cPLA2α (cytosolic phospholipase A2 α) 是位於細胞質的磷脂水解酵素，其水解膜上的phospholipid而形成花生四烯酸(arachidonic acid)的過程中，扮演著速率限制的角色。花生四烯酸會進一步受下游的酵素代謝形成不同脂質產物(eicosanoid)以負責傳達訊息來應付生理功能反應之所需。但是當細胞內產生過度eicosanoid時，常導致細胞不正常的proliferation(增殖)和tumorigenesis(癌化)。根據先前研究我們發現在人類肺癌A549細胞中，PKC的活化劑phorbol 12–myristate 13–acetate (PMA)會增加cPLA2α mRNA和蛋白質的表現，從reporter assay中顯示PMA和c-Jun同樣透過cPLA2α promoter (-53至-35bp)上的2個Sp1 sites來誘導cPLA2α promoter activity增加。利用c-Jun siRNA knock down內生性的c-Jun觀察到cPLA2α promoter activity及蛋白質表現都會下降，而免疫沉澱分析和DNA親合沉澱分析確認了c-Jun及Sp1 有交互作用而c-Jun / Sp1 complex的確會結合在cPLA2α promoter上的Sp1 binding site；利用proteomic assay分析到nucleolin也會結合至Sp1 binding site而且結合的量不會隨著PMA刺激而改變，使用nucleolin siRNA knock down細胞內的nucleolin後，觀察到PMA所誘導的cPLA2α啟動區活性，蛋白質表現和mRNA皆會下降，而pGL3 reporter assay中，我們認為nucleolin會參與在cPLA2α的基因轉錄調控中，免疫沉澱分析的實驗也觀察到nucleolin和c-Jun會形成interaction；另外在訊息傳遞的探討方面，使用ERK抑制劑：U0126會抑制c-Jun蛋白質的生合成進而抑制PMA所誘導的cPLA2α 啟動區活性和蛋白質表現，在此顯示了：PMA誘導cPLA2α基因表現是透過c-Jun/Sp1及nucleolin/c-Jun的交互作用，此複合物會結合至cPLA2α啟動區的Sp1位置並進一步吸引其他cofactor共同調控cPLA2α基因表現，而ERK則參與在PMA誘導cPLA2α基因表現的訊息傳遞過程中。

cPLA2α (cytosolic phospholipase A2 α), major form of PLA2 in the cell, selectively hydrolyzes membrane phospholipids at the sn-2 position, and control the rate-limiting enzyme step in the eicosanoid production. These lipid mediators can be responsible for physiological process. However, marked increased of eicosanoid is involved in the abnormal proliferation and tumorigenesis. According to previous study, we know that PMA, an activator of PKC increased the expression of cPLA2α mRNA and protein synthesis. Reporter assays revealed the PMA response element was in the region of -53 to -35 bp of cPLA2α promoter. Furthermore, the promoter activity of cPLA2α gene induced by c-Jun was also regulated in the same manner as PMA. Small interfering RNA mediated knock down of c-Jun reduced cPLA2α promoter activity and protein expression. Immunoprecipitation and DNA affinity precipitation assays were performed to confirm the interaction between c-Jun and Sp1, and the binding of c-Jun/Sp1 to Sp1 binding sites on cPLA2α promoter. According to proteomic assay, we also observed that nucleolin can bind to Sp1 binding sites. Small interfering RNA mediated knock down of nucleolin also reduced cPLA2α mRNA, promoter activity and protein expression. From the pGL3 reporter assay data, it indicates that nucleolin was involved in the transcriptional regulation of cPLA2α gene expression. Immunoprecipitation assay was performed to confirm the interaction between nucleolin and c-Jun in PMA-treated cells. By using MAPK inhibitors, we identify that PMA treatment requires ERK signaling in regulating cPLA2α transcriptional activity. These findings indicate the cooperation of c-Jun, nucleolin and Sp1 is required for PMA-induced the expression of cPLA2α and the ERK signaling may also be involved in the gene expression.

Ackermann, E. J., and Dennis, E. A. Mammalian calcium- independent phospholipase A2. Biochim. Biophys. Acta. 1259, 125-136,1995

Balsinde, J., Bianoco, I. D., Ackermann, E. J., Conde-Frieboes, K., and Dennis, E. A. Inhibition of Ca2+-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remolding in P388D1 macrophages. Proc. Natl. Acad. Sci. U.S.A. 92, 8527-8531, 1995

Belenguer, P., Baldin, V., Mathieu, C., Prats, H., Bensaid, M., Bouche, G., and Amalric, F. Protein kinase NII and the regulation of rDNA transcription in mammalian cells. Nucleic Acids Res. 17, 6625-6636, 1989

Blaine, S. A., Wick, M., Dessev, C., and Nemenoff, R. A. Induction of cPLA2 in lung epithelial cells and non-small cell lung cancer is mediated by Sp1 and c-Jun. J. Biol. Chem. 276, 42737-42743, 2001

Bonventre, J. V., Huang, Z., Taheri, M. R., O’Leary, E., Li, E., Moskowitz, M. A., and Sapirstein, A. Reduced fertility and postischaemic brain injury in mice descent in cytosolic phospholipase A2. Nature 390: 622–625, 1997

Borer, R. A., Lehner, C. F., Eppenberger, H. M., and Nigg, E. A. Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56, 379-390, 1989

Bouvet, P., Diaz, J. J., Kindbeiter, K., Madjar, J. J., and Amalric, F. Nucleolin interacts with several ribosomal proteins through its RGG domain. J. Biol. Chem. 273, 19025-19029, 1998

Brockstedt, E., Rickers, A., Kostka, S., Laubersheimer, A., Dorken, B., Wittmann-Liebold, B., Bommert, K., and Otto, A. Identification of apoptosis-associated proteins in a human burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3. J. Biol. Chem. 273, 28057-28064, 1998

Chen, B. K., Kung, H. C., Tsai, T. Y., and Chang, W. C. Essential role of mitogen-activated protein kinase pathway and c-Jun induction in epidermal growth factor-induced gene expression of human 12-lipoxygenase. Mol. Pharmacol. 57, 153-161, 2000

Chen, B. K. and Chang, W. C. Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase. Proc. Natl. Acad. Sci. USA 97, 10406 -10411, 2000

Chen, J., Engle, S. J., Seilhamer, J. J., and Tischfield, J. A. Cloning and
characterization of novel rat and mouse low molecular weight Ca2+-
dependent phospholipase A2s containing 16 cysteines. J. Biol. Chem. 269, 23018–23024, 1994

Chen, J., Engle, S. J., Seilhamer, J. J., and Tischfield, J. A. Cloning and
recombinant expression of a novel human low molecular weight Ca2+- dependent phospholipase A2. J. Biol. Chem. 269, 2365–2368, 1994

Clark, J. D., Lin, L. L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin,
A. Y., Milona, N., and Knopf, J. L. A novel arachidonic acid-selective homology to PKC and GAP. Cell 65, 1043-1051, 1991

Clark, J. D., Schievella, A. R., Nalefski, E. A., and Lin, L. L. Cytosolic
phospholipase A2. J. Lipid. Mediat. Cell Signal. 12, 83-117, 1995

Cupillard, L., Koumanov, K., Mattei, M. G., Lazdunski, M., and Lambeau,
G. Cloning, chromosomal mapping, and expression of a novel human
secretory phospholipase A2. J. Biol. Chem. 272, 15745–15752, 1997

Dennis, E. A. Diversity of group types, regulation, and function of
phospholipase. J. Biol. Chem. 269, 13057-13060, 1994

Derenzini, M., Sirri, V., Trere, D., and Ochs, R. L. The quantity of
nucleolar proteins nucleolin and protein B23 is related to cell doubling time in human cancer cells. Lab Invest. 73, 497-502, 1995

Erard, M. S., Belenguer, P., Caizergues-Ferrer, M., Pantaloni, A., and Amalric, F. A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone H1. Eur. J. Biochem. 175, 525-530, 1988

Evans, J. H., Spencer, D. H., Zweifach, A., and Leslie, C. C. Intracellular
calcium signals regulating cytosolic phospholipase A2 translocation to internal membranes. J. Biol. Chem. 276, 30150–30160, 2001

Gelb, M. H., Valentin, E., Ghomashchi, F., Lazdunski, M., and Lambeau,
G. Cloning and recombinant expression of a structurally novel human secreted phospholipase A2. J. Biol. Chem. 275, 39823–39826, 2000

Gill, G., Pascal, E., Tseng, Z. H., and Tjian, R. A glutamine-rich
hydrophobic patch in transcription factor Sp1 contacts the dTAFⅡ110 component of Drosophila TFⅡD complex and mediates transcriptional activation. Proc. Natl. Acad. Sci. U.S.A 91, 192-196, 1994

Ginisty, H., Amalric, F., and Bouvet, P. Nucleolin functions in the first
step of ribosomal RNA processing. EMBO J. 17, 1476-1486, 1998

Han, I. And Kudlow, J. E.: Reduced O glycosylation of Sp1 is associated
with increased proteasome susceptibility. Mol. Cell. Biol. 17, 2550-2558, 1997

Hattori, M., Adachi, H., Tsujimoto, M., Arai, H., and Inoue, K. The
catalytic subunit of bovine brain platelet-activating factor acetylhydrolase is a novel type of serine esterase. J. Biol.Chem. 269, 23150–23155, 1994

Heasley, L. E., Thaler, S., Nicks, M., Price, B., Skorecki, K., and
Nemenoff, R. A. Induction of cytosolic phospholipase A2 by oncogenic Ras in human non-small cell lung cancer J. Biol. Chem. 272, 14501 -14504, 1997

Hefner, Y., Borsch-Haubold, A. G., Murakami, M., Wilde, J. I., Pasquet, S.,
Schieltz, D., Ghomashchi, F., Yates III, J. R., Armstrong, C. G., Paterson,
A., Cohen, P., Fukunaga, R., Hunter, T., Kudo, I., Watson, S. P., and Gelb,
M. H. Serine 727 phosphorylation and activation of cytosolic
phospholipase A2 by MNK1-related protein kinases. J. Biol. Chem. 275,
37542–37551, 2000

Hirabayashi, T., Kume, K., Hirose, K., Yokomizo, T., Iino, M., Itoh, H.,
and Shimizu, T. Critical duration of intracellular Ca2+ response required for continuous translocation and activation of cytosolic phospholipase A2. J. Biol. Chem. 274, 5163–5169, 1999

Huddleson, J. P., Ahmad, N., and Lingrel, J. B. Up-regulation of the KLF2
transcription factor by fluid shear stress requires nucleolin. J. Biol. Chem. 281, 15121-15128, 2006

Hung, J. J., Wang, Y. T., and Chang, W. C. Sp1 deacetylation induced by
phorbol ester recruits p300 to activate 12(S)-lipoxygenase gene transcription. Mol. Cell. Biol. 26, 1770-1785, 2006

Ishizaki, J., Suzuki, N., Higashino, K., Yokota, Y., Ono, T., Kawamoto,
K., Fujii, N., Arita, H., and Hanasaki, K. Cloning and characterization of novel mouse and human secretory phospholipase A2s. J. Biol. Chem. 274, 24973–24979, 1999

Jiang, Y. J., Lu, B., Choy, P. C., and Hatch, G. M. Regulation of cytosolic
phospholipase A2, cyclooxygenase-1 and -2 expression by PMA, TNFalpha, LPS and M-CSF in human monocytes and macrophages. Mol Cell Biochem. 246, 31-38, 2003

Jordan, G. At the heart of the nucleolus. Nature 329, 489-490, 1987

Karlseder, J., Rotheneder, H., and Wintersberger, E. Interaction of Sp1 with the growth– and cell cycle–regulated transcription factor E2F. Mol. Cell. Biol. 16, 1659–1667, 1996

Kibbey, M. C., Johnson, B., Petryshyn, R., Jucker, M., and Kleinman, H. K.
A 110-kD nuclear shuttling protein, nucleolin, binds to the neurite-promoting IKVAV site of laminin-1. J. Neurosci. Res. 15, 314-22, 1995

Kramer, R. M., Hession, C., Johansen, B., Hayes, G., McGray, P., Chow,
E. P., Tizard, R., and Pepinsky, R. B. Structure and properties of a
human non-pancreatic phospholipase A2. J. Biol. Chem. 264, 5768–5775, 1989

Kudo, I., Murakami, M., Hara, S., and Inoue, K. Mammalian non-pancreatic phospholipases A2. Biochim. Biophys. Acta. 177, 217-231, 1993

Kudo, I., and Murakami, M. Phospholipase A2 enzymes. Prostaglandins
Other Lipid Mediat. 68-69, 3-58, 2002

Leggett, R. W., Armstrong, S. A., Barry, D., and Mueller, C. R. Sp1 is
phosphorylated and its DNA binding activity down-regulated upon terminal differentiation of liver. J. Biol. Chem. 270, 25879-25884, 1995

Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R.
J. cPLA2 is phosphorylated and activated by MAP kinase. Cell 72, 269–278, 1993

Masumi A, Fukazawa H, Shimazu T, Yoshida M, Ozato K, Komuro K,
and Yamaguchi K. Nucleolin is involved in interferon regulatory factor-2-dependent transcriptional activation. Oncogene 1-12, 2006

Meyer, A.M., Dwyer-Nield, L. D., Hurteau, G. J., Keith, R. L., O’Leary, E.,
You, M., Bonventre, J. V., Nemenoff, R. A., and Malkinson, A. M.
Decreased lung tumorigenesis in mice genetically deficient in cytosolic phospholipase A2. Carcinogenesis 25, 1517-24, 2004

Miyashita, A., Crystal, R. G., and Hay, J. G. Identification of a 27 bp
5’-flanking region element responsible for the low level constitutive expression of the human cytosolic phosphalipase A2 gene. Nucleic Acids Res. 23, 293-301, 1995

Murakami, M., and Kudo, I. Phospholipase A2. J Biochem. 131, 285-292,
2002

Muthalif, M. M., Hefner, Y., Canaan, S., Harper, J., Zho, H., Parmentier, J.
H., Aebersold, R., Gelb, M. H., and Malik, K. U. Functional interaction of calcium/calmodukin-dependent protein kinase II and cytosolic phospholipase A2. J. Biol. Chem. 276, 39653–39660, 2001

Nagase, T., Uozumi, N., Ishii, S., Kume, K., Izumi, T., Ouchi, Y., and
Shimizu, T. Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2. Nat. Immunol.1, 42–46, 2000

Nishizuka, Y. The Albert Lasker Medical Awards. The family of protein kinase C for signal transduction. JAMA. 262, 1826–1833, 1989

Prekins, N. D., Agranoff, A. B., Pascal, E., and Nabel, G. J. An interaction between the DNA–binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation. Mol. Cell. Biol. 14, 6570–6583, 1994

Pugh, B. F. Mechanisms of transcription complex assembly. Curr. Opin.
Cell. Biol. 8, 303-311, 1996

Putten, V. V., Refaat, Z., Dessev, C., Blaine, S., Wick, M., Butterfield,
L., Han, S. Y., Heasley, L. E., and Nemenoff, R. A. Induction of cytosolic phospholipase A2 by oncogenic Ras is mediated through the JNK and ERK pathways in rat epithelial cells. J. Biol. Chem. 276, 1226-1232, 2001

Rajabi, M. R., and Cybulsky, A. V. Phospolipase A2 activity is increased
in guinea pig uterine cervix in late pregnancy and parturition. Am. J. Physiol. 269, E940-E947, 1995

Roos, M. D., Su, K., Baker, J. R., and Kudlow, J. E. O glycosylation of a
Sp1-derived peptide blocks known Sp1 protein interactions. Mol. Cell. Biol. 17, 6472-6480, 1997

Ryu, S., Zhou, S., Ladurner, A. G., and Tjian, R. The transcriptional
cofactor complex CRSP is required for activity of enhancer-binding protein Sp1. Nature 397, 446-450, 1999

Saluja, D., Vassallo, M. F., and Tanese, N. Distinct subdomains of human
TAFⅡ130 are required for interactions with glutamin-rich transcriptional activators. Mol. Cell. Biol. 18, 5734-5743, 1998

Seto, E., Lewis, B., and Shenk, T. Interaction between transcription factors Sp1 and YY1. Nature 365, 462–464, 1993

Sharp, J. D., White, D. L., Chiou, X. G., Goodson, T., Gamboa, G. C.,
McClure, D., Burgett, S., Hoskins, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M. Molecular cloning and expression of human Ca2+-sensitive cytosolic phospholipase A2. J. Biol. Chem. 266, 14850-14853, 1991

Suzuki, N., Ishizaki, J., Yokota, Y., Higashino, K., Ono, T., Ikeda, M.,
Fujii, N., Kawamoto, K., and Hanasaki, K. Structures, enzymatic properties, and expression of novel human and mouse secretory phospholipase A2s. J. Biol. Chem. 275, 5785–5793, 2000

Tabuchi, S., Uozumi, N., Ishii, S., Shimizu, Y., Watanabe, T., and
Shimizu,T. Mice deficient in cytosolic phospholipase A2 are less susceptible to cerebral ischemia/reperfusion injury. Acta Neurochir Suppl. 86, 169-72, 2003
Tang, J., Kriz, R. W., Wolfman, N., Shaffer, M., Seehra, J., and Jones, S.
S. A novel cytosolic calcium-independent phospholipase A2 contain eight ankyrin motifs. J. Biol. Chem. 273, 8567-8575, 1997

Tjoelker, L. W., Wilder, C., Eberhardt, C., Stafforini, D. M., Dietsch, G.,
Schimpf, B., Hooper, S., Le Trong, H., Cousens, L. S., Zimmerman, G. A., Yamada, Y., McIntyre, T., Prescott, S. M., and Gray, P. W. Anti-inflammatory properties of a platelet-activating factor acetylhydrolase. Nature 374, 549–553, 1995

Uozumi, N., Kume, K., Nagase, T., Nakatani, N., Ishii, S., Tashiro, F.,
Komagata, Y., Maki, K., Ikuta, K., Ouchi, Y., Miyazaki, J., and Shimizu, T. Role of cytosolic phospholipase A2 in allergic response and parturition. Nature 390, 618–622, 1997

Valentin, E., Ghomashchi, F., Gelb, M. H., Lazdunski, M., and Lambeau,
G. On the diversity of secreted phospholipases A2: cloning, tissue distribution, and functional expression of two novel mouse group II enzymes. J. Biol. Chem. 274, 31195–31202, 1999

Verheij, H. M., Slotboom, A. J., and de Haas, G. H. Structure and function of phospholipase A2. Rev. Physiol. Biochem. Pharmacol. 91, 91–203, 1981

Wang, Y. N. and Chang, W. C. Induction of disease-associated Keratin 16 gene expression by epidermal growth factor is regulated through cooperation of transcription factors Sp1 and c-Jun. J. Biol. Chem. 278, 45848-45857, 2003

Westmark, C.J., and Malter, J. S. Up-regulation of nucleolin mRNA and protein in peripheral blood mononuclear cells by extracellular-regulated kinase. J. Biol. Chem. 276, 1119-1126, 2001

Wick, M. J., Blaine, S., Putten, V. V., Saavedra, M., and Nemenoff, R. A.
Lung Kruppel-like factor (LKLF) is a transcriptional activator of the cytosolic phospholipase A2α promoter. Biochem. J. 387, 239-246, 2005

Wu, T., Ikezono, T., Angus, C. W., and Shelhamer, J. H. Characterization
of the promoter for the human 85 kDa cytosolic phospholipase A2 gene. Nucleic Acids Res. 22, 5093-5098, 1994

Yang, T. H., Tsai, W. H., Lee, Y. M., Lei, H. Y., Lai, M. Y., Chen, D. S.,
Yeh, N. H., and Lee, S. C. Purification and characterization of nucleolin and its identification as a transcription repressor. Mol. Cell. Biol. 14, 6068-6074, 1994

You, H. J., Woo, C. H., Cho, E. Y., Cho, S. H..Yoo, Y. J. and Kim, J. H.
Roles of Rac and p38 kinase in the activation of cytosolic phospholipase A2 in response to PMA. Biochem. J. 388, 527-535, 2005

Zaidi, S. H., Denman, R., and Malter J. S. Multiple proteins interact at a
unique cis-element in the 3'- untranslated region of amyloid precursor protein mRNA. J. Biol. Chem. 269, 24000-24006, 1994

Zhang, X., Wrzeszczynska, M. H., Horvath, C. M., and Darnell, J. E. Jr.
Interacting regions in Stat3 and c-Jun that participate in cooperative transcriptional activation. Mol. Cell. Biol. 19, 7138-46, 1999

Zhang, Y., Feng, X. H., and Derynck, R. Smad3 and Smad4 cooperate
with c-Jun/c-Fos to mediate TGF-beta-induced transcription. Nature 394, 909-1013, 1998

Zhang, Y., Bhatia, D., Xia1, H., Castranova, V., Shi, X., and Chen, F.
Nucleolin links to arsenic-induced stabilization of GADD45α mRNA. Nucleic Acids Res. 34, 485–495, 2006