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

SHP-2被认为是MAPK通路的正向调节因子。因此，我们假设SHP-2磷酸酶活性的变化可能影响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wild型、D61N型、Q510E型SHP-2s在ATDC5细胞中短暂表达。定量PCR显示，在表达这两种突变体的细胞中，转录因子sox9（软骨细胞分化的早期标志物）的mRNA水平升高。另一方面，col2a1 mRNA水平没有差异，已知sox9/sox5/sox6复合物上调col2a1 mRNA水平。在使用与荧光素酶报告基因相连接的天然col2启动子的报告基因试验中获得了类似的结果。在海藻酸盐珠中培养的转导细胞表达较低的colX mRNA水平，colX是一种增生性软骨细胞的制造者。(b) MAPK信号分析在ATDC5细胞中瞬时表达野生型、D61N或Q510E SHP-2s后，用phospho-p44/42 MAPK抗体进行Western blotting检测MAPK对50 ng/ml FGF2的活性。而D61N突变表现出增加的活性，Q510E突变表现出降低的活性。(c)软骨细胞增殖分析表达野生型、D61N或Q510E SHP-2s的atdc5细胞培养3天，每天用血细胞计计数。无论基因型如何，细胞数量均无显著差异。这些结果表明突变的SHP-2s通过非mapk途径。诱导sox9表达，延缓软骨细胞成熟，可能是Noonan综合征和LEOPARD综合征生长迟缓的原因之一。

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.