A complex interaction between Wnt signaling and TNF-α in nucleus pulposus cells

Introduction

Increased expression of the proinflammatory cytokine TNF-α in intervertebral discs (IVDs) leads to inflammation, which results in progressive IVD degeneration. We have previously reported that activation of Wnt-β-catenin (hereafter called Wnt) signaling suppresses the proliferation of nucleus pulposus cells and induces cell senescence, suggesting that Wnt signaling triggers the process of degeneration of the IVD. However, it is not known whether cross talk between TNF-α and Wnt signaling plays a role in the regulation of nucleus pulposus cells. The goal of the present study was to examine the effect of the interaction between Wnt signaling and the proinflammatory cytokine TNF-α in nucleus pulposus cells.

Methods

Cells isolated from rat nucleus pulposus regions of IVDs were cultured in monolayers, and the expression and promoter activity of Wnt signaling and TNF-α were evaluated. We also examined whether the inhibition of Wnt signaling using cotransfection with Dickkopf (DKK) isoforms and Sclerostin (SOST) could block the effects of pathological TNF-α expression in nucleus pulposus cells.

Results

TNF-α stimulated the expression and promoter activity of Wnt signaling in nucleus pulposus cells. In addition, the activation of Wnt signaling by 6-bromoindirubin-3′-oxime (BIO), which is a selective inhibitor of glycogen synthase kinase 3 (GSK-3) activity that activates Wnt signaling, increased TNF-α expression and promoter activity. Conversely, the suppression of TNF-α promoter activity using a β-catenin small interfering RNA was evident. Moreover, transfection with DKK-3, DKK-4, or SOST, which are inhibitors of Wnt signaling, blocked Wnt signaling-mediated TNF-α activation; these effects were not observed for DKK-1 or DKK-2.

Conclusions

Here, we have demonstrated that Wnt signaling regulates TNF-α and that Wnt signaling and TNF-α form a positive-feedback loop in nucleus pulposus cells. The results of the present study provide in vitro evidence that activation of Wnt signaling upregulates the TNF-α expression and might cause the degeneration of nucleus pulposus cells. We speculate that blocking this pathway might protect nucleus pulposus cells against degeneration.     

Differential oxidase activity of hepatic and pulmonary microsomal cytochrome P-450 isozymes after treatment with cytochrome P-450 inducers.

Phenobarbital, 3-methylcholanthrene, acetone and pyrazole were used as inducers of cytochrome P450 and the NADPH-dependent oxidase activity (O-2 production) of pulmonary and hepatic microsomes was determined. Oxidase activity of microsomes from 3-methylcholanthrene-treated rats was significantly decreased as compared to that of controls when expressed on the basis of cytochrome P450 content (30% decrease for liver, 60% decrease for lung). The oxidase activity of liver microsomes from pyrazole-treated rats showed a significant increase, whereas phenobarbital treated microsomes had average superoxide-generating activity. The contribution of cytochromes CYP 1A, CYP 2B and CYP 2E1 to superoxide-generating activity was investigated using monoclonal antibodies. Monoclonal antibody 1-91-3 against CYP 2E1 inhibited superoxide generation by 58% in liver microsomes from pyrazole-treated rats. Monoclonal antibodies 1-7-1 and 2-66-3 against CYP 1A and CYP2B, respectively, had no effect on superoxide generation. These results indicate that different cytochrome P450 isoforms are mainly responsible for differential superoxide generating activities of microsomes and complement the reconstitution study of Morehouse and Aust. Furthermore, our study indicates that CYP 1A1, induced by 3-MC, demonstrates an unusually low oxidase activity.     

A case of chondrosarcoma in a free-flying Great Egret

A free-flying Great Egret (Ardea alba) captured in Gifu, central Japan, in May 2006 had a large mass on the right carpal joint. The tumor was diagnosed as chondrosarcoma by histopathologic examination.     

Involvement of p38 mitogen-activated protein kinase in gemcitabine-induced apoptosis in human pancreatic cancer cells

In this study, we investigated the involvement of Akt and members of the mitogen-activated protein kinase (MAPK) superfamily, including ERK, JNK, and p38 MAPK, in gemcitabine-induced cytotoxicity in human pancreatic cancer cells. We found that gemcitabine induces apoptosis in PK-1 and PCI-43 human pancreatic cancer cell lines. Gemcitabine specifically activated p38 MAPK in a dose- and time-dependent manner. A selective p38 MAPK inhibitor, SB203580, significantly inhibited gemcitabine-induced apoptosis in both cell lines, suggesting that phosphorylation of p38 MAPK may play a key role in gemcitabine-induced apoptosis in pancreatic cancer cells. A selective JNK inhibitor, SP600125, failed to inhibit gemcitabine-induced apoptosis in both cell lines. MKK3/6, an upstream activator of p38 MAPK, was phosphorylated by gemcitabine, indicating that the MKK3/6-p38 MAPK signaling pathway is indeed involved in gemcitabine-induced apoptosis. Furthermore, gemcitabine-induced cleavage of the caspase substrate poly(ADP-ribose) polymerase was inhibited by pretreatment with SB203580, suggesting that activation of p38 MAPK by gemcitabine induces apoptosis through caspase signaling. These results together suggest that gemcitabine-induced apoptosis in human pancreatic cancer cells is mediated by the MKK3/6-p38 MAPK-caspase signaling pathway. Further, these results lead us to suggest that p38 MAPK should be investigated as a novel molecular target for human pancreatic cancer therapies.     

Design and synthesis of 2-N-substituted indazolone derivatives as non-carboxylic acid glycogen synthase activators

Glycogen synthase (GS) catalyzes the transfer of glucose residues from UDP-glucose to a glycogen polymer chain, a critical step for glucose storage. Patients with type 2 diabetes normally exhibit low glycogen levels and decreased muscle glucose uptake is the major defect in whole body glucose disposal. Therefore, activating GS may provide a potential approach for the treatment of type 2 diabetes. In order to identify non-carboxylic acids GS activators, we designed and synthesized a series of 2-N-alkyl- and 2-N-aryl-indazolone derivatives and studied their activity in activating human GS.     

Distribution of pink-pigmented facultative methylotrophs on leaves of vegetables

The distribution of pink-pigmented facultative methylotrophs (PPFMs) on the leaves of various vegetables was studied. All kinds of vegetable leaves tested gave pink-pigmented colonies on agar plates containing methanol as sole carbon source. The numbers of PPFMs on the leaves, colony-forming units (CFU)/g of fresh leaves, differed among the plants, although they were planted and grown at the same farm. Commercial green perilla, Perilla frutescens viridis (Makino) Makino, gave the highest counts of PPFMs (2.0-4.1×10(7) CFU/g) of all the commercial vegetable leaves tested, amounting to 15% of total microbes on the leaves. The PPFMs isolated from seeds of two varieties of perilla, the red and green varieties, exhibited high sequence similarity as to the 16S rRNA gene to two different Methylobacterium species, M. fujisawaense DSM5686(T) and M. radiotolerans JCM2831(T) respectively, suggesting that there is specific interaction between perilla and the PPFMs.