Skin exposure to low-dose ultraviolet B (UVB) light up-regulates the expression of matrix metalloproteinase-1 (MMP-1), thus contributing to premature skin aging (photo-aging). Although cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), have been associated with UVB-induced signaling to MMP expression, very little are known about the roles of lipoxygenases and their products, especially leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE), in MMP-1 expression in skin keratinocytes. In the present study, we demonstrate that BLT2, a cell surface receptor for LTB4 and 12(S)-HETE, plays a critical role in UVB-mediated MMP-1 upregulation in human HaCaT keratinocytes. Moreover, our results demonstrated that BLT2-mediated MMP-1 upregulation occurs through a signaling pathway dependent on reactive oxygen species (ROS) production and the subsequent stimulation of ERK. Blockage of BLT2 via siRNA knockdown or with the BLT2-antagonist LY255283 completely abolished the up-regulated expression of MMP-1 induced by low-dose UVB irradiation. Finally, when HaCaT cells were transiently transfected with a BLT2 expression plasmid, MMP-1 expression was significantly enhanced, along with ERK phosphorylation, suggesting that BLT2 overexpression alone is sufficient for MMP-1 up-regulation. Together, our results suggest that the BLT2-ROS-ERK-linked cascade is a novel signaling mechanism for MMP-1 upregulation in low-dose UVB-irradiated keratinocytes and thus potentially contributes to photo-aging.
12-Hydroxy-5,8,10,14-eicosatetraenoic Acid/biosynthesis ; Cell Line ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Humans ; Keratinocytes/metabolism/*radiation effects ; Leukotriene B4/biosynthesis ; Matrix Metalloproteinase 1/*biosynthesis ; Phosphorylation ; Reactive Oxygen Species/metabolism ; Receptors, Leukotriene B4/*physiology ; Signal Transduction ; Ultraviolet Rays/*adverse effects

AIMBLT1 and BLT2 were both recently cloned and identified as two subtypes of leukotrine B4 (LTB4) receptors. With the usage of U-75302 and LY255283, the specific antagonists of BLT1 and BLT2 respectively, the involvement of BLT1 and BLT2 in the inflammatory and immunological responses was in vitro explored.

METHODS(1) To investigate inhibition of U-75302 and LY255283 on the proliferation of rat synovial cells, 3H-TdR incorporation into the cells was quantified. (2) Flow cytometric assay for interferon-gamma (IFN-gamma) and interleukine 4 (IL-4) profiles in CD4+ T lymphocytes from rat spleen was carried out to determine the ratio of Th1/Th2.

RESULTS(1) For inhibition on rat synovial cells proliferation, U-75302 exerted its effect only at a high concentration of 10 micromol/L and LY255283 at the concentrations of 10 micromol/L-10 micromol/L. (2) Both U-75302 and LY255283 could elevate the percentage of Th2, but could not influence that of Th1.

CONCLUSIONBLT1 and BLT2 were involved in the synovial cells proliferation change the ratio of Th1/Th2. Their meaning served as targets for prevention and treatment of infectious diseases should be emphasized.

Animals ; Cell Line ; Cell Proliferation ; drug effects ; Fatty Alcohols ; pharmacology ; Glycols ; pharmacology ; Inflammation ; immunology ; Male ; Rats ; Rats, Wistar ; Receptors, Leukotriene B4 ; antagonists & inhibitors ; physiology ; Synovial Membrane ; cytology ; immunology ; Tetrazoles ; pharmacology ; Th1-Th2 Balance

The incidence rates of urinary bladder cancer continue to rise yearly, and thus new therapeutic approaches and early diagnostic markers for bladder cancer are urgently needed. Thus, identifying the key mediators and molecular mechanisms responsible for the survival of bladder cancer has valuable implications for the development of therapy. In this study, the role of BLT2, a receptor for leukotriene B4 (LTB4) and 12(S)-hydroxyeicosatetraenoic acid (HETE), in the survival of bladder cancer 253J-BV cells was investigated. We found that the expression of BLT2 is highly elevated in bladder cancer cells. Also, we observed that blockade of BLT2 with an antagonist or BLT2 siRNA resulted in cell cycle arrest and apoptotic cell death, suggesting a role of BLT2 in the survival of human bladder cancer 253J-BV cells. Further experiments aimed at elucidating the mechanism by which BLT2 mediates survival revealed that enhanced level of reactive oxygen species (ROS) are generated via a BLT2-dependent up-regulation of NADPH oxidase members NOX1 and NOX4. Additionally, we observed that inhibition of ROS generation by either NOX1/4 siRNAs or treatment with an ROS-scavenging agent results in apoptotic cell death in 253J-BV bladder cancer cells. These results demonstrated that a 'BLT2-NOX1/4-ROS' cascade plays a role in the survival of this aggressive bladder cancer cells, thus pointing to BLT2 as a potential target for anti-bladder cancer therapy.
*Apoptosis ; Blotting, Western ; Cell Proliferation ; Cells, Cultured ; Gene Expression Regulation, Neoplastic/*physiology ; Humans ; Leukotriene Antagonists/pharmacology ; NADPH Oxidase/antagonists & inhibitors/genetics/metabolism ; Phosphorylation ; RNA, Messenger/genetics ; RNA, Small Interfering/genetics ; Reactive Oxygen Species/*metabolism ; Receptors, Leukotriene B4/antagonists & inhibitors/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Tetrazoles/pharmacology ; Up-Regulation ; Urinary Bladder Neoplasms/*genetics/mortality