摘要

纖維母細胞生長因子第一型(FGF-1)在許多生物反應中扮演重要的角色，如細胞分化、傷口修復、血管新生、及組織生長等。FGF-1B 是人類FGF-1基因在腦部組織中主要產生的轉錄片段，先前的研究指出由FGF1-1B 啟動子的-540到 +31所驅動的GFP報導基因可以被用來偵測細胞內FGF-1的表現並且可應用於分離神經幹細胞及神經前驅細胞。FGF-1B 啟動子上有兩個調控區域RR1及RR2，在RR2中有一段18-bp 序列是對於蛋白質與去氧核醣核酸結合最重要的序列，18-bp序列具有調控因子X1 (RFX1)的保守結合位，調控因子X1會藉由與18-bp 結合來調控FGF-1B 啟動子的活性。我們進一步證明調控因子X家族的其他兩個成員調控因子X2 (RFX2)與調控因子X3 (RFX3) 也會與18-bp序列結合。我們藉由電泳移動偏移分析實驗證明RFX1同源二聚體、RFX1-RFX2、 RFX1-RFX3 與 RFX2-RFX3異源二聚體會與與18-bp序列結合。此外，只有RFX2-RFX3異源二聚體會出現在FGF-1B positive而不會出現在FGF-1B negative 細胞。我們使用F1BGFP 報導基因與 siRNA轉染發現FGF-1B 啟動子的活性會在knockdown調控因子X1或調控因子X3後上升，而 knockdown調控因子X2則會降低FGF-1B 啟動子的活性。此外，我們利用蛋白激酶C 抑制劑初步證明蛋白激酶C訊號傳遞路徑參與FGF-1B 啟動子的調控。蛋白激酶C訊號傳遞路徑、調控因子X與FGF-1B 啟動子間的詳細交互作用機制值得進一步深入探討

Abstract

Human fibroblast growth factor 1 (FGF-1) is involved in the regulation of many biological processes such as tissue growth, wound healing, cell differentiation and angiogenesis. FGF-1B is the major transcript of FGF-1 gene in brain tissue. We previously reported that human FGF1 gene 1B promoter (-540 to +31)-driven green fluorescence (F1BGFP) can be used to monitor endogenous FGF1 expression and neural stem/progenitor cells (NSPCs) isolation. The FGF-1B promoter region contains two regulatory elements (RR1 and RR2), and the minimal region required for the DNA-protein interaction in RR2 is the 18-bp sequence (-484 to -467). Regulatory factor X (RFX) 1 binds to the 18-bp sequence, which has the imperfect palindromic RFX1 consensus sequence, and participates in the transcription regulation in FGF-1B. In the present study, we further showed two other members of the RFX family, RFX2 and RFX3, could bind to the 18-bp sequence. Using electrophoretic mobility shift assay (EMSA), we demonstrated that RFX1 homodimers , RFX1-RFX2, RFX1-RFX3 and RFX2-RFX3 heterodimers bind to the 18-bp sequence. In addition, only the RFX2-RFX3 complex could be detected in the nuclear extract of FGF-1B positive cells, but not in FGF-1B negative cells. Using F1BGFP reporter and siRNA transfection, we found that knockdown of RFX1 or RFX3 could increase F1B promoter activity. In contrast, knockdown of RFX2 with siRNA significantly inhibited F1B promoter activity. Furthermore, by using protein kinase C inhibitors, we demonstrate the involvement of protein kinase C signaling in the regulation of FGF-1B promoter. Detailed mechanisms among PKC signaling, RFX transcription factors and F1B promoter are worthy of further investigation.

1. Bottcher RT, Niehrs C (2005) Fibroblast growth factor signaling during early vertebrate development. Endocr Rev 26: 63-77.
2. Murphy M, Drago J, Bartlett PF (1990) Fibroblast growth factor stimulates the proliferation and differentiation of neural precursor cells in vitro. J Neurosci Res 25: 463-475.
3. Itoh N, Ornitz DM (2004) Evolution of the Fgf and Fgfr gene families. Trends Genet 20: 563-569.
4. Mason I (2007) Initiation to end point: the multiple roles of fibroblast growth factors in neural development. Nat Rev Neurosci 8: 583-596.
5. Ornitz DM (2000) FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 22: 108-112.
6. Furdui CM, Lew ED, Schlessinger J, Anderson KS (2006) Autophosphorylation of FGFR1 kinase is mediated by a sequential and precisely ordered reaction. Mol Cell 21: 711-717.
7. Hart KC, Robertson SC, Kanemitsu MY, Meyer AN, Tynan JA, et al. (2000) Transformation and Stat activation by derivatives of FGFR1, FGFR3, and FGFR4. Oncogene 19: 3309-3320.
8. Eswarakumar VP, Lax I, Schlessinger J (2005) Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 16: 139-149.
9. Turner N, Grose R (2010) Fibroblast growth factor signalling: from development to cancer. Nat Rev Cancer 10: 116-129.
10. Yamaguchi TP, Harpal K, Henkemeyer M, Rossant J (1994) fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev 8: 3032-3044.
11. Thisse B, Thisse C (2005) Functions and regulations of fibroblast growth factor signaling during embryonic development. Dev Biol 287: 390-402.
12. Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M (2008) Growth factors and cytokines in wound healing. Wound Repair Regen 16: 585-601.
13. Presta M, Dell'Era P, Mitola S, Moroni E, Ronca R, et al. (2005) Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis. Cytokine Growth Factor Rev 16: 159-178.
14. Gospodarowicz D, Ferrara N, Schweigerer L, Neufeld G (1987) Structural characterization and biological functions of fibroblast growth factor. Endocr Rev 8: 95-114.
15. Gospodarowicz D (1987) Fibroblast growth factor: structural and biological properties. Int J Rad Appl Instrum B 14: 421-434.
16. Zakrzewska M, Marcinkowska E, Wiedlocha A (2008) FGF-1: From Biology Through Engineering to Potential Medical Applications. Critical Reviews in Clinical Laboratory Sciences 45: 45p.
17. Pirvola U, Cao Y, Oellig C, Suoqiang Z, Pettersson R, et al. (1995) The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea. Proceedings of the National Academy of Sciences of the United States of America 92: 9269.
18. Kan M, Huang JS, Mansson PE, Yasumitsu H, Carr B, et al. (1989) Heparin-binding growth factor type 1 (acidic fibroblast growth factor): a potential biphasic autocrine and paracrine regulator of hepatocyte regeneration. Proc Natl Acad Sci U S A 86: 7432-7436.
19. Nabel EG, Yang ZY, Plautz G, Forough R, Zhan X, et al. (1993) Recombinant fibroblast growth factor-1 promotes intimal hyperplasia and angiogenesis in arteries in vivo. Nature 362: 844-846.
20. Jouanneau J, Moens G, Bourgeois Y, Poupon M, Thiery J (1994) A minority of carcinoma cells producing acidic fibroblast growth factor induces a community effect for tumor progression. Proceedings of the National Academy of Sciences of the United States of America 91: 286.
21. Myers R, Payson R, Chotani M, Deaven L, Chiu I (1993) Gene structure and differential expression of acidic fibroblast growth factor mRNA: identification and distribution of four different transcripts. Oncogene 8: 341.
22. Myers RL, Ray SK, Eldridge R, Chotani MA, Chiu IM (1995) Functional characterization of the brain-specific FGF-1 promoter, FGF-1.B. J Biol Chem 270: 8257-8266.
23. Nurcombe V, Ford MD, Wildschut JA, Bartlett PF (1993) Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan. Science 260: 103-106.
24. Stefnik D, Rizkalla L, Soi A, Goldblatt S, Rizkalla W (1991) Acidic and Basic Fibrolast Growth Factors Are Present in Glioblastoma Multiforme. Cancer research 51: 5760.
25. Ornitz D, Xu J, Colvin J, McEwen D, MacArthur C, et al. (1996) Receptor specificity of the fibroblast growth factor family. Journal of Biological Chemistry 271: 15292.
26. Wu DK, Maciag T, de Vellis J (1988) Regulation of neuroblast proliferation by hormones and growth factors in chemically defined medium. J Cell Physiol 136: 367-372.
27. Lipton SA, Wagner JA, Madison RD, D'Amore PA (1988) Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture. Proc Natl Acad Sci U S A 85: 2388-2392.
28. Lin WF, Chen CJ, Chang YJ, Chen SL, Chiu IM, et al. (2009) SH2B1beta enhances fibroblast growth factor 1 (FGF1)-induced neurite outgrowth through MEK-ERK1/2-STAT3-Egr1 pathway. Cell Signal 21: 1060-1072.
29. Malecki J, Wesche J, Skjerpen C, Wiedlocha A, Olsnes S (2004) Translocation of FGF-1 and FGF-2 across vesicular membranes occurs during G1-phase by a common mechanism. Molecular biology of the cell 15: 801.
30. Prudovsky I, Mandinova A, Soldi R, Bagala C, Graziani I, et al. (2003) The non-classical export routes: FGF1 and IL-1alpha point the way. J Cell Sci 116: 4871-4881.
31. Bouleau S, Parvu-Ferecatu I, Rodriguez-Enfedaque A, Rincheval V, Grimal H, et al. (2007) Fibroblast Growth Factor 1 inhibits p53-dependent apoptosis in PC12 cells. Apoptosis 12: 1377-1387.
32. Fu X, Li X, Wang T, Cheng B, Sheng Z (2004) Enhanced anti-apoptosis and gut epithelium protection function of acidic fibroblast growth factor after cancelling of its mitogenic activity. World J Gastroenterol 10: 3590-3596.
33. Guillonneau X, Regnier-Ricard F, Dupuis C, Courtois Y, Mascarelli F (1997) FGF2-stimulated release of endogenous FGF1 is associated with reduced apoptosis in retinal pigmented epithelial cells. Experimental cell research 233: 198-206.
34. Chiu IM, Touhalisky K, Baran C (2001) Multiple controlling mechanisms of FGF1 gene expression through multiple tissue-specific promoters. Prog Nucleic Acid Res Mol Biol 70: 155-174.
35. Hsu YC, Lee DC, Chen SL, Liao WC, Lin JW, et al. (2009) Brain-specific 1B promoter of FGF1 gene facilitates the isolation of neural stem/progenitor cells with self-renewal and multipotent capacities. Dev Dyn 238: 302-314.
36. Chiu IM, Touhalisky K, Liu Y, Yates A, Frostholm A (2000) Tumorigenesis in transgenic mice in which the SV40 T antigen is driven by the brain-specific FGF1 promoter. Oncogene 19: 6229-6239.
37. Ray S, Yang X, Chiu I (1997) Transcriptional activation of fibroblast growth factor 1. B promoter is mediated through an 18-base pair cis-acting element. Journal of Biological Chemistry 272: 7546.
38. Hsu YC, Liao WC, Kao CY, Chiu IM (2010) Regulation of FGF1 gene promoter through transcription factor RFX1. J Biol Chem.
39. Emery P, Durand B, Mach B, Reith W (1996) RFX proteins, a novel family of DNA binding proteins conserved in the eukaryotic kingdom. Nucleic acids research 24: 803.
40. Aftab S, Semenec L, Chu J, Chen N (2008) Identification and characterization of novel human tissue-specific RFX transcription factors. BMC Evolutionary Biology 8: 226.
41. Gajiwala K, Chen H, Cornille F, Roques B, Reith W, et al. (2000) Structure of the winged-helix protein hRFX1 reveals a new mode of DNA binding. Nature 403: 916-921.
42. Iwama A, Pan J, Zhang P, Reith W, Mach B, et al. (1999) Dimeric RFX proteins contribute to the activity and lineage specificity of the interleukin-5 receptor alpha promoter through activation and repression domains. Molecular and cellular biology 19: 3940.
43. Reith W, Ucla C, Barras E, Gaud A, Durand B, et al. (1994) RFX1, a transactivator of hepatitis B virus enhancer I, belongs to a novel family of homodimeric and heterodimeric DNA-binding proteins. Molecular and cellular biology 14: 1230.
44. Siegrist C, Durand B, Emery P, David E, Hearing P, et al. (1993) RFX1 is identical to enhancer factor C and functions as a transactivator of the hepatitis B virus enhancer. Molecular and cellular biology 13: 6375.
45. Safrany G, Perry R (1993) Transcription factor RFX1 helps control the promoter of the mouse ribosomal protein-encoding gene rpL30 by binding to its [alpha] element. Gene 132: 279-283.
46. Ma K, Zheng S, Zuo Z (2006) The transcription factor regulatory factor X1 increases the expression of neuronal glutamate transporter type 3. Journal of Biological Chemistry 281: 21250.
47. Chen L, Smith L, Johnson M, Wang K, Diasio R, et al. (2000) Activation of protein kinase C induces nuclear translocation of RFX1 and down-regulates c-myc via an intron 1 X box in undifferentiated leukemia HL-60 cells. Journal of Biological Chemistry 275: 32227.
48. Liu M, Lee B, Mathews M (1999) Involvement of RFX1 protein in the regulation of the human proliferating cell nuclear antigen promoter. Journal of Biological Chemistry 274: 15433.
49. Sengupta P, Fargo J, Smith B (2002) The RFX family interacts at the collagen (COL1A2) start site and represses transcription. Journal of Biological Chemistry 277: 24926.
50. Nakayama A, Murakami H, Maeyama N, Yamashiro N, Sakakibara A, et al. (2003) Role for RFX transcription factors in non-neuronal cell-specific inactivation of the microtubule-associated protein MAP1A promoter. Journal of Biological Chemistry 278: 233.
51. Zhang Y, Chen B, Li Y, Chen J, Lou G, et al. (2009) Transcriptional regulation of the human gene coding for proline-rich nuclear receptor coactivator (PNRC) by regulatory factor X (RFX1). Molecular Biology 43: 68-75.
52. Maijgren S, Sur I, Nilsson M, Toftgard R (2004) Involvement of RFX proteins in transcriptional activation from a Ras-responsive enhancer element. Archives of Dermatological Research 295: 482-489.
53. Wang K, Nemoto T, Yokota Y (2007) RFX1 mediates the serum-induced immediate early response of Id2 gene expression. Journal of Biological Chemistry 282: 26167.
54. Katan Y, Agami R, Shaul Y (1997) The transcriptional activation and repression domains of RFX1, a context-dependent regulator, can mutually neutralize their activities. Nucleic acids research 25: 3621.
55. Efimenko E, Bubb K, Mak HY, Holzman T, Leroux MR, et al. (2005) Analysis of xbx genes in C. elegans. Development 132: 1923-1934.
56. Senti G, Swoboda P (2008) Distinct isoforms of the RFX transcription factor DAF-19 regulate ciliogenesis and maintenance of synaptic activity. Mol Biol Cell 19: 5517-5528.
57. Feng C, Xu W, Zuo Z (2009) Knockout of the regulatory factor X1 gene leads to early embryonic lethality. Biochem Biophys Res Commun 386: 715-717.
58. El Zein L, Ait-Lounis A, Morle L, Thomas J, Chhin B, et al. (2009) RFX3 governs growth and beating efficiency of motile cilia in mouse and controls the expression of genes involved in human ciliopathies. Journal of Cell Science 122: 3180.
59. Chin K, Mao C, Skinner C, Riley J, Wright K, et al. (1994) Molecular analysis of G1B and G3A IFN [gamma] mutants reveals that defects in CIITA or RFX result in defective class II MHC and li gene induction. Immunity 1: 687-697.
60. Baas D, Meiniel A, Benadiba C, Bonnafe E, Meiniel O, et al. (2006) A deficiency in RFX3 causes hydrocephalus associated with abnormal differentiation of ependymal cells. European Journal of Neuroscience 24: 1020-1030.
61. Ait-Lounis A, Baas D, Barras E, Benadiba C, Charollais A, et al. (2007) Novel function of the ciliogenic transcription factor RFX3 in development of the endocrine pancreas. Diabetes 56: 950.
62. Purvis T, Hearn T, Spalluto C, Knorz V, Piper Hanley K, et al. (2010) Transcriptional regulation of the Alstrom syndrome gene ALMS1 by members of the RFX family and Sp1. Gene.
63. Smith S, Qu H, Taleb N, Kishimoto N, Scheel D, et al. (2010) Rfx6 directs islet formation and insulin production in mice and humans. Nature 463: 775-780.
64. Azzi A, Boscoboinik D, Hensey C (1992) The protein kinase C family. European journal of biochemistry/FEBS 208: 547.
65. Dempsey E, Newton A, Mochly-Rosen D, Fields A, Reyland M, et al. (2000) Protein kinase C isozymes and the regulation of diverse cell responses. American Journal of Physiology- Lung Cellular and Molecular Physiology 279: 429.
66. Mackay H, Twelves C (2007) Targeting the protein kinase C family: are we there yet? Nature Reviews Cancer 7: 554-562.
67. Mellor H, Parker P (1998) The extended protein kinase C superfamily. Biochemical Journal 332: 281-292.
68. Rosse C, Linch M, Kermorgant S, Cameron AJ, Boeckeler K, et al. (2010) PKC and the control of localized signal dynamics. Nat Rev Mol Cell Biol 11: 103-112.
69. Hussain S, Assender J, Bond M, Wong L, Murphy D, et al. (2002) Activation of protein kinase C£a is essential for cytokine-induced metalloproteinase-1,-3, and-9 secretion from rabbit smooth muscle cells and inhibits proliferation. Journal of Biological Chemistry 277: 27345.
70. Kamioka N, Akahane T, Kohno Y, Kuroki T, Iijima M, et al. (2010) Protein kinase C [delta] and [eta] differently regulate the expression of loricrin and Jun family proteins in human keratinocytes. Biochemical and Biophysical Research Communications.
71. Jain N, Zhang T, Kee W, Li W, Cao X (1999) Protein kinase C £_ associates with and phosphorylates Stat3 in an interleukin-6-dependent manner. Journal of Biological Chemistry 274: 24392.
72. 廖偉志 (2008) 調控因子X1 作用於纖維母細胞生長因子第一型之調控機制.
73. Holmberg C, Tran S, Eriksson J, Sistonen L (2002) Multisite phosphorylation provides sophisticated regulation of transcription factors. Trends in biochemical sciences 27: 619-627.
74. Exkenstien F (2004) Fibrolast growth factors in the nervous system. Journal of neurobiology 25: 1467-1480.
75. Lee DC, Hsu YC, Chung YF, Hsiao CY, Chen SL, et al. (2009) Isolation of neural stem/progenitor cells by using EGF/FGF1 and FGF1B promoter-driven green fluorescence from embryonic and adult mouse brains. Mol Cell Neurosci 41: 348-363.
76. Soltoff S (2007) Rottlerin: an inappropriate and ineffective inhibitor of PKC [delta]. Trends in pharmacological sciences 28: 453-458.
77. Swoboda P, Adler H, Thomas J (2000) The RFX-type transcription factor DAF-19 regulates sensory neuron cilium formation in C. elegans. Molecular Cell 5: 411-421.
78. Dubruille R, Laurencon A, Vandaele C, Shishido E, Coulon-Bublex M, et al. (2002) Drosophila regulatory factor X is necessary for ciliated sensory neuron differentiation. Development 129: 5487.
79. Durand B, Vandaele C, Spencer D, Pantalacci S, Couble P (2000) Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors. Gene 246: 285-293.
80. Zhang D, Zeldin D, Blackshear P (2007) Regulatory factor X4 variant 3: A transcription factor involved in brain development and disease. Journal of Neuroscience Research 85: 3515-3522.