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

1. 骨髓造血细胞CCN2产生和分布的研究CCN2产生细胞和靶细胞的鉴定：基于血小板中含有大量CCN2的事实，我们假设巨核细胞是主要的CCN2产生细胞。最初，我们通过巨核细胞CMK细胞系评估了CCN2 mRNA的表达和蛋白的产生，但未能检测到它们。接下来，我们分离了人造血干细胞，并在体外诱导其向巨核细胞分化。因此，我们可以在体外巨核细胞分化的最后阶段检测到ccn2 mRNA。此外，通过骨髓组织化学分析，在巨核细胞上检测到一种假定的CCN2相关的细胞表面受体EphA4，这可能有助于在血小板形成时从外部摄取CCN2。CCN2对造血细胞影响的评估：我们发现，间充质细胞中CCN2表达的下调导致巨噬细胞-单核细胞谱系的破骨细胞发育受到抑制。这一发现强烈提示破骨细胞祖细胞是CCN2的靶细胞之一。骨髓中CCN2与其他细胞因子的相互作用分析。首先，我们首次阐明了CCN2诱导单核细胞系软骨细胞中M-CSF的基因表达，而M-CSF对于单核细胞系细胞的分化至关重要。随后，通过噬菌体展示系统，我们筛选了与四分子CCN2分子每个模块特异性相互作用的十二肽。根据获得的数据，用硅提取常见的肽结合基序。我们合成了一个具有这样一个基序的肽，并发现它实际上在体外抑制CCN2对软骨细胞的作用。

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.