As a result of identifying the regulatory proteins of thioredoxin (TRX), a murine homologue for human vitamin D3 up-regulated protein 1 (VDUP1) was identified from a yeast two-hybrid screen. Cotransfection into 293 cells and precipitation assays confirmed that mouse VDUP1 (mVDUP1) bound to TRX, but it failed to bind to a Cys32 and Cys35 mutant TRX, suggesting the redox-active site is critical for binding. mVDUP1 was ubiquitously expressed in various tissues and located in the cytoplasm. Biochemical analysis showed that mVDUP1 inhibited the insulin-reducing activity of TRX. When cells were treated with various stress stimuli such as H2O2 and heat shock, mVDUP1 was significantly induced. TRX is known to interact with other proteins such as proliferation-associated gene and apoptosis signal-regulating kinase 1. Coexpression of mVDUP1 interfered with the interaction between TRX and proliferation-associated gene or TRX and ASK-1, suggesting its roles in cell proliferation and oxidative stress. To investigate the roles of mVDUP1 in oxidative stress, mVDUP1 was overexpressed in NIH 3T3 cells. When cells were exposed to stress, cell proliferation was declined with elevated apoptotic cell death compared with control cells. In addition, c-Jun N-terminal kinase activation and IL-6 expression were elevated. Taken together, these results demonstrate that mVDUP1 functions as an oxidative stress mediator by inhibiting TRX activity.

Radiotherapy is increasingly used in the treatment of joint diseases, but limited information is available on the effects of radiation on cartilage. Here, we characterize the molecular mechanisms leading to cellular senescence in irradiated primary cultured articular chondrocytes. Ionizing radiation (IR) causes activation of ERK, in turn generating intracellular reactive oxygen species (ROS) with induction of senescence-associated ␤-galactosidase (SA-␤-gal) activity. ROS activate p38 kinase, which further promotes ROS generation, forming a positive feedback loop to sustain ROS-p38 kinase signaling. The ROS inhibitors, nordihydroguaiaretic acid and GSH, suppress phosphorylation of p38 and cell numbers positive for SA-␤-gal following irradiation. Moreover, inhibition of the ERK and p38 kinase pathways leads to blockage of IR-induced SA-␤-gal activity via reduction of ROS generation. Although JNK is activated by ROS, this pathway is not associated with cellular senescence of chondrocytes. Interestingly, IR triggers down-regulation of SIRT1 protein expression but not the transcript level, indicative of post-transcriptional cleavage of the protein. SIRT1 degradation is markedly blocked by SB203589 or MG132 after IR treatment, suggesting that cleavage occurs as a result of binding with p38 kinase, followed by processing via the 26 S proteasomal degradation pathway. Overexpression or activation of SIRT1 significantly reduces the IR-induced senescence phenotype, whereas inhibition of SIRT1 activity induces senescence. Based on these findings, we propose that IR induces cellular senescence of articular chondrocytes by negative post-translational regulation of SIRT1 via ROS-dependent p38 kinase activation.

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We previously demonstrated that the phytosphingosine-induced apoptosis was accompanied by the concomitant induction of both the caspase-8-mediated and mitochondrial activation-mediated apoptosis pathways. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in the activation of these two distinct cell death pathways induced by phytosphingosine in human cancer cells. Phytosphingosine caused strong induction of caspase-8 activity and caspase-independent Bax translocation to the mitochondria. A rapid decrease of phosphorylated ERK1/2 and a marked increase of p38 MAPK phosphorylation were observed within 10 min after phytosphingosine treatment. Activation of ERK1/2 by pretreatment with phorbol 12-myristate 13-acetate or forced expression of ERK1/2 attenuated phytosphingosine-induced caspase-8 activation. However, Bax translocation and caspase-9 activation was unaffected, indicating that down-regulation of the ERK activity is specifically required for the phytosphingosine-induced caspase-8-dependent cell death pathway. On the other hand, treatment with SB203580, a p38 MAPK-specific inhibitor, or expression of a dominant negative form of p38 MAPK suppressed phytosphingosine-induced translocation of the proapoptotic protein, Bax, from the cytosol to mitochondria, cytochrome c release, and subsequent caspase-9 activation but did not affect caspase-8 activation, indicating that activation of p38 MAPK is involved in the mitochondrial activation-mediated cell death pathway. Our results suggest that phytosphingosine can utilize two different MAPK signaling pathways for amplifying the apoptosis cascade, enhancing the understanding of the molecular mechanisms utilized by naturally occurring metabolites to regulate cell death. Molecular dissection of the signaling pathways that activate the apoptotic cell death machinery is critical for both our understanding of cell death events and development of cancer therapeutic agents.

Abstract.We have previously reported that intercellular adhesion molecule-3 (ICAM-3) is associated with an increase of cellular radio-resistance and cancer cell proliferation. In this study, we hypothesized that ICAM-3 has an additional effect on cancer cell migration and invasion because molecules induced by ICAM-3 are known as regulators of cell migration and invasion. To examine this hypothesis, we used NCI-H1299 non-small cell lung cancer (NSCLC) cell line (p53 and PTEN null cell) and constructed an ICAM-3-over-expressing stable transfectant, which exhibited increased cell migration and invasion. The increased migration and invasion resulted from up-regulation of expression and activities of MMP-2 and MMP-9. ICAM-3 also increased Akt phosphorylation, which caused an increase in cellular migration/invasion and MMP activities. Activity of several transcriptional factors located downstream in the Akt pathway was also tested, and constitutive activation of adenosine 3', 5'-monophosphate response element-binding protein (CREB) by ICAM-3 was detected. Blockage of the Akt pathway attenuated CREB activation, and a decrease in CREB expression reduced cellular migration/invasion and activity of MMPs. This result indicates that CREB functions in the signaling pathway between Akt and MMP. We also showed ICAM-3-induced cell migration and invasion in NCI-H460 NSCLC cells (wild-type p53 and PTEN cell) through the same signaling pathway. Taken together, our findings suggest that ICAM-3 stimulates cancer cell migration/ invasion via ICAM-3/Akt/CREB/MMP pathway regardless of p53 and PTEN status, and this reflects the possibility that ICAM-3 could be considered as a candidate for anti-cancer drug development and as a cancer diagnostic marker.