The Correlation Study Of Altered Expression Of Connexin43 And Phosphorylated Protein In Human Glioma Tumors

Background and Objection:Malignant gliomas are a particularly lethal form of cancer. Glioma in adults has been traditionally classified by the World Health Organization (WHO) into 4 grades, of which grade IV is the most malignant, basing only on morphological criteria. Their lethality is largely due to diffuse invasiveness. Individual glioma cells migrate into the surrounding brain parenchyma, while other cancers, metastatic to this site, do not intermingle with host cells and grow as circumscribed destructive masses. This diffuse infiltration is also the origin of the failure of surgical treatment. By the time the tumour is diagnosed, glioma cells have migrated over large distances well beyond the main bulk of the tumour. Thus, following resection, they usually reconstitute a tumour within a few months. Invasiveness will probably compromise newer therapeutic modalities such as antiangiogenic drugs, which reduce the tumour bulk but at the same time could stimulate glioma cell migration.Connexins are highly regulated integral membrane proteins that contain four transmembrane domains, two extracellular loops containing six conserved cysteine residues, a cytoplasmic loop and cytoplasmic N- and C-termini. The C-terminal domain varies widely in length and is thought to play key regulatory roles and provide for sites of protein-protein interactions. Gap junctions are composed of proteins from the connexin gene family (21 members in humans). These collections of intercellular channels permit passage of ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP 3) between cells while macromolecules are excluded (although small RNAs may pass). Gap junction intercellular communication is critically important in many cell processes including control of cell proliferation, embryonic development, cell differentiation and the coordinated contraction of heart and smooth muscle. GJs exist in almost all cell types except mature skeletal muscles, spermatozoa, and erythrocytes. Although GJs possess some general features, they also exhibit specific characteristics depending on the subtypes, cell types and tissues. So far,21 subtypes of Cxs have been found. In the brain, neurons (Cx43, Cx32, Cx36), oligodendrocytes (Cx32, Cx47, Cx29), astrocytes (Cx43, Cx30, Cx26), and microglia (Cx43, Cx36, Cx32) express different Cxs; Genetic linkage analysis has implicated connexins in at least 14 human diseases-many of which can be recapitulated in mutant connexin mouse models. Connexins are expressed in a tissue specific manner allowing them to fulfill a variety of physiological roles.Recent studies have revealed a new emerging role of Cx43 in promoting cell migration, such as in normal brain development and in enhancing glioma invasion. The GJIC, the C-tail of Cx43, or the intercellular adhesion conferred by the extracellular loops of Cx43, have all been suggested to be critical in promoting cell motility. Incidentally, the C-tail of Cx43 has been shown to interact directly with various cytoskeleton proteins implicated in cell motility, including actin, microtubules, actin binding proteins, tight junction and adherens junction proteins. In particular, the microtubule-binding domain of Cx43 is required for epicardial cell migration. In tumor tissue, decreasing Cx43 expression is associated with increasing proliferation and higher tumor grades generally. In a larger screen of 44 tumors, decreasing Cx43 protein and mRNA expression was correlated with increasing Ki67 proliferative index in high-grade gliomas. However, a recent study with 32 human samples reported high levels of Cx43 mRNA but low levels of Cx43 protein in high-grade gliomas, suggesting a post-transcriptional regulation of Cx43 protein.In most cases examined, connexins are modified post-translationally by phosphorylation. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the connexin "lifecycle", such as the trafficking, assembly/disassembly, degradation, as well as, the gating of gap junction channels.A variety of protein kinases (PKC, PKA, PKG, MAPK and PTK, etc.) play important roles in regulating phosphorylation level of GJ protein and changing conformation of the protein, which will due to function change by depolymerization and distortion of matching proteins. It is the most important regulatory mechanism of intercellular communication in the pathological state. Different kinases can be applied to the same binding site, and the same kinases may also bind different site. The phosphorylation levels of different kinases are avarying depending on different tissues and even diseases and kinases interactions, forming a complex regulatory network. For example, accumulating studies demonstrated PKC, MAPK and PTK regulated GJ protein phosphorylation:1, PKC:PKC is a serine/threonine protein kinase that phosphorylates Cx43 on S368 and S372. Although some exceptions may exist in specific cell types, most experiments indicate that these phosphorylation events correlate with the reduction of gap junctional communication mediated by Cx43. For example, chemical tumor promoters that stimulate PKC activity (e.g. phorbol esters) can reduce Cx43 gap junction production, assembly, and half-life. In rat myocardial cells, the TPA can activate PKC, reduce the single channel conductivity, decrease the permeability of GJ, but increase the total GJ conductivity. 2, MAPK:Ser279 and Ser282 of Cx43 can be phosphorylated by MAPK, causing a rapid transient reduction of gap junction communication. In addition, MAPK can enhance the extent of phosphorylation of Cx43, promote electrical conductivity between myocardial cells; however, decrease GJ permeability in the non-myocardial cells.3, PTK:PTK increases phosphorylation of Cx43 and destructs of gap junction communication brief and fast by MAPKs indirectly.As a phosphoprotein Cx43, it has many potential phosphorylation sites. Studies have shown that 21 serine were in the C-terminal (amino acids 250-382) of Cx43 protein and at least 12 of which were phosphorylated by different protein kinase. Protein kinase-induced phosphorylation of Cx43, as the regulater of intercellular communication, affect the synthesis, transport, assembly and channel gating, etc. So the amount of phosphorylation and protein expression of Cx43 is closely related to the function of the GJ. So, it is urgent to reveal the mechanism of phosphorylation pathways.In conclusion, accumulating studies demonstrated the mechanism of Cxs phosphorylation and metastasis capacity of gliomas, the effects of Cxs phosphorylation on metastasis capacity of gliomas is still unclear. In this study, we revealed the relationship between Cx43 phosphorylation and proliferation and tumor grades of glioma. Furthermore,the inhibitor of PKC, PTK and MAPK kinase were used to incubate cells and investigate their effects on Cx43 phosphorylation in glioma. Further functional analysis revealed the effects of Cxs phosphorylation on cell viability, apoptosis and invasion of gliomas cells, providing new ideas and targets for the treatment of gliomas.Methods and materials:Part 1:Analyze Cx43 phosphorylation levels in different grade gliomasCollection intraoperative pathological diagnosis of gliomas diagnosed (WHO Ⅰ, Ⅱ, Ⅲ, Ⅳ grade) as the experimental group and the brain tissue after traumatic brain decompression collected as a control group. Remove glioma tissue/control organization for preparing tissue sections. Using immunohistochemical to test the expression levels of Cx43, Cx43 phosphorylation in the experimental group and the control group. Western blot was used to detect the expression of Cx43, Cx43 phosphorylation in the experimental and control group. Analysis the correlation among Cx43 and Cx43 phosphorylation at different levels of glioma. The data from all procedures were expressed as "mean±standard error mean (S.E.M. )". For different grades of glioma tissues, the expression levels of Cx43, p-Cx43 pro tein were analyzed with two-way ANOVA of repeated measures. Data among the th ree groups and those at different time points within one group were processed with o ne-way analysis of variance or Welch test; LSD or Dunnett’s T3 test was used for pai redcomparison;Part 2:Inhibitors of PTK, MAPK and PKC influence phosphorylation levels of Cx43 in human glioma cell line U251In heart diseases, PTK, PKC and MAPK affect the function of the GJ channel via phosphorylating GJ; In this study, we showed that PKC and MAPK, PTK regulates the GJ protein phosphorylation levels in human glioma cell line U251, clearing its signal transduction mechanism in glioma cell proliferation. To do various experimental analysis afer recoveried U251 glioma cells and normal precultured cells for a week. 1μM PKC inhibitor (Staurosporine),0.5μM MAPK inhibitor (SB 203580) and 50nM PTK inhibitors (Vatalanib 2HCl PTK787) were used to incubated cells for 12,24 and 48 hours. The expression levels of Cx43 and phosphorylation Cx43 was detected by western blot analysis. The differences of the effects of inhibitors on Cx43 and p-Cx43 total protein at differ ent processing times were compared using a factorial analysis. When analyzing the effect of time and inhibitor alone, the use of single-factor analysis of variance when t he variance correction method used welch test, and analyzed by one-way ANOVA us ing LSD test or Dunnett’s T3 test.Part 3:Phosphorylation of Cx43 effect cells proliferation, invasion and apoptosis in U251 glioma1μM PKC inhibitor (Staurosporine),0.5μM MAPK inhibitor (SB 203580) and 50nM PTK inhibitors (Vatalanib 2HCl PTK787) used to incubated cell for 12,24 and 48 hours. And then MTT assay was used to examine the cell activity, transwell assay was used to analyze cell invasion and migration ability and flow cytometry method is used to discriminate apoptosis. In conclusion, we demonstrated the correlation between Cx43 phosphorylation level with glioma cell proliferation, invasive,andcellapoptosis.The different effects of inhibitors on cell viability and tran swell cell invasion ability were compared using a factorial analysis. When analyzin g the effect of time and inhibitor alone, the use of single-factor analysis of variance when the variance correction method used welch test, and analyzed by one-way AN OVA using LSD test or Dunnett’s T3 test.Results: 1.Immunohistochemistry and western blot results showed that, Cx43 protein were observed in all normal brain tissue and in glioma tissue. Indeed, it appears that the phosphorylation state and localization of Cx43 vary with tumor grades.Decreasing Cx43 expression is associated with increasing proliferation and higher tumor grades(P<0.01). In low grade gliomas (WHO Ⅰ-Ⅱ level), Cx43 expression levels were high; high degree of evil gliomas (WHO Ⅲ-Ⅳ) Cx43 expression were low. In addition, different expression levels of p-Cx43 in different grade of gliomas and normal tissues and Ⅰ-Ⅱ glioma tissue showed obvious p-Cx43 protein and class Ⅲ and Ⅳ glioma immunohistochemistry increased significantly, indicating increasing p-Cx43 expression is associated with increasing proliferation and higher tumor grades(P<0.01).2.PKC inhibitors (Staurosporine), MAPK inhibitors (SB 203580) and the PTK inhibitors (Vatalanib 2 HCL PTK787) were applied respectively after 12,24 and 48 hours incubation, and found that three kinds of inhibitors not significantly affect the total of Cx43 protein in 12 h and 24 h (P>0.05), while Cx43 was significantly reduce with treatment of 48 h(P<0.01). After MAPK inhibitor (SB 203580) or PKC inhibitors (Staurosporine) incubated cells for 12 h and the expression of P-Cx43-368 was significantly decreased in glioma(P<0.01). MAPK inhibitor (SB 203580) or PKC inhibitors (Staurosporine) can obviously inhibit the expression of P-Cx43-368 protein in glioma at 48 h. (P<0.01) 3.MAPK inhibitors (SB 203580) incubated cells for 12 h,24 h and 48 h can significantly decrease the activity of U251 cells and cell invasion, and increase cells apoptosis(P<0.01). PKC inhibitors (Staurosporine) can also reduce the activity of U251 cells and increase cell apoptosis and inhibit cell invasion at 12 h, 24 h(P<0.01), however, apoptosis and invasion did not significantly affect the activity of cells at 48 h. PTK inhibitors (Vatalanib 2 HCL PTK787) had no significant effect on cell activity, apoptosis and invasion at 12 h,24 h and 48 h (P >0.05).Conclusion1、 Cx43 protein and p-Cx43 protein were distributed in all normal brain tissue and in glioma. It appears that the phosphorylation state and localization of Cx43 vary with tumor grades.Decreased Cx43 expression and increased p-Cx43 expression is associated with increasing proliferation and higher tumor grades.2、PKC, MAPKand other signal transduction pathways regulate GJ function and play an important role in tumor cell proliferation, invasion and apoptosis by regulating balance between Cx43 and phosphorylated Cx43. Inhibition of the PKC, MAPK pathway reduce the total protein Cx43 and phosphorylation of Cx43 and significantly decreased cell viability and invasion capacity, and increased cell apoptosis, implicating its potential in glioma therapy.