Research On The Roles Of MEPE/OF45 In DNA Damage Response

Since MEPE/OF45 (Matrix Extracellular Phospho-glycoprotein/ Osteoblast/ Osteocyte Factor 45) was first cloned in 2000, however, its role as one member of SIBLING (Small Integrin-Binding Ligand N-linked Glycoprotein) family had been limited to regulation of bone metabolism and phosphate homeostasis. Our previous research has shown a new function of MEPE/OF45. That is, MEPE/OF45 is involved in cellular DNA damage response and affects cellular resistance to DNA damage inducers including ionising radiation (IR) and camptothecin (CPT) through its interaction with CHK1 (Checkpoint kinase 1) which reducing CHK1 interaction with E3 ligase and protecting CHK1 from the ubiquitin-mediated degradation. Wang's group found hMEPE/OF45 (human MEPE/OF45) was generally expressed in tumour cell lines derived from almost all human tissues and expression level of hMEPE/OF45 correlated to the resistance of tumour cells to DNA damage inducers. hMEPE/OF45 increased the G2 checkpoint response and increased resistance of tumour cells to DNA damage inducers treatment. They proposed hMEPE/OF45 as a new target for sensitizing tumour cells to radiotherapy or chemotherapy.However, is MEPE/OF45 regulating cellular checkpoint response through interacting with CHK1 the only mechanism by which MEPE/OF45 is involved in DNA damage response? What roles of MEPE/OF45 as a extracellular matrix protein are linked to its function affecting cellular DNA damage response?Many questions remain unsettled.To further research signaling molecules affected directly or indirectly by MEPE/OF45 and uncover the potential molecular mechanisms through which MEPE/OF45 increasing cellular resistance to DNA damage inducers, gene microarray experiments were performed to analyze the differentially expressed genes between cells MEPE/OF45 highly expressed and ones MEPE/OF45 poorly expressed and between cells treated by DNA damage inducers and ones untreated by DNA damage inducers. Thousands of differentially expressed genes were found. To provide independent confirmation of microarray data, semi-quantitative PCR was performed on a selection of genes which were expressed significantly differentially, related to DNA damage response and researched generally. 24 genes among 29 genes show consistency between RT-PCR and microarray. To avoid specificity of cell lines, semi-quantitative PCR was performed in another two couples of cells (A1-5 highly expressing MEPE/OF45 and B4 poorly expressing MEPE/OF45, A1-5 transfected with pREP10 vector and A1-5 stably expressing antisense MEPE/OF45). 16 genes out of 18 showed consistent results.To screen genes involved in DNA damage response, semi-quantitative PCR was performed to detect kinetic mRNA expression of candidate genes at different time points post-irradiation in different cells sensitive or resistant to IR. The mRNA expressions of Serpinb2 and Tgfβ2 were affected by IR exposure. The kinetic expressions of protein products were detected and SERPINB2 were identified to be induced by IR. Cell line stably expressing SERPINB2 was constructed and MTT assay showed that SERPINB2 increased cellular resistance to DNA damage inducers.Microarray data analysis hinted that cell lines highly or poorly expressing MEPE/OF45 were induced apoptosis after DNA damage inducer treatment and the most possible function of MEPE/OF45 is inhibiting apoptosis. To identify whether MEPE/OF45 was involved in apoptosis response, we detected 3 couples of cells with different level of MEPE/OF45. We found that the level of MEPE/OF45 is correlated to cellular resistance to apoptosis. Over-expressing MEPE/OF45 increased the apoptosis resistance of cells while knocking down the expression of MEPE/OF45 made cells sensitive to apoptosis. Accordingly, we concluded that MEPE/OF45 made cells more resistant to apoptosis, which is coherent with the microarray assay.Further study detected if Caspase 8 in extrinsic apoptosis pathway and/or Caspase 9 in intrinsic apoptosis pathway were/was activated after DNA damage inducer treatment. We found that only Caspase 9 was cleaved. Thus it is intrinsic apoptosis pathway by which MEPE/OF45 affected apoptosis induced by DNA damage inducer.But what are the mechanisms underlying the anti-apoptosis roles of MEPE/OF45?We previously showed that MEPE/OF45 stabilized CHK1 by its interaction with CHK1. It had been also reported CHK1 phosphorylated RB at S612 after DNA damage to promote RB binding to transcriptional factor E2F-1 which inhibited apoptosis. Then we detected the apoptotic sensitivity of couples of cells and found that cell line stably expressing MEPE/OF45 and cell line stably expressing CHK1 showed resistance to apoptosis while cell line stably transfected with pREP10 vector and cell line stably expressing mutant MEPE/OF45 (the interaction between mutant MEPE/OF45 and CHK1 is significant impaired compared to the interaction between wild MEPE/OF45 and CHK1) were sensitive to apoptosis. Conclusively, the anti-apoptosis role of MEPE/OF45 was related to its interaction with CHK1.We also noted that SERPINB2 had been reported to protect RB from degradation by binding to RB and influence apoptosis. We found cells highly expressing MEPE/OF45 would highly express SERPINB2 simultaneously at transcriptional level and translation-level. Over-expressing MEPE/OF45 gave rise to upregulation of SERPINB2 and RB. Furthermore, highly expressing SERPINB2 protected cells from apoptosis. Therefore MEPE/OF45 promoting the expression of SERPINB2 which stabilizes RB may be another mechanism responsible for the anti-apoptosis role of MEPE/OF45.Taken together, this study identified Serpinb2 as a gene related to DNA damage response, demonstrated that MEPE/OF45 increased cellular resistance to apoptosis induced by CPT through intrinsic apoptosis pathway and revealed that MEPE/OF45 inhibited apoptosis depending on its interaction with CHK1 and being related to it upregulating the level of SERPINB2 and RB.Our study together with previous research suggested that MEPE/OF45 could protect cells from being killed by DNA damage inducers at different level including regulating cell cycles and inhibiting apoptosis. Our data will not only increase our knowledge on DNA damage response but also give a clue to future research concerned on targets of radiotherapy.