Annexin Ⅱ Receptor Induces Apoptosis Independent Of Annexin ⅡTmed2Accelerates Murine MC3T3-E1Pre-osteoblast Proliferation

Apoptosis, also called programmed cell death, is a tightly regulated form of cell death that plays a critical role in normal growth development tissue homeostasis. Apoptosis is still one of the hot spots of research in life science. Great progress has been made in apoptotic signal transduction, the biological mechanism and the gene regulation. However, apoptosis is a very complex physiological progress, so there are still many questions need to be resolved.The subject of our study is human Annexin Ⅱ receptor (AXIIR), also known as chromosome5open reading frame39(C5orf39), which is a protein with193amino acids, and its ecoding gene is located in human chromosome5p12region. Sequence alignment for human AXIIR and PCR analysis implied that AXIIR only present in human chromosome5and is peculiar to human. In2006, Annexin Ⅱ receptor (AXIIR), a type Ⅰ membrane receptor encoding gene was cloned from human marrow cDNA library and identified for the first time. The authors also demonstrate that AXIIR is expressed on the surface of bone marrow stromal cells and mediates Annexin Ⅱ signal for osteoblast formation. So far, the only known function about AXIIR is mediating Annexin Ⅱ signal. The study of AXIIR is in early stage, there is no any evidence to show its correlation with apoptosis.Annexin Ⅱ is a member of the Annexin family. It is well-known that Annexin Ⅱ interact with a number of extracellular matrix molecules involved in several membrane-related events, such as exocytotic and endocytotic pathways, cell-cell adhesion, plasminogen receptor activation, and tumor metastasis and invasion.In our study, firstly we over-expressed AXIIR in chronic myeloid leukemic cells K562to see how AXIIR affected cellular function, we discovered that AXIIR induced K562cells apoptosis. Furthermore AXIIR was found to induce apoptosis in multiple human cell types. But the apoptotic rates in these cell type were lower than its in K562cells, which indicated the phenomenon that over-expressed AXIIR induce apoptosis is universal to some extent, but is associated with certain cell type. So far, As AXIIR is known to be a receptor for Annexin II, so we analyzed whether AXIIR-induced apoptosis was an event triggered by Annexin Ⅱ. Through immunofluorescence staining, AXIIR-myc was examined to locate in cytoplasm (Fig.7A). Moreover, inhibition of ubiquitin and lysosome protein degradation pathways by MG-132did not elevate AXIIR expression, indicating that the cytoplasmic AXIIR was not on their way to degradation. This observation implied that AXIIR might not function as a cell surface receptor. Annexin Ⅱ did not affect AXIIR-induced apoptosis in a wide range of concentration. These data suggested that AXIIR did not act as an Annexin Ⅱ receptor to induce apoptosis.We next examined which components of apoptotic pathway were regulated by AXIIR. The activity of Caspase-8and-3/7were dramatically up-regulated, in response to over-expression of AXIIR. Caspase-8inhibitor was used to indicate that Caspase-8played a key role in AXIIR-induced apoptosis. Iimmunoprecipitation (IP) experiments showed AXIIR bound to and activated pro-Caspase-8independent of FADD. Moreover, knockdown of FADD did not affect the capacity of AXIIR to up-regulate Caspase-8activity. In addition, over-expressed truncated AXIIR indicated C-terminus was indispensable for AXIIR to activate Caspase-8and N-terminus also contributed to the function. Also we found that AXIIR induced apoptosis independent of death receptor signal.In our study, Caspase9was also activated, and the protein level of BCL2and BCL-XL was remarkably decreased in response to over-expression of AXIIR, while the expression of BAX remained unchanged. We over-express BCL-XL to block mitochondrial function, but MTS assay showed that AXIIR-induced apoptosis was unaffected. This suggested that mitochondrial pathway is dispensable in AXIIR-induced apoptosis.To investigate the expression level of AXIIR, we found that the mRNA level of AXIIR was considerably high in multiple human cell types. However, AXIIR protein was hardly detectable in these cells. These results implied that the expression of AXIIR is generally tightly inhibited in translational level. Further study demonstrated that inhibition of the translation of AXIIR was regulated by the5'UTR region of AXIIR. In addition, cell death receptors, anti-tumor drugs and UVA could not elevate the protein expression level of AXIIR. The ways to activate the translation of AXIIR need further investigation.In summary, we found that besides being a cell surface receptor of Annexin II, AXIIR can also be located in cytoplasm and act as a novel activator of apoptosis mainly through activation of Caspase-8. Our findings reveal a new function of AXIIR and a novel pathway for the activation of Caspase-8, and also provide new data for mechanism studies of cell apoptosis. Our labolatory discovered some novel cell proliferation-enhancing genes by random siRNA library based combinatorial screening. Tmed2gene was verified to be able to enhance murine pre-osteoblast MC3T3-E1cell proliferation through Over-expressing and knocking down Tmed2by expression plasmids. Through analysis of cell viability, cell cycle distribution and the expression level of cyclins, we found that Tmed2accelerates MC3T3-E1proliferation through up-regulating Cyclin A expression and therefore increasing the proportion of cells in S phase. In addition, the expression of Tmed2is regulated by β-Estradiol, implying that Tmed2is possibly involved in the process of MC3T3-E1proliferation promoted by P-Estradiol.The study will provide some new data to cell proliferation.