摘要
目的：CD82(KAI1)以及同源蛋白CD9均属于四跨膜蛋白家族的成员,二者均可抑制体外培养的肿瘤细胞的运动迁移能力。临床研究也发现,多种肿瘤的转移能力(尤其是淋巴结转移能力)与这两个蛋白的表达成负相关,因此,CD82和CD9被认为是广谱的肿瘤转移抑制因子。对其作用机制研究表明,二者主要是通过作抑制一些生长因子受体,如肝细胞生长因子受体(HGFR,c-Met).表皮细胞生长因子受体(EGFR)以及成纤维细胞生长因子受体(FGFR)等的活化,下调细胞内一些与肿瘤转移相关的信号转导途经的信号传递,从而发挥对肿瘤转移的抑制作用。已知细胞内一些与肿瘤转移相关的信号转导途经有PI3K/AKT和PLCγ／DAG／PKC信号转导途经,它们通过调控与转移有关的一些蛋白,如细胞粘连分子、细胞基质分子、金属蛋白酶,与细胞骨架蛋白等的表达,从而影响肿瘤的侵润转移能力。近年来研究又发现,质膜上的神经节苷脂GM2/GM3与CD82和CD9具有协同作用。CD82、CD9分别与GM2、GM3形成复合体而发挥对肿瘤转移抑制作用。因此,弄清楚GM2／GM3→CD82／CD9→GFR→P13K／AKT和PLCγ／DAG／PKC调控轴(regulating axes)的作用机制,对阐明肿瘤转移的调控机制具用重要的意义。
在我们的前期工作中,我们曾采用两个淋巴结不同转移能力的细胞株做为模型,比较分析了两株细胞神经节苷脂、CD82/CD9的表达以及P13 K／AKT和P LCγ／DAG／PKC信号通路的活化情况。结果发现两个细胞株神经节苷脂的表达明显不同,低转移以GM3为主,高转移的以GM2为主；两细胞株CD82表达没有显著差别,但高转移的CD9表达显著降低；高转移的细胞PLCγ／DAG／PKC信号通路活性高于低转移的细胞株。我们又分析了两个细胞株肝细胞生长因子受体的表达和磷酸化活化情况,发现两个细胞株肝细胞生长因子的磷酸化程度没有显著差别。已知肝细胞生长因子受体的激活机制,首先是配体与受体结合,受体发生二聚化,胞内区酪氨酸残基磷酸化形成活性的磷酸化酪氨酸位点,不同的磷酸化酪氨酸位点可激活下游不同的细胞内信号通路,从而,发挥不同的调控作用,产生不同的生物学效应。因此,受体胞内区磷酸化的酪氨酸残基,决定了受体活化以后激活细胞的哪些信号通路以及发挥什么样的调控作用。那么,肝细胞生长因子受体活化以后,酪氨酸残基磷酸化的方式是全或无的方式,还是选择磷酸化某些特定的酪氨酸残基。如果是选择性磷酸化的方式,那影响选择性的因素是什么,机制是什么,目前尚没有人提出和研究,虽然这两个细胞株肝细胞生长因子受体磷酸化水平相同,我们认为这两个细胞株肝细胞生长因子受体活化后磷酸化的酪氨酸残基不同,这可能是导致两个信号通路活性不同的原因,因此,我们在下面的研究工作中,检测了不同处理条件下,两细胞株肝细胞生长因子受体酪氨酸残基位点磷酸化的活化情况。
方法：体外培养不同淋巴道转移能力的小鼠腹水型肝癌细胞HCa-F(高转移株)和HCa-P(低转移株)。细胞在无血清1640培养基中饥饿10小时后,将细胞分为对照组和处理组。采用SDS-PAGE、Western Blotting技术及计算机扫描定量分析,比较分析了在不同配体(肝细胞生长因子、层粘连蛋白、纤维粘连蛋白)处理情况下,两细胞株肝细胞生长因子受体的磷酸化酪氨酸残基位点以及细胞内PLCγ1／DAG／PKC和P13 K／AKT信号通路的活化情况。
结果：(1)HGF处理后,高转移HCa-F细胞的c.Met第1313位和1365位酪氨酸残基的磷酸化程度均高于低转移的HCa-P细胞；低转移的HCa-P细胞c-Met第1349位酪氨酸残基磷酸化程度高于高转移HCa-F细胞株；同时高转移HCa-F细胞plcγ1的磷酸化程度高于低转移株的HCa-P细胞,低转移的HCa-P细胞AKT磷酸化程度高于高转移株的HCa-F细胞。说明高转移HCa-F细胞PLCγ1／DAG／PKC信号通路的活性大于低转移的HCa-P细胞；低转移HCa-P细胞P13K/AKT信号通路的活性大于高转移的HCa-F细胞。(2)FN处理后,高转移HCa-F细胞的c-Met的第1313位酪氨酸残基磷酸化程度显著大于低转移的HCa-P细胞；两细胞株c-Met的第1349位和1365位酪氨酸残基磷酸化程度无明显差别；同时高转移HCa-F细胞plcγ1的磷酸化程度高于低转移株的HCa-P细胞,说明高转移HCa-F细胞PLCγ1／DAG／PKC信号通路的活性大于低转移的HCa-P细胞；两细胞株PI3 K／AKT信号通路的活性无明显差别。(3)LN处理后,高转移HCa-F细胞的c-Met第1365位酪氨酸残基的磷酸化程度高于低转移的HCa-P细胞;两细胞株c-Met的第1349位和1313位酪氨酸残基磷酸化程度无明显差别；同时高转移HCa-F细胞plcγ1的磷酸化程度高于低转移株的HCa-P细胞,说明高转移HCa-F细胞PLCγ1／DAG／PKC信号通路的活性大于低转移的HCa-P细胞；两细胞株P13 K／AKT信号通路的活性无明显差别。
结论：(1)肝细胞生长因子受体与配体结合后,酪氨酸残基的磷酸化不是全或无的方式,而是选择性磷酸化。(2)不同的配体可使肝细胞生长因子受体不同位点的酪氨酸残基磷酸化。(3)肝细胞生长因子受体第1313位和1365酪氨酸残基磷酸化可能参与PLCγ／DAG／PKC信号通路活化；肝细胞生长因子受体第1349位酪氨酸残基磷酸化可能和P13K／AKT信号通路活化相关。(4)HCa-F细胞淋巴结转移能力可能与PLCγ／DAG／PKC信号通路活化相关。
Objective:CD82 (KAI1), as well as homologous proteins CD9 belong to the four transmembrane protein family, can inhibit both tumor cells in vitro movement and migration. Clinical studies have found that the metastatic rate of a variety of tumor (in particular, lymph node metastasis) and the expression of these two proteins have a negative correlation, therefore, CD82 and CD9 are considered broad-spectrum inhibitor of tumor metastasis. Studies of its mechanism of action have shown that the CD82 and CD9 mainly inhibit the activation of a number of growth factor receptor, such as hepatocyte growth factor receptor (HGFR, c-Met), epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) and so on, down-regulate the activity of some signaling parthway which are involved in regulation of the tumor metastasis. As we known, two signaling parthway, PI3K/AKT and PLCγ/DAG/PKC signaling parthway in cells play a crucial role in tumor metastasis, which regulate the expression of a number of tumor metastasis-related metastasis protein factors, such as cell adhesion molecules, extracellular matrix molecules, metalloproteinases, and cytoskeletal proteins, thus affecting tumor invasion and metastasis. Recent studies have also found that gangliosides GM2/GM3 on the plasma membrane have synergistic effect with CD82 and CD9. GM2 and GM3 could form dimmer with CD82 and CD9, respectively and enhance the inhibiting action of tumor metastasis. Therefore, it is important to illuminate molecular mechanism of GM2/GM3→CD82/CD9→GFR→PI3K/AKT and PLCγ/DAG/PKC regulating axes.
In our previous work, we have compared the expression of gangliosides and CD82/CD9 between two cell lines with different metastatic potential in lymph node and found that the expression of gangliosides and CD82/CD9 in two cell lines are significantly different, Hca-F cell line with high metastatic potentia express mainly ganglioside GM2, but Hca-P cell line with poor metastatic potential express mainly ganglioside GM3; Hca-P cells express CD9 higher than that of Hca-P cells; There is no significant difference in expression of CD82 between two cells. We compared further the activity of the PI3K/AKT and PLCγ/DAG/PKC signaling pathway in two cells. The results showed that the content of phosphorylated PLCyl is higher in Hca-F cells that in Hca-P cells. It is surgested that the activity of PLCγ/DAG/PKC signaling pathways in Hca-F cells is higher than that of low metastatic Hca-P cells. We also analyzed comparatively the expression and phosphorylation of hepatocyte growth factor receptor between two cell lines and found that there was no significant difference in the phosphorylation of c-Met between two cell lines. It is well known that, after binding with ligand, the c-Met receptors are dimerized and phosphorylated at some tyrosine residues. The phosphorylated tyrosine residues can be recognized and bind by some signal molecules with SH2 doman in cells. Different phosphorylated tyrosine residues can be recognized by different signal molecules and activate different downstream signaling pathway, thus, play a different regulatory role, have different biological effects. Therefore, it is the phosphorylate tyrosine residues that could determine which signaling pathway can be activated and what kind of regulating action can be generated. But, it is not known that the tyrosine residues of receptor are phosphorylated by all or none or select phosphorylation manner. If the tyrosine residues of receptor are phosphorylated by the selective phosphorylation manner, what are the factors that affect the selectivity and what is the mechanism are not known. Although the phosphorylation of c-Met between two cell lines is at the same level, we suppose that the tyrosine residues which can be phosphorylated should be different, which lead to the higher avtivity of PLCγ/DAG/PKC signaling pathway in Hca-F cells than in Hca-P cells. In this work, to Validate the presumption, we analyzed comparatively the phosphorylated tyrosine residues of c-Met and activity of PLCγ/DAG/PKC and PI3K/AKT signaling pathway between the two cell lines under different treatment conditions.
Method:The murine ascitic hepatic cancer cell lines HCa-F cells(high metastatic potential) and HCa-P cells(low metastatic potential) were cultured in 1640 medium containing 10%, after 10 hours of hunger in serum-free 1640 medium, cells were divided into control group and treatment group. After treatment of HGF, laminin, fibronectin, the phosphorylated tyrosine residues of c-Met and activity of PLCγ/DAG/ PKC and PI3K/AKT signaling pathway between the two cell lines were analyzed by SDS-PAGE, Western Blotting technology and computer quantitative.
Result:(1) After treatment of HGF, the level of phosphorylation of 1365 tyrosine residue and 1313 tyrosine residue is higher in high metastatic Hca-F cells than in low metastatic Hca-P cells; The level of phosphorylation of 1349 tyrosine residue is higher in low metastatic Hca-P cells than in high metastatic Hca-F cells; The content of p-plcyl (Tyr783) in high metastatic Hca-F cells is higher than that in low metastatic Hca-P cells; The content of p-Akt (Thr308) and p-Akt (ser473) in low metastatic Hca-P cells is higher than that in high metastatic Hca-F cells.(2) After treatment of FN, the level of phosphorylation of 1313 tyrosine residue is higher in high metastatic Hca-F cells than in low metastatic Hca-P cells; The level of phosphorylation of 1365 tyrosine residue and 1349 tyrosine residue has no significant difference between two cell lines; The content of p-plcyl (Tyr783) in high metastatic Hca-F cells is higher than that in low metastatic Hca-P cells; The content of p-Akt (Thr308) and p-Akt (ser473) between two cell lines has no significant difference. (3) After treatment of LN, the level of phosphorylation of 1365 tyrosine residue is higher in high metastatic Hca-F cells than in low metastatic Hca-P cells; The level of phosphorylation of 1313 tyrosine residue and 1349 tyrosine residue has no significant difference between two cell lines; The content of p-plcyl (Tyr783) in high metastatic Hca-F cells is higher than that in low metastatic Hca-P cells; The content of p-Akt (Thr308) and p-Akt (ser473) between two cell lines has no significant difference.
Conclusion:(1) After cMet is activated, the tyrosine residues in cMet is phosphorylated by the manner of selective phosphorylation, but not by the all or none manner. (2) Different ligands could cause phosphorylation at different tyrosine residues of cMet. (3) The phosphorylation of 1313 and 1365 tyrosine residues of cMet may be involved in avtivation of PLCγDAG/PKC signaling pathway; Thr phosphorylation of 1349 tyrosine residues of cMet may be closely related to PI3K/AKT signaling pathway activation. (4) The metastatic potential of HCa-F cells in lymph node may be associated with PLCγ/DAG/PKC signal pathway activation.
引文
1. Jiang WG, Martin TA, Parr C, et al. Hepatocyte growth factor, its receptor, and their potential value in cancer therapies [J]. Hematology,2005,53:35-61.
2. Edakuni G, Sasatomi E, Satoh T, et al. Expression of the hepatocyte growth factor/c-Met path-way is increased at the cancer front in breast carcinomafJ]. Pathol Int,2001,51(3):172-178.
3. Yama guchi R, Yano H, Lemura A, et al. Expression of vascular endothelial growthfactor in hu-man hepatocell ularcinoma[J]. Hepatology,1998,28(1):68-77.
4. Matsumoto K, Nakamura T, Kramer RH, et al. Hepatocyte growth factor/scatter factor in-duces tyrosine phosphorylation of focal adhesion kinase (p125FAK) and promotes migration and invasion by oral squamous cell carcinoma cells. [J]. BiolChem,1994,269(50):31807-31813.
5. DongG, Chen Z, Li ZY, etal. Hepatocyte growth factor/scatter factor-induced activation of MEK and PI3K signal pathways contributes to expression of proangiogenic cytokines interleuk-in-8 and vascular endothelial growth factor in head and neck squaruous cell catcinoma [J].Can-cer Res,2001,61:5911-5918.
6. 汪进国,耿小平等。HGF/cMet信号转导对肝细胞肝癌的侵袭和转移的作用[J]。临床肝胆病杂志,2002,18(3)：731-732.
7. Nakanishi K, Fujimoto I,Ueki T, et al.Hepatocyte growth factor promotes migra-tion of human hepatocellular carcinoma via phosphatidylinositaI3-kinase[J].Clin Exp Metastasis,1999,17 (6):507-514.
8. Lee HS, Huang AM, Huang GT.et al.Hepaticyte growth factor stimulate the growth and active-tes mitogen-activated protein kinase in human heptomacells[J].JBiomed Sci,1998,5(3):180-184.
9. Hili K, Weiti S, Yu J, et al. Specific requirement for the p85 p1 10 alpha phosphatidylinositol 3-kinase during epidermal growth factor-stimulated actin nucleation in breast cancer cells [J].J-ol Chem,2000,275(6):3741-3744.
10.姜虹,吴焕明等,HGF/c-Met信号转导与肿瘤侵袭和转移的关系[J]。国外医学生理、病理学与临床分册,2003,3(23)532,632,732.
11. Adams JM, Cory S, at el.The bcl-2 family:arbiters of cell survival[J]. Science, 1998,281:1322-1326.
12. Borner C, Guadagno SN, Sh iao WWL, et al. Expression of four protein kinase C isoforms in rat fibroblasts[J]. J B io Chem,1992,267:129.
13. Dumont JA, Jones WD, Bilonti AJ, et al. Inhibition of experimental metastasis and cell adhesion of B16F3 melanoma cells by inhibitors of PKC[J].Cancer Research1992,52:1195-1200.
14.尹玉新,司履生,刘绍康等.胃肠道肿瘤中纤维粘连蛋白的分布及其与肿瘤生物学特性的关系,中华病理学杂志,1989;18(2)：125～127.
15. Sorokin AV,Mikhailov AM,Kachko AV et al.Human recombinant laminin-binding protein:isolation purification and crystallization Binchemistry(Mosc),2000 May; 65(5):546-53
16. Miranti CK, Brugge JS. Sensing the environment:a historical perspective on integrin signal transduction. Nat Cell Biol,2002,4:E83-E89.
17. Comoglio PM,Boccaccio C,Trusolino L.Interactions between growth factor receptors and adhsion molecules:breaking the rules[J].Curr Opin Cell Biol, 2003,15(5):565-571.
18. Leitinger B,Hogg N. The involvement of lipid rafts in the regulation of integrin function[J].J Cell Sci,2002,115(Pt 5):963-972.
19. Schneller M,Vuori K,Ruoslahti E.Alphavbeta3 integrin associates with activated insulin and PDGFbeta receptors and Potentiates the biological activity of PDGF[J].EMBO J,1997,16(18):5600-5607.
20. Giancotti FG,Tarone G. Positional control of cell fate therough joint integrin/ receptor protein kinase signaling[J].Annv Rev Cell Dev Biol,2003,19:173-206.
21. Mariotti A,Kedeshian PA,Dans M,et al.EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes:role in epithet-lial cell migration and carcinoma invasion[J].J Cell Biol,2001,155(3):447-458.
22. Trusolino L.Bertotti A,Comoglio PM. A signaling adapter function for alpha6beta4 integrin in the control of HGF-dependent in-vasive growth[J]. Cell,2001,107(5):643-654.
23. Liu WM, Zhang XA. KAI1/CD82, a tumor metastasis suppressor. Cancer Lett. 2006; 240(2):183-94.
24. Miyake M, Koyama M, Seno M, et al. Identification of the motility-related protein(MRP-1),recognized by monoclonal antibody M31-15, which inhibits cell motility[J].J Exp Med,1991; 174(12):1347-1354
25. Sridhar SC, Miranti CK. Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene.2006; 25(16):2367-78.
26. Ono M, Handa K, Withers DA, Hakomori S. Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation. Cancer Res.1999; 59(10):2335-9.
27. Takahashi M, Sugiura T, Abe M, Ishii K, Shirasuna K. Regulation of c-Met signaling by the tetraspanin KAI-1/CD82 affects cancer cell migration. Int J Cancer.2007; 121 (9):1919-29
28. Todeschini AR, Dos Santos JN, et al. Ganglioside GM2-tetraspanin CD82 complex inhibits met and its cross-talk with integrins, providing a basis for control of cell motility through glycosy-napse[J]. J Biol Chem.2007; 282 (11):8123-33.
29. Ono M, Handa K, et al. GM3 ganglioside inhibits CD9-facilitated haptotatic cell motility:coexpression of GM3 and CD9 is essential in the downregulation of tumor cell motility and malignancy. Biochemistry.2001; 40(21):6414-6421.
30. Iwabuchi K, Yamamura S, Prinetti A, Handa K, Hakomori S. GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells. J Biol Chem.1998 Apr 10;273(15):9130-8.
31. Hakomori S. Carbohydrate-to-carbohydrate interaction, through glycosynapse, as a basis of cell recognition and membrane organization. Glycoconj J.2004; 21(3-4):125-37.
32. Regina Todeschini A, Hakomori SI. Functional role of glycosphingolipids and gangliosides in control of cell adhesion, motility, and growth, through glycosynaptic microdomains. Biochim Biophys Acta.2008 Mar;1780(3):421-33.
33. Kojima N, Hakomori S. Cell adhesion, spreading, and motility of GM3-expressing cells based on glycolipid-glycolipid interaction. J Biol Chem.1991 Sep 15;266(26):17552-8.
34. Todeschini AR, Dos Santos JN, et al. Ganglioside GM2-tetraspanin CD82 complex inhibits met and its cross-talk with integrins, providing a basis for control of cell motility through glycosy-napse[J]. J Biol Chem.2007 Mar 16;282(11):8123-33.
35. Kawakami Y, Kawakami K, et al. Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of Iaminin-5-dependent cell motility[J]. J Biol Chem.2002 Sep 13;277(37):34349-58.
36. Toledo MS, Suzuki E, Handa K, Hakomori S. Effect of ganglioside and tetraspanins in microdomains on interaction of integrins with fibroblast growth factor receptor. J Biol Chem.2005 Apr 22;280(16):16227-34.
1. Edakuni G, Sasatomi E, Satoh T, et al. Expression of the hepatocyte growth factor/c-Met path-way is increased at the cancer front in breast carcinoma. Pathol Int,2001; 51(3):172-178.
2. Yama guchi R, Yano H, Lemura A, et al. Expression of vascular endothelial growth factor in hu-man hepatocell ularcinoma[J]. Hepatology,1998; 28(1):68-77.
3.汪进国,耿小平等。HGF/cMet信号转导对肝细胞肝癌的侵袭和转移的作用。临床肝胆病杂志,2002,18(3)：731-732。
4.李学农,丁彦青。HGF/SF-met信号通路与肿瘤转移。 医学综述,2001,7(11)：346
5.赖人旭,袁世珍。HGF/c2Met系统在肿瘤发生和发展中的作用。国外医学内科学分册,2002；29(1)：815
6. Liu WM, Zhang XA. KAI1/CD82, a tumor metastasis suppressor. Cancer Lett. 2006; 240(2):183-94.
7. Miyake M, Koyama M, Seno M, et al. Identification of the motility-related proteinC MRP-1),recognized by monoclonal antibody M31-15, which inhibits cell motility[J].J Exp Med,1991; 174(12):1347-1354
8. Sridhar SC, Miranti CK. Tetraspanin KAI1/CD82 suppresses invasion by inhibiting integrin-dependent crosstalk with c-Met receptor and Src kinases. Oncogene.2006; 25(16):2367-78.
9. Ono M, Handa K, Withers DA, Hakomori S. Motility inhibition and apoptosis are induced by metastasis-suppressing gene product CD82 and its analogue CD9, with concurrent glycosylation. Cancer Res.1999; 59(10):2335-9.
10. Takahashi M, Sugiura T, Abe M, Ishii K, Shirasuna K. Regulation of c-Met signaling by the tetraspanin KAI-1/CD82 affects cancer cell migration. Int J Cancer.2007; 121 (9):1919-29
11. Todeschini AR, Dos Santos JN, et al. Ganglioside GM2-tetraspanin CD82 complex inhibits met and its cross-talk with integrins, providing a basis for control of cell motility through glycosy-napse[J]. J Biol Chem.2007; 282(11):8123-33.
12. Ono M, Handa K, et al. GM3 ganglioside inhibits CD9-facilitated haptotatic cell motility. coexpression of GM3 and CD9 is essential in the downregulation of tumor cell motility and malignancy. Biochemistry.2001; 40(21):6414-6421.
13.马克里朱正美、凌茂英崔肇春。具不同淋巴道转移潜能小鼠肝癌瘤株细胞膜鞘糖脂比较研究。生物化学杂志,1996；12(3)360-364。
1 4.刘希风,龙翔,杨凌,郑怀祖,于青南。小鼠肝癌不同转移力克隆的分离及其特性的研究.中华医学杂志,1990,70(6)315-318。
15.凌茂英,王明辉,郭伶伶,王波。HCa/16A3— F及HCa/A2 — P两株小鼠淋巴道转移肝癌细胞系的特性研究。中华医学杂志,1995；75(3)170-171。
16. Dennis-Sykes CA, Miller WJ and McAleer WJ:A quantitative Western Blot method for protein measurement. J Biol Stand 1985,13:309-314.
17. Todeschini AR, Dos Santos JN, Handa K, Hakomori SI. Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway. Proc Natl Acad Sci USA.2008,105(6):1925-30.
18. Todeschini AR, Dos Santos JN, Handa K, Hakomori SI. Ganglioside GM2-tetraspanin CD82 complex inhibits met and its cross-talk with integrins, providing a basis for control of cell motility through glycosynapse. J Biol Chem. 2007; 282(11):8123-33.
19. Kawakami Y, Kawakami K, Steelant WF, Ono M, Baek RC, Handa K, Withers DA, Hakomori S. Tetraspanin CD9 is a "proteolipid," and its interaction with alpha 3 integrin in microdomain is promoted by GM3 ganglioside, leading to inhibition of laminin-5-dependent cell motility. J Biol Chem.2002; 277(37):34349-58.