Role of CCN2 as a trans-modulator in the integrated development of bone, cartilage and hematopoietic systems

1. CCN 2在骨髓造血细胞中的产生和分布研究CCN 2产生者和靶细胞的鉴定：基于血小板含有大量CCN 2的事实，我们假设巨核细胞是CCN 2的主要产生者。最初，我们评估了巨核细胞CMK细胞系的CCN 2 mRNA表达和蛋白质产生，但未能检测到它们。接下来，我们分离了人造血干细胞，并在体外诱导其分化为巨核细胞。因此，我们可以在巨核细胞体外分化的最后阶段检测到ccn 2 mRNA。此外，通过分析骨髓组织化学，一个假定的CCN 2相关的细胞表面受体，EphA 4，检测到巨核细胞，这可能有助于从外面吸收CCN 2后形成的platelet.Evaluation的影响CCN 2对造血细胞：我们发现，间充质敲低的ccn 2表达导致的抑制破骨细胞的发展从巨噬细胞-单核细胞谱系。这一发现有力地表明破骨细胞祖细胞是CCN 2的靶细胞之一。分析CCN 2和骨髓中其他细胞因子之间的相互作用首先，我们首次阐明了CCN 2引起M-CSF的基因表达，这是软骨细胞中单核细胞谱系细胞分化所必需的。随后，通过使用噬菌体展示系统，我们筛选出与四模块CCN 2分子的每个模块特异性相互作用的十二肽。利用获得的数据，在计算机中提取常见的肽结合基序。我们合成了一种具有这样一个基序的肽，并发现它实际上抑制了CCN 2对体外软骨细胞的影响。

1. Investigation on the production and distribution of CCN2 in hematopoietic cells in bone marrowIdentification of CCN2 producers and target cells : Based on the fact that platelets harbor a vast amount of CCN2, we hypothesized megakaryocytes as the major CCN2 producer. Initially, we evaluated the CCN2 mRNA expression and protein production by a megakaryocytic CMK cell line, but failed to detect them. Next, we isolated human hematopoietic stem cells and drove them differentiate into megakaryocytes in vitro. As a result, we could detect ccn2 mRNA at the final stage of the differentiation of megakaryocytes in vitro. In addition, by analyzing bone marrow histochemically, a putative CCN2-associated cell surface receptor, EphA4, was detected on megakaryocytes, which may contribute to the uptake of CCN2 from outside upon the formation of platelets.Evaluation of the effects of CCN2 on hematopoietic cells : We showed that mesenchymal knocking-down of ccn2 expression resulted in the repression of osteoclast development from the macrophage-monocyte lineage. This finding strongly indicates that osteoclast progenitor is one of the CCN2 target cells.2. Analysis of the interaction between CCN2 and other cytokines in bone marrow.First of all, we for the first time clarified that CCN2 provoked the gene expression of M-CSF, which is critically required for the differentiation of the cells in the monocyte lineage, in chondrocytes. Subsequently, by using a bacteriophage-display system, we screened dodecapeptides that specifically interacted with each module of tetramodular CCN2 molecule. With the data obtained, common peptide binding motifs were extracted in silico. We synthesized a peptide with one such motif and found that it actually repressed the effects of CCN2 on chondrocytes in vitro.