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

点击翻译按钮获取中文摘要

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.