過去實驗室研究發現， IP致效劑 berapost 和 BMY45778 會誘導經 PMA 處理24小時後的 HEL 細胞分化成棘狀細胞，且此分化現象為 MEK/ERK-dependent 。因 MEK/ERK1/2 上游為 cRaf (即Raf-1)。因此當我們投予 cRaf抑制劑 ZM 336372或 cRaf kinase 抑制劑 GW 5074 時，不僅不會抑制 IP 致效劑所誘導棘狀細胞的生成，反而會促進。此外從 western blot分析中可知， PMA 會誘導 cRaf 活性磷酸化位置 ser338 持續磷酸化以及 c-Raf抑制性磷酸化位置 ser259 持續去磷酸化，推測 cRaf 可能處於持續活化態；而當給予 IP 致效劑 beraprost 刺激於經 PMA 處理24小時後的 HEL 細胞發現， beraprost 不影響 c-Raf ser338 磷酸化，但 c-Raf ser259磷酸化則再度被誘導出來；從這些結果推測 c-Raf inhibition可能在參與在 IP 致效劑所誘導棘狀細胞的分化過程中。因此我們便想要了解 cRaf inhibition 對於在前處理 PMA 的 HEL 細胞中所扮演的角色；在單獨投予 cRaf / cRaf kinase 抑制劑，是否也也會促使棘狀細胞的形成。
　　實驗結果發現，加入 cRaf / cRaf kinase 抑制劑於 PMA 前處理過之 HEL 細胞中，由外觀發現，cRaf / cRaf kinase 抑制劑會誘導其分化成棘狀細胞的細胞型態；但其並不影響棘狀細胞專一性之表面抗原 CD83 及 HLA-DR 的表現。而在功能性試驗中，其細胞吞噬能力下降，這也是棘狀細胞的象徵。另一方面， cRaf / cRaf kinase 抑制劑也不會抑制 IP 致效劑所誘導的 ERK1/2 磷酸化；而當我們單獨投予 cRaf / cRaf kinase 抑制劑時，即可誘導 MEK 及 ERK1/2 磷酸化且呈現時間以及劑量相關性，這顯示出 MEK / ERK1/2 的活化為同步的。此外，比較其活化 ERK 的程度(即量)發現， IP 致效劑 BMY45778 其 ERK 磷酸化表現量為 ZM336372 和 GW5074 的三倍。好奇的是為何當 cRaf 受到抑制後，ERK 會活化，活化 ERK 的訊號又是從哪來的？從 cRaf 抑制劑 ZM 336372 劑量與 ERK 磷酸化表現結果或許可以說明，當其隨者抑制 cRaf 濃度增加， ERK1/2 磷酸化也遞增，其 peak 為 300nM ，但隨者抑制 B-Raf 濃度增加，ERK1/2 磷酸化也隨之遞減，在 10μM 達 B-Raf 完全抑制效果時 (10x IC50 of B-Raf) 降低至基本背景值 (ZM 336372：IC50 of cRaf = 70nM ； IC50 of B-Raf = 800nM)；而在 MEK 磷酸化上，也可以看到同樣的現像。由此可知當 cRaf 受到抑制後，所誘導 MEK / ERK1/2 磷酸化，可能是經由 B-Raf 所媒介的。
　　在抑制劑的篩選方面發現， ZM 336372 或 GW 5074 誘導 ERK1/2 磷酸化為 MEK- 和 PKCδ- dependent，但為 PKA- 和 classical PKC - independent；至於其它的抑制劑，例如 non-selective PKC 的抑制劑 Ro 318425 及 GF109203x ，會使貼壁細胞失去貼壁的能力，而 PI3K 抑制劑 Wortmannin 以及 LY 294002 則是會影響細胞的 viability 。
　　近來許多文獻指出，在許多細胞中， B-RAF 扮演者 MEK 的主要活化者，其主要功能是活化 MEK ；透過直接活化 MEK ，或者間接藉由 recruit C-RAF 的方式進而活化 MEK ；至於 C-RAF 和 A-RAF 則扮演者微調 ERK 的訊號，藉由調整其 ERK 活性的強度 (intensity) 或持續活化時間 (duration) ，進而決定細胞未來的命運。此外也有研究指出， wild-type C-RAF 會與 B-RAF 形成 heterodimer ；特別的是，isolated cRaf autoinhibitory domain 會去互相作用、並且抑制 B-Raf kinase domain 的活性。
　　因此我們便推測 C-RAF 和 B-RAF 之間可能存在著交叉調控的機制；而在我們的系統中， cRaf 似乎會去 associate B-Raf 並抑制 B-Raf 的活性，因此當 cRaf 受到抑制後，便解除對 B-Raf 活性的束縛，而 B-Raf 活性的增加便會去誘導 MEK / ERK1/2 的持續磷酸化及棘狀細胞分化。

　Previous studies we found that prostacyclin receptor agonists, berapost , BMY45778 induced a dendritic cell differentiation on PMA-pretreated HEL cells, and MEK / ERK is involved. It was found that selective cRaf / cRaf kinase inhibitor ZM 336372 or GW5074, they did not inhibit beraprost or BMY 45778 induced dendrite outgrowth but promoted the formation. Moreover, PMA induced dephosphorylation of inactivating site of cRaf ser259 was reversed by IP agonist while PMA induced sustained phosphorylation of activating site of cRaf ser338 was not. Therefore, cRaf may be in a constitutive active form after PMA treatment and inhibition of cRaf may be involved in cell differentiation.
　In our finding, addition of each of selective cRaf inhibitor ZM336372 or cRaf kinase inhibitor GW5074 to PMA-pretreated HEL cells also caused dendrite formation alone. Further characterized concluded that cRaf / cRaf kinase inhibitors induced a DC differentiation from PMA-pretreated HEL cells, by which reduced the phargocytosis capacity, but without phenotype changes CD83, HLA-DR. cRaf / cRaf kinase inhibitors also induced MEK/ERK1/2 phosphorylation in a time and dose dependent manner on PMA-pretreated cells; moreover, comparison of the intensity of ERK it is found that IP agonist BMY45778 induced 3-fold more potently than ZM336372 or GW5074. Curiously, where was ERK signal come from? According to ZM336372 (IC50 = 70 nM for inhibition of human c-Raf ; IC50 = 800 nM for inhibition of human B-Raf).dose-response curve may explain that why ERK activation while cRaf inhibition. ZM336372 induced ERK phosphorylation with increased concentration of cRaf inhibition, whereas it reduced ERK phosphorylation with increased concentration of B-Raf inhibition instead. Thus, it may indicate that B-Raf play an important role in mediate cRaf / cRaf kinase inhibitors induced ERK1/2 phosphorylation.
　By inhibitors’ screening, it was found that ZM336372 or GW5074 induced ERK1/2 phosphorylation were MEK- and PKCδ- dependent but PKA- and classical PKC- independent; as to others, like non-selective PKC inhibitors (Ro318426, GF109203x) would cause cells to lose adhesion ability after treatment; and PI3K inhibitors (LY294002, wortmannin) would affect cell viability.
　Recent studies indicate that B-RAF is the main MEK activator in most cells and that C-RAF and A-RAF modulate the signal to fine-tune the intensity and/or duration of ERK activity and therefore to determine cell fate.Furthermore, it is noteworthy that wild-type cRaf and B-Raf can heterodimerize and, more specifically, that the isolated autoinhibitory domain of cRaf can interact with, and inhibit, the catalytic domain of B-Raf.
　We propose that cRaf and B-Raf may cross-regulate each other; in our system cRaf seems to inhibit B-Raf activity. Therefore, cRaf inhibition caused by cRaf / cRaf kinase inhibitors would relieve the inhibition toward B-Raf activity, which induce low-intensity ERK activation and dendritic cell differentiation.

1. Claudia Wellbrock ,Maria Karasarides and Richard Marais THE RAF PROTEINS TAKE CENTRE STAGE NATURE REVIEWS | MOLECULAR CELL BIOLOGY 5 875-885 (2004)
2. Manuela Baccarini* Second nature: Biological functions of the Raf-1 ‘‘kinase’’ FEBS Letters 579, 3271–3277 (2005)
3. Amardeep S. Dhillon and Walter Kolch Untying the regulation of the Raf-1 kinase Archives of Biochemistry and Biophysics 404, 3–9 (2002)
4. Huira Chonga, Haris G. Vikisa, Kun-Liang Guan Mechanisms of regulating the Raf kinase family Cellular Signalling 15, 463–469 (2003)
5. Barnier, J. V., Papin, C., Eychene, A., Lecoq, O. & Calothy, G. The mouse B-raf gene encodes multiple protein isoforms with tissue-specific expression. J. Biol. Chem. 270, 23381–23389 (1995).
6. Storm, S. M., Cleveland, J. L. & Rapp, U. R. Expression of raf family proto-oncogenes in normal mouse tissues. Oncogene 5, 345–351 (1990).
7. Pritchard, C. A., Bolin, L., Slattery, R., Murray, R. & McMahon, M. Post-natal lethality and neurological and gastrointestinal defects in mice with targeted disruption of the A-Raf protein kinase gene. Curr. Biol. 6, 614–617 (1996).
8. Wojnowski, L. et al. Endothelial apoptosis in Braf-deficient mice. Nature Genet. 16, 293–297 (1997).
9. Wojnowski, L. et al. Craf-1 protein kinase is essential for mouse development. Mech. Dev. 76, 141–149 (1998).
10. Huser, M. et al. MEK kinase activity is not necessary for Raf-1 function. EMBO J. 20, 1940–1951 (2001).
11. Mikula, M. et al. Embryonic lethality and fetal liver apoptosis in mice lacking the c-raf-1 gene. EMBO J. 20, 1952–1962 (2001).
12. Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002).
13. Allen, L. F., Sebolt-Leopold, J. & Meyer, M. B. CI-1040 (PD184352), a targeted signal transduction inhibitor of MEK (MAPKK). Semin. Oncol. 30, 105–116 (2003).
14. Nicolas Dumaz and Richard Marais Integrating signals between cAMP and the RAS/RAF/MEK/ERK signalling pathways FEBS Journal 272, 3491–3504 (2005)
15. E O’Neill,1 and W Kolch Conferring specificity on the ubiquitous Raf/MEK signaling Pathway British Journal of Cancer 90, 283 – 288 (2004)
16. Anthony J. Muslin*Role of Raf Proteins in Cardiac Hypertrophy and Cardiomyocyte Survival Trends Cardiovasc Med. 15, 225 – 229 (2005)
17. Philip J. S. Stork and John M. Schmitt Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation TRENDS in Cell Biology ,12 258-266 (2002)
18. Kao, S., Jaiswal, R. K., Kolch, W. & Landreth, G. E. Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PC12 cells. J. Biol. Chem. 276, 18169–18177 (2001).
19. Corbit, K. C. et al. Different protein kinase C isoforms determine growth factor specificity in neuronal cells. Mol. Cell. Biol. 20, 5392–5403 (2000).
20. Bogoyevitch, M. A., Marshall, C. J. & Sugden, P. H. Hypertrophic agonists stimulate the activities of the protein kinases c-Raf and A-Raf in cultured ventricular myocytes. J. Biol. Chem. 270, 26303–26310 (1995).
21. Alavi, A., Hood, J. D., Frausto, R., Stupack, D. G. & Cheresh, D. A. Role of Raf in vascular protection from distinct apoptotic stimuli. Science 301, 94–96 (2003).
22. Sutor, S. L., Vroman, B. T., Armstrong, E. A., Abraham, R. T. & Karnitz, L. M. A phosphatidylinositol 3-kinase-dependent pathway that differentially regulates c-Raf and A-Raf. J. Biol. Chem. 274, 7002–7010 (1999).
23. Marshall, C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signalregulated kinase activation. Cell 80, 179–185 (1995).
24. Weber, J. D., Raben, D. M., Phillips, P. J. & Baldassare, J. J. Sustained activation of extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin D1 in G1 phase. Biochem. J. 326, 61–68 (1997).
25. Woods, D. et al. Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1. Mol. Cell. Biol. 17, 5598–5611 (1997).
26. Kerkhoff, E. & Rapp, U. R. High-intensity Raf signals convert mitotic cell cycling into cellular growth. Cancer Res. 58, 1636–1640 (1998)
27. Pritchard, C. A., Samuels, M. L., Bosch, E. & McMahon, M. Conditionally oncogenic forms of the A-Raf and B-Raf protein kinases display different biological and biochemical properties in NIH 3T3 cells. Mol. Cell. Biol. 15, 6430–6442 (1995).
28. Clare A Hall-Jackson et al. Paradoxical activation of Raf by a novel Raf inhibitor Chemistry & Biology 6, 559-568 (1999)
29. Maria Wartenberg, et al. Down-regulation of Intrinsic P-glycoprotein Expression in Multicellular Prostate Tumor Spheroids by Reactive Oxygen Species THE JOURNAL OF BIOLOGICAL CHEMISTRY 276, 17420–17428, (2001)
30. Maria WARTENBERG et al. REACTIVE OXYGEN SPECIES-MEDIATED REGULATION OF eNOS AND iNOS EXPRESSION IN MULTICELLULAR PROSTATE TUMOR SPHEROIDS Int. J. Cancer: 104, 274–282 (2003)
31. Karen Lackey et al. The Discovery of Potent cRaf1 Kinase Inhibitors Bioorganic & Medicinal Chemistry Letters 10, 223-226 (2000)
32. Ming-Shyan Chang, et al. Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-nB, but not p38 MAPK, pathways Cellular Signalling 17, 299–310 (2005)
33. Paul C. Chin, et al. The c-Raf inhibitor GW5074 provides neuroprotection in vitro and in an animal model of neurodegeneration through a MEK-ERK and Akt-independent mechanism Journal of Neurochemistry 90, 595–608 (2004)
34. JEROME W. , et al. VEGF increases endothelial permeability by separate signaling pathways involving ERK-1/2 and nitric oxide Am J Physiol Heart Circ Physiol 284,: H92–H100 (2003)
35. Banchereau J. and Steinman RM. Dendritic Cells and the Control of Immunity. Nature 392, 245-251, 1998.
36. Pierre P., Turley SJ., Gatti E., Hull M., Meltzer J., Mirza A., Inaba K., Steinman RM. and Mellman I. Developmental Regulation of MHC class II Transport in Mouse Dendritic Cells. Nature 388, 787-792, 1997.
37. Faries MB., Bedrosian I., Xu S., Koski G., Roros JG., Moise MA., Nguyen HQ., Eegels FC., Cohen PA. and Czerniecki BJ. Calcium Signaling Inhibits Interleukin-12 Production and Activates CD83+ Dendritic Cells that Induce Th2 Cell Development. Blood 98, 2489-2497, 2001.
38. Warren MK., Rose WL., Cone JL., Rice WG. and Turpin JA. Differential Infection of CD34+ Cell-Derived Dendritic Cells and Monocyte-Tropic HIV-1 Strains. J. Immunol. 158, 5035-5042, 1997.
39. Smyth MJ., Godfrey DI. and Trapani JA. A Fresh Look at Tumor Immunosurveillance and Immunotherapy. Nature Immunology 2, 293-299, 2001.
40. Biragyn A. and Kwak LW. Designer Cancer Vaccines Are Still in Fashion. Nature Medicine 6, 966-968, 2000.
41. Nestle FO. Dendritic Cell Vaccination for Cancer Therapy. Oncogene 19, 6673-6679, 2000.
42. Hart DJ. Dendritic Cells: Unique Leukocyte Populations Which Control the Primary Immune Response. Blood 90, 3245-3287, 1997.
43. Shortman K. and Liu YJ. Mouse and Human Dendritic Cell Subtypes. Nature Immunology. 2, 151-161, 2002.
44. Despars G. and O’Neill HC. A Role for Niches in the Developmant of A Multiplicity of Dendritic Cell Subsets. Exp. Hematol. 32, 235-243, 2004.
45. Davis TA., Saini AA., Blair PJ., Levine BL., Craighead N., Harian DM., June CH., and Lee KP. Phorbol Easters Induce Differentiation of Human CD34+ Hemopoietic Progenitors to Dendritic Cells: Evidence for Protein Kinase C-Mediated Signaling. Blood 160, 3689-3697, 1998.
46. Lindner I., Kharfan-Dabaja MA., Ayala E., Kolonias D., Carlson LM., Beazer-Barclay Y., Scherf U., Hnatyszyn JH. and Lee KP. Induced Dendritic Cell Differentiation of Chronic Myeloid Leukemia Blasts Is Associated with Down-Regulation of BCR-ABL. J. Immunol. 171, 1780-1791, 2003.

47. Ramadan G., Schmidt RE., and Schubert J. In Vitro Generation of Human CD86+ Dendritic Cells from CD34+ Haematopoietic Progenitors by PMA and in Serum-Free Medium. Clin. Exp. Immunol. 125, 237-244, 2001.
48. Blandine de SV., Isabelle FV., Massacrier C., Gaillard C., Vanbervliet B., Aït-Yahia S., Banchereau J., Liu YJ., Lebecque S. and Caux C. The Cytokine Profile Expressed by Human Dendritic Cells Is Dependent on Cell Subtype and Mode of Activation. J. Immunol. 160, 1666-1676, 1998.
49. Mellman I. and Steinman RM. Cell 106, 255-258, 2001.
50. Yanagawa Y. and Onoé K. CCR7 Ligands Induce Rapid Endocytosis in Mature Dendritic Cells with Concomitant Up-Regulation of Cdc42 and Rac Activities. Blood 101, 4923-4929, 2003.
51. MacDonald KA., Munster DJ., Clark GJ., Dzionek A., Schmitz J. and Hart DN. Characterization of Human Blood Dendritic Cell Subsets. Blood 100, 4512-4520, 2002.
52. Yanagawa Y. and Onoé K. CCR7 Ligands Induce Rapid Endocytosis in Mature Dendritic Cells with Concomitant Up-Regulation of Cdc42 and Rac Activities. Blood 101, 4923-4929, 2003.
53. Inaba K., Turley S., Iyoda T., Yamaide F., Shimoyama S., Sousa CR., Germain RN., Mellman I, and Steinman RM. The Formation of Immunogenic Major Histocompatibility Compartments of Dendritic Cells Is Regulated by Inflammatory Stimuli. J. Exp. Med. 191, 927-936, 2000.
54. Martin P. HEL Cells: A New Human Erythroleukemia Cell Line with Spontaneous and Induced Globin Expression. Science 216, 1233-1235, 1982.
55. Tsuji T., Waga I., Tezuka K., Kamada M., Yatsunami K. and Kodama H. Integrin β2 (CD18)-Mediated Cell Proliferation of HEL Cells on a Hematopoietic-Supportive Bone Marrow Stromal Cell Line, HESS-5 Cells. Blood 91, 1263-1271, 1998.
56. Long MW., Heffner CH., Williams JL., Peters C., and Prochownik EV. Regulation of Megakaryocyte Phenotype in Human Erythroleukemia Cells. J. Clin. Invest. 85, 1072-1084, 1990.
57. Sheth S. B. et al. Distribution of prostaglandin IP and EP receptor subtypes and isoforms in platelets and human umbilical artery smooth muscle cells. Biritish J. Haema.. 102, 1204-1211,1998.
58. Feoktistov, I., Breyer, R. M., Biaggioni, I. Prostanoid Receptor with a Novel Pharmacological Profile in Human Erythroleukemia Cells. Biochem. Pharmacol. 54, 917-926, 1997.
59. Papayannopoulou T., Yokochi T., Nakamoto B. and Martin P. The Surface Antigen Profile of HEL Cells. Globin Gene Expression and Hematopoietic Differentiation , 277-292,1983.
60. Papayannopoulou T., Yokochi T., Chait A., and Kannagi R. Human Erythroleukemia Cell Line (HEL) Undergoes a Drastic Macrophage-Like Shift With TPA. Blood 62, 832-845, 1983.
61. Papayannopoulou T., Nakamoto B., Kurachi S., Tweeddale M. and Messner H. Surface Antigenic Profile and Globin Phenotype of Two New Human Erythroleukemia Lines：Characterization and Interpretataions. Blood 72, 1029-1038, 1988.
62. Berthier R., Chapel A., Schweitzer A., and Andrieux A. β2 Integrins mediate adherent phenotype of human erythroblastic cell lines after phorbol 12-myristate 13-acetate induction. Biochem. J. 309, 491-497, 1995.
63. Auwerx JH., Chait A., Wolfbauer G.. and Deeb SS. Loss of Copper-Zinc Superoxide Dismutase Gene Expression in Differentiated Cells of Myelo-Monocytic Origin. Blood 74, 1807-1810, 1989.
64. Berlanga O., Bobe R., Becker M., Murphy G., Leduc M., Bon C., Barry FA., Gibbins JM., Garcia P., Frampton J. and Watson SP. Expression of the Collagen Receptor Glycoprotein VI During Megakaryocyte Differentiation. Blood 96, 2740-2745, 2000.
65. Yeo E., Furie BC. and Furie B. PADGEM Protein in Human Erythroleukemia Cells. Blood 73, 722-728, 1989.
66. Zauli G., Bassini A., Catani L., Gibellini D., Celeghini C., Borgatti P., Caramelli E., Guidotti L. and Capitani S. PMA-Induced Megakaryocytic Differentiation of HEL Cells is Accompanied by Striking Modifications of Protein Kinase C Catalytic Activity and Isoform Composition at the Nuclear Level. Br. J. Haematol. 92, 530-536, 1996.
67. Jiang F., Jia Y., and Cohen I. Fibronectin- and Protein Kinase C-Mediated Activation of ERK/MARK are Essential for Proplateletlike Formation. Blood 99, 3579-3584, 2002.
68. 莊惠評，“鑑別前列腺環素致效劑對於PMA誘導HEL細胞分化特性影響之研究”，國立成功大學碩士論文，2004
69. 柯夢昀，“EP2與EP4受體致效劑對於PMA前處理HEL細胞之影響”，國立成功大學碩士論文，2005
70. Silvano C. et al. PMA-induced megakaryocytic of HEL cells is accompanied by striking modifications of protein kinase C catalytic activity and isoform composition at the nuclear level. British Journal of Haematology 92, 530-536,1996.
71. Wan, P. T. et al. Mechanism of activation of the RAF–ERK signaling pathway by oncogenic mutations of B-RAF. Cell 116, 855–867 (2004).
72. Ikenoue, T. et al. Functional analysis of mutations within the kinase activation segment of B-Raf in human colorectal tumors. Cancer Res. 63, 8132–8137 (2003).
73. Houben, R. et al. Constitutive activation of the Ras–Raf signaling pathway in metastatic melanoma is associated with poor prognosis. J. Carcinog. 3, 6 (2004).
74. Mathew J. Garnett1 and Richard Marais1 Guilty as charged: B-RAF is a human oncogene CANCER CELL 6, 313-319 (2004)
75. Walter KOLCH1 Meaningful relationships : the regulation of theRas/Raf/MEK/ERK pathway by protein interactions Biochem. J. 351, 289-305 (2000)
76. OLIVIER ZUGASTI. et al. Raf-MEK-Erk Cascade in Anoikis Is Controlled by Rac1 and Cdc42 via Akt MOLECULAR AND CELLULAR BIOLOGY 21, 6706–6717 (2001)
77. Maude Le Gall. et al. The p42/p44 MAP Kinase Pathway Prevents Apoptosis Induced by Anchorage and Serum Removal Molecular Biology of the Cell. 11, 1103–1112 (2000)
78. Weber, C.K., Slupsky, J.R., Kalmes, H.A. and Rapp, U.R. Active Ras induces heterodimerization of cRaf and BRaf. Cancer Res. 61, 3595–3598 (2001)
79. Tran, N.H., Wu, X. and Frost, J.A. B-Raf and Raf-1 are regulated by distinct autoregulatory mechanisms. J. Biol. Chem. 280, 16244-16253 (2005)