selective inhibitors of this enzyme. Toxoflavin derivatives appeared to exhibit a clear structure-activity relationship (SAR) for TDP2 enzymatic inhibition. However, we observed a key redox liability of this series, and this, alongside early in vitro drug metabolism and pharmacokinetics (DMPK) issues, precluded further exploration. The deazaflavins were developed from a singleton HTS hit. This series showed distinct SAR and did not display redox activity; however low cell permeability proved to be a challenge.
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