UV irradiation has been reported to induce p21WAF1/CIP1 protein degradation through a ubiquitinproteasome pathway, but the underlying biochemical mechanism remains to be elucidated. Here, we show that ser-114 phosphorylation of p21 protein by glycogen synthase kinase 3␤ (GSK-3␤) is required for its degradation in response to UV irradiation and that GSK-3␤ activation is a downstream event in the ATR signaling pathway triggered by UV. UV transiently increased GSK-3␤ activity, and this increase could be blocked by caffeine or by ATR small interfering RNA, indicating ATR-dependent activation of GSK-3␤. ser-114, located within the putative GSK-3␤ target sequence, was phosphorylated by GSK-3␤ upon UV exposure. The nonphosphorylatable S114A mutant of p21 was protected from UV-induced destabilization. Degradation of p21 protein by UV irradiation was independent of p53 status and prevented by proteasome inhibitors. In contrast to the previous report, the proteasomal degradation of p21 appeared to be ubiquitination independent. These data show that GSK-3␤ is activated by UV irradiation through the ATR signaling pathway and phosphorylates p21 at ser-114 for its degradation by the proteasome. To our knowledge, this is the first demonstration of GSK-3␤ as the missing link between UV-induced ATR activation and p21 degradation.

SUMMARY Transforming growth factor (TGF)‐β1 is expressed abundantly in the rheumatoid synovium. In this study, the inflammatory effect of TGF‐β1 in rheumatoid arthritis (RA) was investigated using cultured fibroblast‐like synoviocytes (FLS) from RA and osteoarthritis (OA) patients, as well as non‐arthritic individuals. mRNA expressions of IL‐1β, tumour necrosis factor (TNF)‐α, IL‐8, macrophage inflammatory protein (MIP)‐1α and metalloproteinase (MMP)‐1 were increased in RA and OA FLS by TGF‐β1 treatment, but not in non‐arthritic FLS. Enhanced protein expression of IL‐1β, IL‐8 and MMP‐1 was also observed in RA FLS. Moreover, TGF‐β1 showed a synergistic effect in increasing protein expression of IL‐1β and matrix metalloproteinase (MMP)‐1 with TNFα and IL‐1β, respectively. Biological activity of IL‐1 determined by mouse thymocyte proliferation assay was also enhanced by 50% in response to TGF‐β1 in the culture supernatant of RA FLS. DNA binding activities of nuclear factor (NF)‐κB and activator protein (AP)‐1 were shown to increase by TGF‐β1 as well. These results suggest that TGF‐β1 contributes for the progression of inflammation and joint destruction in RA, and this effect is specific for the arthritic synovial fibroblasts.

Lung cancer is one of the deadliest and commonly diagnosed neoplasms. Early diagnosis of this disease is critical for improving clinical outcome and prognosis. Because the early stages of lung cancer often produce no symptoms, it is necessary to identify biomarkers for early detection, prognostic evaluation, and recurrence monitoring of the cancer. To identify potential lung cancer biomarkers, we analyzed the differential protein secretion from transformed bronchial epithelial cells (1198 and 1170-I) as compared to immortalized normal bronchial epithelial cells (BEAS-2B) and non-transformed cells (1799) all of which are derived from BEAS-2B and represent multistage bronchial epithelial carcinogenesis. The proteins recovered from the conditioned media of the cells were separated on two-dimensional gels. There was little difference between the secretome of the BEAS-2B and 1799 cells, whereas the patterns between the transformed 1198 and 1170-I cells and non-transformed 1799 cells were significantly different. Using mass spectrometry and database search, we identified 20 proteins including protein gene product 9.5 (PGP9.5), translationally controlled tumor protein (TCTP), tissue inhibitors of metalloproteinases-2 (TIMP-2), and triosephosphate isomerase (TPI), that were either increased or decreased simultaneously in conditioned media of both 1198 and 1170-I cells. Furthermore, levels of PGP9.5, TCTP, TIMP-2, and TPI were significantly increased not only in the conditioned media of both transformed cell lines when compared to those of BEAS-2B and 1799 cells, but also in plasmas and tissues from lung cancer patients when compared to those in normal controls. We suggest the PGP9.5, TCTP, TIMP-2, and TPI as promising candidates for lung cancer serum biomarkers.

In inflamed joints of rheumatoid arthritis, PGE2 is highly expressed, and IL-10 and IL-6 are also abundant. PGE2 is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE2 on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE2 significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE2 suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE2-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE2-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE2 selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE2 may modulate immune responses by alteration of cytokine signaling in THP-1 cells.

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Panaxydol, a polyacetylenic compound derived from Panax ginseng roots, has been shown to inhibit the growth of cancer cells. In this study, we demonstrated that panaxydol induced apoptosis preferentially in transformed cells with a minimal effect on non-transformed cells. Furthermore, panaxydol was shown to induce apoptosis through an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), activation of JNK and p38 MAPK, and generation of reactive oxygen species (ROS) initially by NADPH oxidase and then by mitochondria. Panaxydol-induced apoptosis was caspase-dependent and occurred through a mitochondrial pathway. ROS generation by NADPH oxidase was critical for panaxydol-induced apoptosis. Mitochondrial ROS production was also required, however, it appeared to be secondary to the ROS generation by NADPH oxidase. Activation of NADPH oxidase was demonstrated by the membrane translocation of regulatory p47(phox) and p67(phox) subunits and shown to be necessary for ROS generation by panaxydol treatment. Panaxydol triggered a rapid and sustained increase of [Ca(2+)](i), which resulted in activation of JNK and p38 MAPK. JNK and p38 MAPK play a key role in activation of NADPH oxidase, since inhibition of their expression or activity abrogated membrane translocation of p47(phox) and p67(phox) subunits and ROS generation. In summary, these data indicate that panaxydol induces apoptosis preferentially in cancer cells, and the signaling mechanisms involve a [Ca(2+)](i) increase, JNK and p38 MAPK activation, and ROS generation through NADPH oxidase and mitochondria.

No abstract

We reported previously that panaxydol, a component of Panax ginseng roots, induced mitochondria-mediated apoptosis preferentially in transformed cells. This study demonstrates that EGFR activation and the resulting ER stress mediate panaxydolinduced apoptosis, and that panaxydol suppresses in vivo tumor growth in syngeneic and xenogeneic mouse tumor models. In addition, we elucidated that CaMKII and TGF-b-activated kinase (TAK1) Ginsenoside and C17 polyacetylenic compounds of Panax ginseng (P. ginseng C.A. Meyer, Korean ginseng) roots possess anticancer activities. [1][2][3][4] It has been shown that panaxydol (heptadeca-1-en-4,6-diyn-9,10-epoxy-3-ol), one of the C17 polyacetylenic compounds of P. ginseng, induces G1 cell cycle arrest or apoptosis in cancer cells depending on the concentration. 4,5 We also previously reported that the mechanisms of panaxydol-induced apoptosis involve a rapid increase in the cytoplasmic Ca 21 concentration ([Ca 21 ]c), activating NADPH oxidase via p38/JNK. NADPH oxidase activation induces oxidative stress and triggers mitochondria-dependent apoptosis. 5Transfer of excess Ca 21 from the endoplasmic reticulum (ER) to the mitochondria is an important mechanism in mitochondria-dependent apoptosis. It is postulated that ER Ca 21 is transferred via the mitochondria-associated ER