权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Mechanisms of Growth Plate Regulation by Protein Tyrosine Phosphatase SHP-2

蛋白酪氨酸磷酸酶SHP-2调控生长板的机制

基本信息

批准号：
17591086
负责人：
NAMBA Noriyuki
金额：
$ 2.24万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17591086/
关键词：
SHP-2 chondrocyte growth plate growth retardation Noonan syndrome LEOPARD syndrome

项目摘要

SHP-2 is thought to be a positive regulator of the MAPK pathway. We thus hypothesized that changes in SHP-2 phosphatase activity might affect MAPK activity and result in retardation of growth seen in Noonan or LEOPARD syndromes. To this end we performed the following experiments.(a) Analysis of chondrocyte differentiationWild type, D61N, or Q510E SHP-2s were transiently expressed in ATDC5 cells. Quantitative PCR revealed that mRNA levels of the transcription factor sox9, an early marker of chondrocyte differentiation, were increased in cell expressing either of the mutants. On the other hand, no difference in col2a1 mRNA levels, known to be upregulated by sox9/sox5/sox6 complexes, could be demonstrated. Similar results were obtained in reporter assays using a native col2al promoter linked to a luciferase reporter gene. Transduced cells cultured in alginate beads expressed lower mRNA levels of colX, a maker of hypertrophic chondrocytes.(b) Analysis of MAPK signalingFollowing transient expression of wild type, D61N, or Q510E SHP-2s in ATDC5 cells, MAPK activity in response to 50 ng/ml FGF2 was determined by Western blotting with a phospho-p44/42 MAPK antibody. While the D61N mutation demonstrated increased activity, the Q510E mutation exhibited reduced activity.(c) Analysis of chondrocyte proliferationATDC5 cells expressing wild type, D61N, or Q510E SHP-2s were cultured up to 3 days and counted every day using a hemocytometer. No significant difference in cell numbers could be demonstrated regardless of genotype.These results imply that mutant SHP-2s, via non-MAPK pathways. induce sox9 expression and delay chondrocyte maturation, possibly leading to one of the causes of growth retardation in Noonan and LEOPARD syndromes.

SHP-2 被认为是 MAPK 通路的正调节因子。因此，我们假设 SHP-2 磷酸酶活性的变化可能会影响 MAPK 活性，并导致 Noonan 或 LEOPARD 综合征中出现的生长迟缓。为此我们进行了以下实验。(a)软骨细胞分化分析野生型、D61N或Q510E SHP-2在ATDC5细胞中瞬时表达。定量 PCR 显示，在表达任一突变体的细胞中，转录因子 sox9（软骨细胞分化的早期标志物）的 mRNA 水平增加。另一方面，没有证明 col2a1 mRNA 水平存在差异，已知该水平会被 sox9/sox5/sox6 复合物上调。使用与荧光素酶报告基因连接的天然 col2al 启动子的报告测定中获得了类似的结果。在藻酸盐珠中培养的转导细胞表达较低的 colX mRNA 水平，colX 是肥大软骨细胞的标志物。(b) MAPK 信号传导分析在 ATDC5 细胞中瞬时表达野生型、D61N 或 Q510E SHP-2 后，通过使用磷酸化 p44/42 MAPK 的蛋白质印迹法测定响应 50 ng/ml FGF2 的 MAPK 活性抗体。虽然 D61N 突变表现出活性增加，但 Q510E 突变表现出活性降低。(c) 软骨细胞增殖分析将表达野生型、D61N 或 Q510E SHP-2 的 ATDC5 细胞培养长达 3 天，并每天使用血细胞计数器进行计数。无论基因型如何，细胞数量都没有显着差异。这些结果表明突变型 SHP-2 通过非 MAPK 途径发生。诱导 sox9 表达并延迟软骨细胞成熟，可能是导致 Noonan 和 LEOPARD 综合征生长迟缓的原因之一。