Sang-Gu Hwang

Regulation of β-Catenin Signaling and Maintenance of Chondrocyte Differentiation by Ubiquitin-independent Proteasomal Degradation of α-Catenin

Accumulation of β-catenin and subsequent stimulation of β-catenin-T cell-factor (Tcf)/lymphoid-enhancerfactor (Lef) transcriptional activity causes dedifferentiation of articular chondrocytes, which is characterized by decreased type II collagen expression and initiation of type I collagen expression. This study examined the mechanisms of α-catenin degradation, the role of α-catenin in β-catenin signaling, and the physiological significance of α-catenin regulation of β-catenin signaling in articular chondrocytes. We found that both α- and β-catenin accumulated during dedifferentiation of chondrocytes by escaping from proteasomal degradation. β-Catenin degradation was ubiquitination-dependent, whereas α-catenin was proteasomally degraded in a ubiquitination-independent fashion. The accumulated α- and β-catenin existed as complexes in the cytosol and nucleus. The complex formation between α- and β-catenin blocked proteasomal degradation of α-catenin and also inhibited β-catenin-Tcf/Lef transcriptional activity and the suppression of type II collagen expression associated with ectopic expression of β-catenin, the inhibition of proteasome, or Wnt signaling. Collectively, our results indicate that ubiquitin-independent degradation of α-catenin regulates β-catenin signaling and maintenance of the differentiated phenotype of articular chondrocytes.

Wnt-3a regulates chondrocyte differentiation via c-Jun/AP-1 pathway

Our previous study indicated that interleukin (IL)‐1β induces expression of several Wnt proteins in chondrocytes and causes chondrocyte dedifferentiation via the c‐Jun/activator protein‐1 (AP‐1) pathway. This study examined whether Wnt‐3a causes chondrocyte dedifferentiation via the c‐Jun/AP‐1 pathway. Wnt‐3a inhibited chondrogenesis of mesenchymal cells by stabilizing cell–cell adhesion in a manner independent of β‐catenin transcriptional activity. Wnt‐3a also induced dedifferentiation of articular chondrocytes by stimulating the transcriptional activity of β‐catenin‐T cell‐factor/lymphoid‐enhancer‐factor (Tcf/Lef) complex. In chondrocytes, Wnt‐3a caused the expression of c‐Jun and its phosphorylation by c‐Jun N‐terminal kinase (JNK), resulting in activation of AP‐1. AP‐1 activation suppressed the expression of Sox‐9, a major transcription factor regulating type II collagen expression. Collectively, our results suggest that Wnt‐3a inhibits chondrogenesis by stabilizing cell–cell adhesion and that it causes dedifferentiation of chondrocytes by activating of β‐catenin‐Tcf/Lef transcriptional complex and the c‐Jun/AP‐1 pathway.

Claudin-1 Acts through c-Abl-Protein Kinase Cδ (PKCδ) Signaling and Has a Causal Role in the Acquisition of Invasive Capacity in Human Liver Cells

Claudins are identified as members of the tetraspanin family of proteins, which are integral to the structure and function of tight junction. Recent studies showed an increase in expression of claudins during tumorigenesis, which is associated with loss of cell-cell contact, dedifferentiation, and invasiveness. However, the molecular basis for the causal relationship between claudin expression and cancer progression is not fully understood yet. In this study, we show that claudin-1 plays a causal role in the acquisition of invasive capacity in human liver cells and that c-Abl-protein kinase Cδ (PKCδ) signaling is critical for the malignant progression induced by claudin-1. Overexpression of claudin-1 clearly induced expression of matrix metalloproteinase-2 (MMP-2) and cell invasion and migration in normal liver cells as well as in non-invasive human hepatocellular carcinoma (HCC) cells. Conversely, small interfering RNA targeting of claudin-1 in invasive HCC cells completely inhibited cell invasion. Both c-Abl and PKCδ are found to be activated in normal liver cell line clones that stably overexpress claudin-1. Inhibition of either c-Abl or PKCδ alone clearly attenuated MMP-2 activation and impeded cell invasion and migration in both human HCC and normal liver cells expressing claudin-1. These results indicate that claudin-1 is both necessary and sufficient to induce invasive behavior in human liver cells and that activation of c-Abl-PKCδ signaling pathway is critically required for the claudin-1-induced acquisition of the malignant phenotype. The present observations raise the possibility of exploiting claudin-1 as a potential biomarker for the spread of liver cancer and might provide pivotal points for therapeutic intervention in HCC.

β-Catenin regulates expression of cyclooxygenase-2 in articular chondrocytes ☆

Pro-inflammatory cytokine such as interleukin (IL)-1beta causes inflammation of articular cartilage via induction of cyclooxygenase (COX)-2 expression. We investigated in this study the role of beta-catenin in the IL-1beta regulation of COX-2 expression in articular chondrocytes. IL-1beta increased expression of COX-2 and induced accumulation and nuclear translocation of transcriptionally competent beta-catenin. Inhibition of beta-catenin degradation by the treatment of cells with LiCl or proteasome inhibitor stimulated expression of COX-2, indicating that transcriptionally active beta-catenin is sufficient to induce COX-2 expression. This was demonstrated further by the observation that ectopic expression of transcriptionally competent beta-catenin stimulated expression of COX-2. Levels of beta-catenin and COX-2 protein were increased in osteoarthritic and rheumatoid arthritic cartilage, suggesting that beta-catenin may play a role in the inflammatory responses of arthritic cartilage. Taken together, our data suggest that accumulation of transcriptionally active beta-catenin contributes to the expression of COX-2 in articular chondrocytes.

Phospholipase A2 Activity of Peroxiredoxin 6 Promotes Invasion and Metastasis of Lung Cancer Cells

Peroxiredoxins (PRDX) are a family of thiol-dependent peroxidases. Among the six mammalian members of this family, PRDX6 is the only protein that additionally exhibits phospholipase A2 (PLA2) activity. The physiologic role of this interesting PRDX6 feature is largely unknown at present. In this study, we show that PRDX6 increases the metastatic potential of lung cancer cells. Functional analyses of the enzymatic activities of PRDX6, using specific pharmacologic inhibitors and mutagenesis studies, reveal that both peroxidase and PLA2 activities are required for metastasis. Specifically, peroxidase activity facilitates the growth of cancer cells, and PLA2 activity promotes invasiveness. Further investigation of the latter event discloses that PLA2 activity promotes accumulation of arachidonic acid, which, in turn, induces the invasive pathway involving p38 kinase, phosphoinositide 3-kinase, Akt, and urokinase-type plasminogen activator. This study is the first to define the functions of the enzymatic activities of PRDX6 in metastasis and to show the involvement of arachidonic acid in PRDX6 action in intact cells. These novel findings provide a significant step toward elucidating the role of PRDX6 in cancer and the mechanism of its action. Mol Cancer Ther; 9(4); 825–32. ©2010 AACR.

Bcl‐XL and STAT3 mediate malignant actions of γ‐irradiation in lung cancer cells

Previous reports suggest that, in addition to its therapeutic effects, ionizing radiation (IR) increases the invasiveness of surviving cancer cells. Here, we demonstrate that this activity of IR in lung cancer cells is mediated by a signaling pathway involving p38 kinase, phosphoinositide 3‐kinase, Akt, and matrix metalloproteinase (MMP‐2). The invasion‐promoting doses of IR also increased and reduced the levels of vimentin and E‐cadherin, respectively, both of which are markers for the epithelial–mesenchymal transition (EMT). Interestingly, all of these malignant actions of IR were mimicked by the overexpression of Bcl‐XL, a pro‐survival member of the Bcl‐2 family, in lung cancer cells. Moreover, both RNA and protein levels of Bcl‐XL were elevated upon irradiation of the cells, and the prevention of this event using small‐interfering RNAs of Bcl‐XL reduced the ability of IR to promote invasion signals and EMT‐associated events. This suggests that Bcl‐XL functions as a signaling mediator of the malignant effects of IR. It was also demonstrated that IR enhances signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the reduction of STAT3 levels via RNA interference prevented IR‐induced Bcl‐XL accumulation, and thus all the tested Bcl‐XL‐dependent events. Overall, the data suggest that IR induces Bcl‐XL accumulation via STAT3, which then promotes cancer cell invasion and EMT‐associated markers. Our findings demonstrate a novel function of Bcl‐XL in cancer, and also advance our understanding of the malignant actions of IR significantly. (Cancer Sci 2010)

Nicotinamide Phosphoribosyltransferase Is Essential for Interleukin-1β-mediated Dedifferentiation of Articular Chondrocytes via SIRT1 and Extracellular Signal-regulated Kinase (ERK) Complex Signaling

Although much is known about interleukin (IL)-1β and its role as a key mediator of cartilage destruction in osteoarthritis, only limited information is available on IL-1β signaling in chondrocyte dedifferentiation. Here, we have characterized the molecular mechanisms leading to the dedifferentiation of primary cultured articular chondrocytes by IL-1β treatment. IL-1β or lipopolysaccharide, but not phorbol 12-myristate 13-acetate, retinoic acid, or epidermal growth factor, induced nicotinamide phosphoribosyltransferase (NAMPT) expression, showing the association of inflammatory cytokines with NAMPT regulation. SIRT1, in turn, was activated NAMPT-dependently, without any alteration in the expression level. Activation or inhibition of SIRT1 oppositevely regulates IL-1β-mediated chondrocyte dedifferentiation, suggesting this protein as a key regulator of chondrocytes phenotype. SIRT1 activation promotes induction of ERK and p38 kinase activities, but not JNK, in response to IL-1β. Subsequently, ERK and p38 kinase activated by SIRT1 also induce SIRT1 activation, forming a positive feedback loop to sustain downstream signaling of these kinases. Moreover, we found that the SIRT1-ERK complex, but not SIRT1-p38, is engaged in IL-1β-induced chondrocyte dedifferentiation via a Sox-9-mediated mechanism. JNK is activated by IL-1β and modulates dedifferentiation of chondrocytes, but this pathway is independent on NAMPT-SIRT1 signaling. Based on these findings, we propose that IL-1β induces dedifferentiation of articular chondrocytes by up-regulation of SIRT1 activity enhanced by both NAMPT and ERK signaling.

WD repeat-containing mitotic checkpoint proteins act as transcriptional repressors during interphase

WD repeats are implicated in protein–protein interactions and regulate a wide variety of cellular functions, including chromatin remodeling and transcription. The WD repeats of the Bub3 and Cdc20 kinetochore proteins are important for the physical interactions of these proteins with Mad2 and BubR1 to yield a kinetochore protein complex capable of delaying anaphase by inhibiting ubiquitin ligation via the anaphase‐promoting complex/cyclosome. Here, we show that Bub3 and Cdc20 form a complex with histone deacetylases; this interaction appears to confer transcriptional repressor activity in a heterologous DNA‐binding context. In addition, inhibition of Bub3 and Cdc20 expression significantly impairs interphase cell cycle. These results indicate that Bub3 and Cdc20 play additional roles in the integration of cell cycle arrest as transcriptional repressors.

CIP2A Modulates Cell-Cycle Progression in Human Cancer Cells by Regulating the Stability and Activity of Plk1

Abnormal cell-cycle control can lead to aberrant cell proliferation and cancer. The oncoprotein cancerous inhibitor of protein phosphatase 2A (CIP2A) is an inhibitor of protein phosphatase 2A (PP2A) that stabilizes c-Myc. However, the precise role of CIP2A in cell division is not understood. Herein, we show that CIP2A is required for mitotic progression by regulating the polo-like kinase (Plk1). With mitotic entry, CIP2A translocated from the cytoplasm to the nucleus, where it was enriched at spindle poles. CIP2A depletion delayed mitotic progression, resulting in mitotic abnormalities independent of PP2A activity. Unexpectedly, CIP2A interacted directly with the polo-box domain of Plk1 during mitosis. This interaction was required to maintain Plk1 stability by blocking APC/C-Cdh1-dependent proteolysis, thereby enhancing the kinase activity of Plk1 during mitosis. We observed strong correlation and in vivo interactions between these two proteins in multiple human cancer specimens. Overall, our results established a novel function for CIP2A in facilitating the stability and activity of the pivotal mitotic kinase Plk1 in cell-cycle progression and tumor development.

Bmal1 suppresses cancer cell invasion by blocking the phosphoinositide 3-kinase-Akt-MMP-2 signaling pathway

Bmal1 is a core factor in the regulation of circadian rhythms. Previous studies have shown that Bmal1 suppresses tumor growth in cell culture and animal models and is down-regulated in certain types of cancer. The aim of the present study was to investigated whether Bmal1 influences the invasiveness of cancer cells. We demonstrated that knockdown of Bmal1 by RNA interference promoted cancer cell invasion, whereas its overexpression reduced cellular invasiveness. These effects were observed in lung cancer and glioma cells, and occurred regardless of p53 status. Therefore, it appears that Bmal1 suppresses the invasion of multiple cancer types in a p53-independent manner. Bmal1 knockdown-induced cancer cell invasion was accompanied by activation of the PI3K-Akt-MMP-2 pathway, and was prevented by inhibitors of PI3K, Akt or MMP-2. This suggests that Bmal1 suppresses cell invasion by blocking the PI3K-Akt-MMP-2 pathway. Since this invasion pathway is activated by the oncogene Bcl-w, we investigated whether Bmal1 affects the activity of Bcl-w. As expected, Bmal1 attenuated the ability of Bcl-w to promote MMP-2 accumulation and cell invasion, supporting the idea that Bmal1 antagonizes Bcl-w activity. Collectively, our data suggest that Bmal1 is a tumor suppressor, capable of suppressing cancer cell growth and invasiveness, and support the recent proposal that there is a tight molecular link between circadian rhythms and tumor formation/progression.