摘要
关于肿瘤转移机制的报道很多,本课题主要针对Ca2+相关通路,以人成骨肉瘤细胞(MG63)为平台,研究钙调磷蛋白磷酸酶(PP2B)、钙调素依赖蛋白激酶Ⅱ(CaMKII)和LIM激酶(LIMK1)对丝切蛋白磷酸酶(SSH1L)的活性调控,从而观察肿瘤细胞伪叶的形成及细胞迁移能力的变化。
本研究将不同方法标记的丝切蛋白磷酸酶重组载体(YFP-SSH1L, Myc-SSH1L等)分别转染到MG63细胞中,并加入Ca2+运输载体A23187,通过共聚焦显微镜和免疫印迹来观察发现5μM的A23187可通过调节钙调磷蛋白磷酸酶(PP2B)的活性,激活SSH1L,促使P-cofilin的水平降低。当用1μM的A23187载体作用MG63细胞时, CaMKII和LIMK1活性达到了最大,同时P-cofilin水平升高,当在高钙环境时(A23187为5μM),CaMKII和LIMK1活性则降至最低。当P-cofilin水平较低时,促进了肌动蛋白(actin)的动力学作用,从而促进细胞的移动和趋化性,诱导骨肉瘤细胞细胞形成伪叶,并且其作用可被PP2B的抑制剂氯氰菊酯(cypermethrin)完全抑制。通过体外激酶测定及SSH1L磷酸酶活性测定实验显示CaMKII可使SSH1L磷酸化,但是对其的2个丝氨酸突变体无作用。
这些结果表明Ca2+信号介导PP2B对SSH1L有激活作用,而CaMKII和LIMK1则对SSH1L起着负相调节的作用,通过对这些Ca2+的相关通路的研究有助于揭示骨肉瘤细胞侵袭、转移的机制,并为临床治疗提供了一定的理论基础。
Osteosarcoma is the second most common primary malignancy of bone, espacialy for the children and adolescents. Osteosarcoma accounts for 20% of primary bone malignancies. The age of onset of this tumors mainly in 14- 25 years and above 60 years, slightly lower than the incidence of chondrosar- coma, but has higher mortality, accounting for 2/5 of bone sarcoma, pathogenesis is unknown. Osteosarcoma is a bone tumor that can occur in any bone. The major of shcolar believe that The tumour may be localised at the end of the long bone. Most often it affects the upper end of tibia or humerus, or lower end of femur. When the tumor occures or the spread under the periosteum, the periosteum from the bone surface stripping and arising reactive new bone, like daylight radiation. New bone was triangular at The junction between tumors and the backbone. Grossly, osteosarcomas are big bulky tumors that are gritty, are gray-white, and often contain areas of hemorrhage and cystic degeneration. When joint invasion occurs, the tumor grows into it along tendinoligamentous structures or through the insertion site of the joint capsule. The mortality of osteosarcoma is high, survival rate of only 3-5 years after an amputation of 5-20%. It can occures lung metastasis within a few months. The percents Occurred in the distal femur and proximal tibia osteosarcoma is about three-quarters of all the other premises such as the humerus, femur, fibula, spine, iliac bone, etc can also occur.
Calcineurin was extracted from binding protein of cyclic nucleotide phosphodiesterase, a kind of calmodulin (CaM). Calcineurin (CaN) is one member of the serine/threonine protein phosphatase family and the only known phosphatase activated by Ca2+ and CaM. The Ca2+ dependence of the phosphatase activity of CaN is controlled by two structurally similar but functionally different Ca2+ binding proteins, CaM and a regulatory subunit CaNB. Calcineurin is widely distributed in various mammalian tissues,but its concentration in brain is 10-20 times that found in other tissues . In T lymphocytes, CaN has been found intranuclearly, colocalized with one of its substrates, NFAT (Nuclear Factor of Activated T cell),which is not able to translocate into the nucleus unless it is dephosphorylated. CaN has also been found in nuclei from different species. It is also a heterodimer of a catalytic subunit (CNA) and a regulatory subunit (CNB).The catalytic subunit contains four functional domains: a catalytic domain, a CNB-binding domain (BBH), a calmodulin binding domain (CBD), and an autoinhibitory domain (AI). Some studies have shown that calcium (Ca2+) / PP2B activating downstream effector SSH-1L, and further activate its downstream Cofilin. Slingshot (SSH) is a cofilin phosphatase, its function can lead to abnormal phosphorylation of - Wire-factor (P-cofilin) level rises. Studies have shown that SSH family can play an important role through cofilin phosphorylation in the regulation of the actin cytoskeleton. Calcium / calmodulin dependent protein kinase family is a serine / threonine protease, by calmodulin (CaM) regulation. Ca2+/CaM binding to CaMKⅡsubunit, it first triggers the phosphorylation of substrate, then promoting CaMKⅡmolecules on the first 286 self-threonine phosphorylation, leading to Ca2+/ CaM dissociation rate decrease and prevent the CaM dissociation of enzymes in post- inactivation. Some studies have shown that calcium (Ca2+) / PP2B pathway can activate downstream effector SSH-1L, which can activate its downstream Cofilin.
1 Calmodulin phosphoprotein phosphatase regulation of Slingshot 1L
Calcium(Ca2+)/calmodulin phosphoprotein phosphatase (Calcineurin/ PP2B/ CnA/Cn) is currently the only one known phosphoprotein phosphatase depends on the Ca2+/ CaM-Ser / Thr. Our preliminary work has proven that calcium (Ca2+) / PP2B pathway can activate SSH-1L, and further activate its downstream Cofilin. However, Ca2+/ PP2B/SSH/ Cofilin pathway activated on cell biological behavior has not been unknown. In this study,we set the human osteosarcoma MG63 cells as object of study, focused on the above-mentioned pathway activation on cell morphology and its mechanism.
We Applicate the mothed of liposome-containing YFP-SSH1L recombinant plasmid transfected into MG63 cells (human into osteoma cells) by calcium ionophore A23187 in 10-minute incubation then carried out immunocytochemical staining and cell morphology; In Vivo and In Vitro Experimental Detection of the activity of PP2B regulation on SSH1L, and also application of the immune co-precipitation experiments testing the interaction with PP2B and SSH1L situation. And we find that Ca2+ signals can induce human osteosarcoma cells to form pseudo-leaves, But was completely blocked by PP2B inhibitor of Cypermethrin; In Vitro, SSH1L phosphatase activity measurement experiments and SSH1L PP2B binding assay confirmed that SSH1L and PP2B in the cell are a pair of mutually-binding protein, while PP2B has a role in promoting SSH1L activity.
2 CaMKII regulation activity of SSH1L
CaMKⅡ,beling to the calcium/calmodulin-dependent protein kinase family (Ca2+/CaMK) has been becoming more hot. Calcium / calmodulin -dependent protein kinase family is a serine/threonine protease,can be regulation by calmodulin (CaM). Ca2+/ CaM binds to CaMKⅡsubunit, the first role of its is triggering the phosphorylation of substrat, while promoting CaMKⅡmolecules within the 286threonine self-phosphorylation, leading to Ca2+/ CaM dissociation rate decrease and keep the activity of enzymes while CaM dissociation. This study focused on CaMKII’s regulation on Slingshot 1L (SSH1L) activity.
Transfect the recombinant plasmids containing Myc-SSH1L to cell MG63 through liposome method and incubate for ten minutes via calcium ionophore A23187 of different concentrations; then observe the changes of P-cofilin, P-SSH1L and P-CaMKII with Western Blot . In Vivo and In Vitro assays test the phosphorylation and activity regulation of CaMKⅡon SSH1L. Results: When the concentration of A23187 is 1μM, CaMKII activity gets to the highest and the levels of P-cofilin and phosphorylation on Ser 978 of SSH1L rise too. In vitro assays have proved that CaMKII can phosphorylate SSH1L(WT) and (NP)、SSH1L(C), but has no effect on its mutant SSH1L (2SA)); apart from this, CaMKII inhibits the activity of SSH1L (WT) obviously, but has no effect on SSH1L(2SA).
3 Ca2+ mediated signal pathway on the regulation of LIMK1
LIM Kinase (LIMK) is a serine / threonine protein kinase. Human LIMK family, were found two subtype, LIMK1 and LIMK2. They both have two N-terminal LIM domain, a spacer region and a C-terminal of the kinase domains. LIMK1 shuttle between the cytoplasm and the nucleus. LIMK1, in cytoplasm, can be directly phosphorylated Cofilin protein 3 serine residue and inactivate them, mainly play a role in the assembly thereby reversing the cofilin-induced actin depolymerization. Rho and other proteins can activate the phosphatase ROCK or PAK, etc., and then take the role of its by activation on downstream sub-LIMK1, at last inactivate Cofilin. Studies have shown that Rho, Rac pathway induces tumor cell invasion and metastasis by activated through the regulation of Actin rearrangements; So if blocking Rho, Rac pathway, then we can inhibition of tumor cell invasion and metastasis.
This study focuses on the LIM Kinase 1 (LIMK1)’s activity change and effect in the Ca2+ mediated signaling pathway. We incubate MG63 cells with 5μM A23187 for 1,3,5,10-minute, then test P-LIMK1 (Thr508, on behalf of LIMK1 activity) level by Western Blot Detection; incubating MG63 cell respectively 0.5, 1, 2.5, 5μM concentrations for 10 min, detection LIMK1 activity through the P-cofilin phosphorylation level in vitro by kinase assay experiments; transfecting green fluorescent protein labeled SSH1L (YFP-SSH1L) into MG63 cells by liposome, immunofluorescence staining after 1μM of A23187 incubation. The results showed that the concentration of 5μM of A23187 induced P-LIMK1 show the trend of transient increase on MG63 cells in the initial (1 min) and then gradually decline; it reached the minimum when 10min; LIMIK1 level rise when the A23187 concentration is 1μM, then fall, reached to a minimum as 5μM; In addition, the distribution of SSH1L and F-actin from the cells were evenly became irregular aggregation, and cell morphology became irregular, by the A23187 induced of 1μM.
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