AVP(4-8)调控基因的新途径研究及大鼠CCTβ在昆虫细胞中的表达
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摘要
第一部分、AVP4-8调控基因的新途径
AVP4-8是精氨酸加压素在脑内的天然酶解产物，具有促进学习记忆的功能，它能在大鼠脑内引起一系列生理生化反应，RT－PCR结合Southern杂交结果表明，AVP4-8能提高大鼠海马神经元内CTP:磷酸胆碱胞苷转移酶（CCT）的mRNA水平，以及大鼠海马胶质细胞中c-fos的mRNA水平。其拮抗剂ZDC(C)PR能够抑制这一作用。用放线菌素D中止细胞mRNA合成后发现，AVP4-8能提高CCT mRNA和c-fos mRNA的稳定性。这提示转录后水平的调控是AVP4-8影响一些基因mRNA水平的重要手段。
此外，AVP4-8上调海马神经元内CCT mRNA水平导致了其胞内CCT酶活性的上升，从而进一步证明AVP4-8的某些功能是通过与CCT相关的途径来完成的。
Part1. The New Pathways by Which AVP4-8 Regulate Genes
Xu Kanyan ( Biochemistry )
Directed by Prof. Du Yucang
The neuropeptide AVP4-8 is a metabolite of argipressin which has been shown to have potent memory-enhancing activity. It can evoke a series of biophysical and biochemical events in rat brain. Using RT-PCR plus Southern Blot, we found that AVP4-8 could upregulate the mRNA level of CTP: phosphocholine cytidylyltransferase in rat hippocampal neurons and the mRNA level of c-fos in rat hippocampal astrocytes. It's antagonist, ZDC(C)PR could inhibit the process. Using Actinomycin-D to inhibit the cell's transcription, we found AVP4-8 could prolong the halflife of CCT mRNA and c-fos mRNA, which suggesting the role of posttranscriptional regulation in AVP4-8's effect on some genes' mRNA levels.
We also found the upregulation of CCT mRNA led to the increase of enzymatic activity, this further proved the speculation that CCT may play an
important role in some of the AVP4-8 's functions.
AVP4-8是精氨酸加压素在脑内的天然酶解产物，具有促进学习记忆的功能，它能在大鼠脑内引起一系列生理生化反应，RT－PCR结合Southern杂交结果表明，AVP4-8能提高大鼠海马神经元内CTP:磷酸胆碱胞苷转移酶（CCT）的mRNA水平，以及大鼠海马胶质细胞中c-fos的mRNA水平。其拮抗剂ZDC(C)PR能够抑制这一作用。用放线菌素D中止细胞mRNA合成后发现，AVP4-8能提高CCT mRNA和c-fos mRNA的稳定性。这提示转录后水平的调控是AVP4-8影响一些基因mRNA水平的重要手段。
此外，AVP4-8上调海马神经元内CCT mRNA水平导致了其胞内CCT酶活性的上升，从而进一步证明AVP4-8的某些功能是通过与CCT相关的途径来完成的。
Part1. The New Pathways by Which AVP4-8 Regulate Genes
Xu Kanyan ( Biochemistry )
Directed by Prof. Du Yucang
The neuropeptide AVP4-8 is a metabolite of argipressin which has been shown to have potent memory-enhancing activity. It can evoke a series of biophysical and biochemical events in rat brain. Using RT-PCR plus Southern Blot, we found that AVP4-8 could upregulate the mRNA level of CTP: phosphocholine cytidylyltransferase in rat hippocampal neurons and the mRNA level of c-fos in rat hippocampal astrocytes. It's antagonist, ZDC(C)PR could inhibit the process. Using Actinomycin-D to inhibit the cell's transcription, we found AVP4-8 could prolong the halflife of CCT mRNA and c-fos mRNA, which suggesting the role of posttranscriptional regulation in AVP4-8's effect on some genes' mRNA levels.
We also found the upregulation of CCT mRNA led to the increase of enzymatic activity, this further proved the speculation that CCT may play an
important role in some of the AVP4-8 's functions.
引文
1， Lin C, Liu R Y and Du Y C. Cysteinyl methyyl ester of AVP(4-8), a potent agonist on the maintenance of passive avoidance in rats. Peptides, 1990, 11(4): 633-9.
2， De Jong W O, et al. Differentiation of behvioral and peripheral actions of neuropeptides generated from vasopressin in the brain. In: Schrier R W, ed. Vasopressin. New York: Racen Press, 1985, 189-94.
3， Burbach J P H, Kovacs G L and de Wied D. A major metabolite of arginine vasopressin in the brain is a highly potent neuropeptide. Science 1983; 221: 1310 - 1312
4， Liu R Y, Lin C and Du Y C. Facilitation of arginine-vasopressin analogs on learning and memory in rats. Acta Pharmacologica Sinica, 1990, 11: 97 - 100
5， Du Y C, Guo N N and Chen Z F. Autoradiographic approach to the developmental study on the bindingsites of AVP4-8 in rat hippocampus, Acta Physiologica Sinica, 46(1994): 435-440.
6， Gu B X and Du Y C. Arginine-vesopressin C-terminal peptide stimilates inositol phospholipid metabolism in rat hippocampus, Acta Biochimica et Biophysica Sinica, 24(1992): 287-294.
7， Gu B X. and Du Y C. The neuropeptide ZNC(C)PR can induce c-fos and c-src transcriptions in the hippocampus of newborn rats, Acta Biochimica et Biophysica Sinica, 23(1991): 537-542.
8， Zhou A W, Li W X, Guo J and Du Y C. Facilitation of AVP4-8 on gene expression of BDNF and NGF in rat brain, Peptides, 18(1997): 1179-1187.
9， Li W X, Gu B X and Du Y C. Effects of ZNC(C)PR and its analogs on CDNF mRNA expression in the rat brain, Acta Biochimica et Biophysica Sinica, 31(1999) 249-253.
10， Chen X F, Tang T, Zhang J W, Miao H H, Wang T X and Du Y C. ZNC(C)PR affects developmental changes of p46 phosphorylation in rat hippocampus, Mol Reprod Dev, 35(1993): 251-256.
11， Qiao L Y and Du Y C. Involvement of a putative G-protein-coupled receptor and a branching pathway in argipressin(4-8) signal transduction in rat hippocampus. Atca Pharmacologica Sinica, 1998, 19: 15-20.
12， Dong M, Xiong Y, Xu K Y and Du Y C. ZNC(C)PR induces phosphorylation of CREB in rat hippocampus. Acta Biochimica et Biophysica Sinica, 2000, 32: 575-80.
13， He M, Chen X F and Du Y C, Effect of arginine-vasopressin short analogs on the growth of C6 cells, Chin J Cell Biol, 17(1995) 176-180.
14， Yan Q W and Du Y C. AVP(4-8) may stimulate a G protein-coupled receptor in rat hippocampal synaptosomal membranes. Acta Biochimica et Biophysica Sinica, 1998, 30: 505-9.
15， Dong M, Xu K Y, Zheng X G and Du Y C. Arginine vesopressin(4-8) mobilizes intracellular calcium in C6 gliom cells. Acta Biochimica et Biophysica Sinica, 2000, 32: 533-6.
16， Rong X W, Chen X F and Du Y C. Potentiation of synaptic transmission by neuropeptide AVP(4-8) in rt hippocampus. Neuroreport, 1993, 4:1135-8.
17， Zhen X G, Dong M and Du Y C. Effect of arginine-vasopressin(4-8) on PKC and PKA activities in rat brain. Chinese Journal of Biochemistry and Molecular Biology, 2000,16: 529-32.
18， Qiao L Y and Du Y C. Increase of mitogen-activated protein kinase activity in rat brain after injection of argipressin(4-8). Atca Pharmacologica Sinica, 1997, 18: 380-4.
19， Guhaniyogi J and Brewer G. Regulation of mRNA stability in mammlian cells. Gene, 2001, 265: 11-23.
20， Decker C J and Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev, 1993, 7: 1632-43.
21， Culbertson M R. RNA surveillance. Unforseen consequences for gene expression, inherited genetic disorders and cancer. Trends Genet, 1999, 15: 74-80.
22， Beelman C A and Parker R. Degradation of mRNA in eukaryotes. Cell, 1995, 81: 179-83.
23， Chen C Y and Shyu A B. AY-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci, 1995, 20: 465-70.
24， Peng S S, Chen C Y, Xu N and Shyu A B. RNA stabilization by the AU rich element binding protein, HuR, an ELAV protein. EMBO J, 1998, 17: 3461-70.
25， Loflin P, Chen C Y and Shyu A B. Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element. Genes Dev, 1999, 13: 1884-97.
26， Schiavi S C, Wellington C L, Shyu A B, Chen C Y, Greenberg M E and Belasco J G. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. J Biol Chem, 1994, 269: 3441-8.
27， Oliveira C C and McCarthy J E. The relationship between eukaryotic translation and mRNA stability. A short upstream open reading frame strongly inhibits translational initiation and greatly accelerates mRNAA degradation in the yeast Saccharomyces cerevisiae. J Biol Chem, 1995, 270: 8936-43.
28， Chen C Y, Cherzi R, Anderson J S, Gaietta G, Jurchott K, Royer H D et al. Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation. Genes Dev, 2000, 14: 1236-48.
Rouault T and Klausner R. Regulation of iron metabolism in
29， eukaryote. Curr Top Cell Regul, 1997, 35: 1-19.
30， Short S, Tian D, Short M L and Jungmann R A. Structural determinants for post-transcriptional stbilization of lactate dehydrogenase A mRNAA by protein kinase C signal pathway. J Biol Chem, 2000, 275: 12963-9.
31， Pages G, Berra E, Milanini J, Levy A P and Pouyssegur J. Stress-activated protein kinase(JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability. J Biol Chem, 2000, 275: 26484-91.
32， Baburina I and Jackowski S. Cellular Responses to excess phospholipid. J Biol Chem, 1999, 274(12): 9400-8.
33， Chung S Y, Moriyama T, Uezu K, Hirata R, Yohena N, Masuda Y, Kokubu T and Yamamoto S. Administration of phosphatidylylcholine increases brain acetylcholine concentration and improves memory in mice with dementia. J Nutr, 1995, 125(6): 1484-9.
34， Cacabelos R, Caamano J, Gomez M J, Fernandez-Novoa L, Franco-Maside A and Alvarez X A. Therapeutic effects of CDP-choline in Alzheimer's disease. Cognition, brain mpping, cerebrovascular dynmics, and immune factors. Ann NY Acad Sci, 1996, 777(2): 399-403.
35， Tessner T G, Rock C O, Kalmar G B, Cornell R B and Jackowski S. Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels. J Biol Chem, 1991, 266(25): 16261-4.
36， Garbay B and Cassagne C. Expression of the ceramide galactisyltransferase gene during myelination of the mouse nervous system, Comparison with the genes encoding myelin basic proteins, choline kinase and CTP: phosphocholine cytidylyltransferase. Brain Res Dev Brain Res, 1994, 83(1): 119-24.
Xiong Y, Liu X L, Wang Y and Du Y C. Cloning of cytidine
37， triphosphte: phosphocholine cytidylyltransferase mRNA upregulated by a neuropeptide arginine-vesopressin(4-8) in rat hippocampus. Neurosci Lett. 2000, 283(2): 129-32.
38， Qiao L Y, Chen X F, Gu B X, et al. Effect of AVP(4-8) administration on Ca2+/CaM-dependent protein kinase autophosphorylation in rat brain. Acta Physiologica Sinica, 1998, 50: 132-8.
39， Yan Q W, Zhen X G, Wang T X, Dong M and Du Y C. Indirectly neurotrophic effect of neuropeptide ZNC(C)PR on PC12 cells via peptide-stimulation of C6 cell. Acta Pharmacologica Sinica, 2000, 21: 410-4.
40， Horie M and Broxmeyer H E. Involvement of immediate-early gene expression in the synergistic effects of steel factor in combination with granulocyte-macrophage colony-stimulating factor or interleukin-3 on proliferation of a human factor-dependent cell line. J Biol Chem, 1993, 268: 968-73.
41， Radzioch D and Varesio L. c-fos mRNA expression in macrophages is downregulated by interferon-γ at posttranscriptional level. Mol Cell Biol, 1991, 11: 2718-22.
2， De Jong W O, et al. Differentiation of behvioral and peripheral actions of neuropeptides generated from vasopressin in the brain. In: Schrier R W, ed. Vasopressin. New York: Racen Press, 1985, 189-94.
3， Burbach J P H, Kovacs G L and de Wied D. A major metabolite of arginine vasopressin in the brain is a highly potent neuropeptide. Science 1983; 221: 1310 - 1312
4， Liu R Y, Lin C and Du Y C. Facilitation of arginine-vasopressin analogs on learning and memory in rats. Acta Pharmacologica Sinica, 1990, 11: 97 - 100
5， Du Y C, Guo N N and Chen Z F. Autoradiographic approach to the developmental study on the bindingsites of AVP4-8 in rat hippocampus, Acta Physiologica Sinica, 46(1994): 435-440.
6， Gu B X and Du Y C. Arginine-vesopressin C-terminal peptide stimilates inositol phospholipid metabolism in rat hippocampus, Acta Biochimica et Biophysica Sinica, 24(1992): 287-294.
7， Gu B X. and Du Y C. The neuropeptide ZNC(C)PR can induce c-fos and c-src transcriptions in the hippocampus of newborn rats, Acta Biochimica et Biophysica Sinica, 23(1991): 537-542.
8， Zhou A W, Li W X, Guo J and Du Y C. Facilitation of AVP4-8 on gene expression of BDNF and NGF in rat brain, Peptides, 18(1997): 1179-1187.
9， Li W X, Gu B X and Du Y C. Effects of ZNC(C)PR and its analogs on CDNF mRNA expression in the rat brain, Acta Biochimica et Biophysica Sinica, 31(1999) 249-253.
10， Chen X F, Tang T, Zhang J W, Miao H H, Wang T X and Du Y C. ZNC(C)PR affects developmental changes of p46 phosphorylation in rat hippocampus, Mol Reprod Dev, 35(1993): 251-256.
11， Qiao L Y and Du Y C. Involvement of a putative G-protein-coupled receptor and a branching pathway in argipressin(4-8) signal transduction in rat hippocampus. Atca Pharmacologica Sinica, 1998, 19: 15-20.
12， Dong M, Xiong Y, Xu K Y and Du Y C. ZNC(C)PR induces phosphorylation of CREB in rat hippocampus. Acta Biochimica et Biophysica Sinica, 2000, 32: 575-80.
13， He M, Chen X F and Du Y C, Effect of arginine-vasopressin short analogs on the growth of C6 cells, Chin J Cell Biol, 17(1995) 176-180.
14， Yan Q W and Du Y C. AVP(4-8) may stimulate a G protein-coupled receptor in rat hippocampal synaptosomal membranes. Acta Biochimica et Biophysica Sinica, 1998, 30: 505-9.
15， Dong M, Xu K Y, Zheng X G and Du Y C. Arginine vesopressin(4-8) mobilizes intracellular calcium in C6 gliom cells. Acta Biochimica et Biophysica Sinica, 2000, 32: 533-6.
16， Rong X W, Chen X F and Du Y C. Potentiation of synaptic transmission by neuropeptide AVP(4-8) in rt hippocampus. Neuroreport, 1993, 4:1135-8.
17， Zhen X G, Dong M and Du Y C. Effect of arginine-vasopressin(4-8) on PKC and PKA activities in rat brain. Chinese Journal of Biochemistry and Molecular Biology, 2000,16: 529-32.
18， Qiao L Y and Du Y C. Increase of mitogen-activated protein kinase activity in rat brain after injection of argipressin(4-8). Atca Pharmacologica Sinica, 1997, 18: 380-4.
19， Guhaniyogi J and Brewer G. Regulation of mRNA stability in mammlian cells. Gene, 2001, 265: 11-23.
20， Decker C J and Parker R. A turnover pathway for both stable and unstable mRNAs in yeast: evidence for a requirement for deadenylation. Genes Dev, 1993, 7: 1632-43.
21， Culbertson M R. RNA surveillance. Unforseen consequences for gene expression, inherited genetic disorders and cancer. Trends Genet, 1999, 15: 74-80.
22， Beelman C A and Parker R. Degradation of mRNA in eukaryotes. Cell, 1995, 81: 179-83.
23， Chen C Y and Shyu A B. AY-rich elements: characterization and importance in mRNA degradation. Trends Biochem Sci, 1995, 20: 465-70.
24， Peng S S, Chen C Y, Xu N and Shyu A B. RNA stabilization by the AU rich element binding protein, HuR, an ELAV protein. EMBO J, 1998, 17: 3461-70.
25， Loflin P, Chen C Y and Shyu A B. Unraveling a cytoplasmic role for hnRNP D in the in vivo mRNA destabilization directed by the AU-rich element. Genes Dev, 1999, 13: 1884-97.
26， Schiavi S C, Wellington C L, Shyu A B, Chen C Y, Greenberg M E and Belasco J G. Multiple elements in the c-fos protein-coding region facilitate mRNA deadenylation and decay by a mechanism coupled to translation. J Biol Chem, 1994, 269: 3441-8.
27， Oliveira C C and McCarthy J E. The relationship between eukaryotic translation and mRNA stability. A short upstream open reading frame strongly inhibits translational initiation and greatly accelerates mRNAA degradation in the yeast Saccharomyces cerevisiae. J Biol Chem, 1995, 270: 8936-43.
28， Chen C Y, Cherzi R, Anderson J S, Gaietta G, Jurchott K, Royer H D et al. Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation. Genes Dev, 2000, 14: 1236-48.
Rouault T and Klausner R. Regulation of iron metabolism in
29， eukaryote. Curr Top Cell Regul, 1997, 35: 1-19.
30， Short S, Tian D, Short M L and Jungmann R A. Structural determinants for post-transcriptional stbilization of lactate dehydrogenase A mRNAA by protein kinase C signal pathway. J Biol Chem, 2000, 275: 12963-9.
31， Pages G, Berra E, Milanini J, Levy A P and Pouyssegur J. Stress-activated protein kinase(JNK and p38/HOG) are essential for vascular endothelial growth factor mRNA stability. J Biol Chem, 2000, 275: 26484-91.
32， Baburina I and Jackowski S. Cellular Responses to excess phospholipid. J Biol Chem, 1999, 274(12): 9400-8.
33， Chung S Y, Moriyama T, Uezu K, Hirata R, Yohena N, Masuda Y, Kokubu T and Yamamoto S. Administration of phosphatidylylcholine increases brain acetylcholine concentration and improves memory in mice with dementia. J Nutr, 1995, 125(6): 1484-9.
34， Cacabelos R, Caamano J, Gomez M J, Fernandez-Novoa L, Franco-Maside A and Alvarez X A. Therapeutic effects of CDP-choline in Alzheimer's disease. Cognition, brain mpping, cerebrovascular dynmics, and immune factors. Ann NY Acad Sci, 1996, 777(2): 399-403.
35， Tessner T G, Rock C O, Kalmar G B, Cornell R B and Jackowski S. Colony-stimulating factor 1 regulates CTP: phosphocholine cytidylyltransferase mRNA levels. J Biol Chem, 1991, 266(25): 16261-4.
36， Garbay B and Cassagne C. Expression of the ceramide galactisyltransferase gene during myelination of the mouse nervous system, Comparison with the genes encoding myelin basic proteins, choline kinase and CTP: phosphocholine cytidylyltransferase. Brain Res Dev Brain Res, 1994, 83(1): 119-24.
Xiong Y, Liu X L, Wang Y and Du Y C. Cloning of cytidine
37， triphosphte: phosphocholine cytidylyltransferase mRNA upregulated by a neuropeptide arginine-vesopressin(4-8) in rat hippocampus. Neurosci Lett. 2000, 283(2): 129-32.
38， Qiao L Y, Chen X F, Gu B X, et al. Effect of AVP(4-8) administration on Ca2+/CaM-dependent protein kinase autophosphorylation in rat brain. Acta Physiologica Sinica, 1998, 50: 132-8.
39， Yan Q W, Zhen X G, Wang T X, Dong M and Du Y C. Indirectly neurotrophic effect of neuropeptide ZNC(C)PR on PC12 cells via peptide-stimulation of C6 cell. Acta Pharmacologica Sinica, 2000, 21: 410-4.
40， Horie M and Broxmeyer H E. Involvement of immediate-early gene expression in the synergistic effects of steel factor in combination with granulocyte-macrophage colony-stimulating factor or interleukin-3 on proliferation of a human factor-dependent cell line. J Biol Chem, 1993, 268: 968-73.
41， Radzioch D and Varesio L. c-fos mRNA expression in macrophages is downregulated by interferon-γ at posttranscriptional level. Mol Cell Biol, 1991, 11: 2718-22.