LPS, the primary constituent of the outer membrane of Gram-negative bacteria, is recognized by TLR4. Binding of TLR4 to LPS triggers various cell signaling pathways including NF-κB activation and reactive oxygen species (ROS) production. In this study, we present the data that LPS-induced ROS generation and NF-κB activation are mediated by a direct interaction of TLR4 with (NAD(P)H oxidase 4 (Nox) 4), a protein related to gp91phox (Nox2) of phagocytic cells, in HEK293T cells. Yeast two hybrid and GST pull-down assays indicated that the COOH-terminal region of Nox4 interacted with the cytoplasmic tail of TLR4. Knockdown of Nox4 by transfection of small interference RNA specific to the Nox4 isozyme in HEK293T cells expressing TLR4 along with MD2 and CD14 resulted in inhibition of LPS-induced ROS generation and NF-κB activation. Taken together, these results indicate that direct interaction of TLR4 with Nox4 is involved in LPS-mediated ROS generation and NF-κB activation.

Nox4 plays a central role in LPS-induced proinflammatory responses by endothelial cells in an ROS-dependent manner.

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/ threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H 2 O 2 or tumor necrosis factor ␣ treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr 838 phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H 2 O 2 -mediated apoptosis by enhancing the ASK1 activity through Thr 838 phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.Apoptosis signal-regulating kinase 1 (ASK1) 2 is one of the mitogen-activated protein kinase kinase kinases (MAPKKK) that is stimulated in response to various cellular stresses, including reactive oxygen species, tumor necrosis factor ␣ (TNF-␣), Fas, ischemia insult, and anti-tumor agents. ASK1 stimulation leads to activation of the c-Jun NH 2 -terminal kinase (JNK)/p38 signaling cascade by phosphorylating and activating mitogen-activated protein kinase kinases (MAPKK) such as MKK3, -4, -6, and -7 (1-3). Emerging evidence indicates that ASK1 activity is regulated by its interaction with several cellular partners (3-8), including thioredoxin (Trx), glutaredoxin, heat shock protein 72 (Hsp72), 14-3-3, and protein serine/threonine phosphatase 5 (PP5). For example, Trx and glutaredoxin bind to the NH 2 -and COOH-terminal domains of ASK1, respectively, and inhibit ASK1 kinase activity, and Hsp72 inhibits ASK1 activation through direct interaction. These findings suggest that other ASK1-interacting proteins could be involved in the regulation of ASK1 activity.Murine protein serine/threonine kinase 38 (MPK38), also known as maternal embryonic leucine zipper kinase (Melk), is a member of the AMP-activated protein kinase-related serine/ threonine kinase family (9, 10). MPK38 was originally identified as a murine counterpart for its human homolog, HPK38/ hMelk/KIAA175, that may be involved in the proliferation of interleukin-4-induced normal human keratinocytes (9). The importance of MPK38 in oncogenesis is also underscored by the finding that MPK38 expression is increased in tumor-derived progenitor cells as well as in cancers of nondifferentiated cells (11-13). However, the physiological regulation and functions of MPK38 have r...

TMPRSS4 is a novel type II transmembrane serine protease found at the cell surface that is highly expressed in pancreatic, colon and gastric cancer tissues. However, the biological functions of TMPRSS4 in cancer are unknown. Here we show, using reverse transcription-PCR, that TMPRSS4 is highly elevated in lung cancer tissues compared with normal tissues and is also broadly expressed in a variety of human cancer cell lines. Knockdown of TMPRSS4 by small interfering RNA treatment in lung and colon cancer cell lines was associated with reduction of cell invasion and cell-matrix adhesion as well as modulation of cell proliferation. Conversely, the invasiveness, motility and adhesiveness of SW480 colon carcinoma cells were significantly enhanced by TMPRSS4 overexpression. Furthermore, overexpression of TMPRSS4 induced loss of E-cadherin-mediated cell-cell adhesion, concomitant with the induction of SIP1/ZEB2, an Ecadherin transcriptional repressor, and led to epithelialmesenchymal transition events, including morphological changes, actin reorganization and upregulation of mesenchymal markers. TMPRSS4-overexpressing cells also displayed markedly increased metastasis to the liver in nude mice upon intrasplenic injection. Taken together, these studies suggest that TMPRSS4 controls the invasive and metastatic potential of human cancer cells by facilitating an epithelial-mesenchymal transition; TMPRSS4 may be a potential therapeutic target for cancer treatment.

Similarly, NM23-H1 and STRAP stimulated p53-induced apoptosis and growth inhibition, whereas the NM23-H1(C145S) and STRAP(C152S/C270S) mutants had no effect. We also demonstrated that p53 activation by NM23-H1 and STRAP was mediated by removing Mdm2, a negative regulator of p53, from the p53-Mdm2 complex. These results suggest that NM23-H1 and its interacting partner STRAP physically interact with p53 and positively regulate its functions, including p53-induced apoptosis and cell cycle arrest.

Osteoporosis is the most common chronic metabolic bone disease. It has been estimated that more than 10 million people in the United States and 200 million men and women worldwide have osteoporosis. Given that the aging population is rapidly increasing in many countries, osteoporosis could become a global challenge with an impact on the quality of life of the affected individuals. Osteoporosis can be defined as a condition characterized by low bone density and increased risk of fractures due to the deterioration of the bone architecture. Thus, the major goal of treatment is to reduce the risk for fractures. There are several treatment options, mostly medications that can control disease progression in risk groups, such as postmenopausal women and elderly men. Recent studies on the basic molecular mechanisms and clinical implications of osteoporosis have identified novel therapeutic targets. Emerging therapies targeting novel disease mechanisms could provide powerful approaches for osteoporosis management in the future. Here, we review the etiology of osteoporosis and the molecular mechanism of bone remodeling, present current pharmacological options, and discuss emerging therapies targeting novel mechanisms, investigational treatments, and new promising therapeutic approaches.

To gain more insights about the biological roles of PDK1, we have used the yeast two-hybrid system and in vivo binding assay to identify interacting molecules that associate with PDK1. As a result, serine-threonine kinase receptor-associated protein (STRAP), a transforming growth factor-␤ (TGF-␤) receptor-interacting protein, was identified as an interacting partner of PDK1. STRAP was found to form in vivo complexes with PDK1 in intact cells. Mapping analysis revealed that this binding was only mediated by the catalytic domain of PDK1 and not by the pleckstrin homology domain. Insulin enhanced a physical association between PDK1 and STRAP in intact cells, but this insulin-induced association was prevented by wortmannin, a phosphatidylinositol 3-kinase inhibitor. In addition, the association between PDK1 and STRAP was decreased by TGF-␤ treatment. Analysis of the activities of the interacting proteins showed that PDK1 kinase activity was significantly increased by coexpression of STRAP, probably through the inhibition of the binding of 14-3-3, a negative regulator, to PDK1. Consistently, knockdown of the endogenous STRAP by the transfection of the small interfering RNA resulted in the decrease of PDK1 kinase activity. PDK1 also exhibited an inhibition of TGF-␤ signaling with STRAP by contributing to the stable association between TGF-␤ receptor and Smad7. Moreover, confocal microscopic study and immunostaining results demonstrated that PDK1 prevented the nuclear translocation of Smad3 in response to TGF-␤. Knockdown of endogenous PDK1 with small interfering RNA has an opposite effect. Taken together, these results suggested that STRAP acts as an intermediate signaling molecule linking between the phosphatidylinositol 3-kinase/PDK1 and the TGF-␤ signaling pathways.

Natural killer (NK) cells play important roles in immune surveillance. However, the tumor microenvironment suppresses NK cell function and allows cancer cells to evade immune detection. In this study, we investigated whether the thyroid cancer cell microenvironment has this effect on NK cells. We found that prostaglandin (PG) E2 produced by thyroid cancer cells suppressed the cytolytic activity of NK cells by inhibiting the expression of the natural cytotoxicity receptors NKp44 and NKp30 and the death receptor tumor necrosis factor-related apoptosis-inducing ligand. PGE2 and cyclooxygenase-2 were highly expressed in thyroid cancer cells; moreover, anaplastic thyroid cancer cells released higher amounts of PGE2 than the papillary subtype, which was associated with suppression of NK cell-inducing nuclear factor-κB and mitogen-activated protein kinase/extracellular signal-regulated kinase pathways via PGE2 receptor (EP) 2 and EP4 expressed on the NK cell surface. In addition, PGE2 inhibited the functional maturation of NK cells and reduced their cytotoxicity against target cells. These results indicate that PGE2 promotes thyroid cancer progression by inhibiting NK cell maturation and cytotoxicity. Thus, therapeutic strategies that target PGE2 in thyroid cancer could potentiate the immune response and improve patient prognosis.