Modulatory role of phospholipase D in the activation of signal transducer and activator of transcription (STAT)-3 by thyroid oncogenic kinase RET/PTC

Abstract

Background: RET/PTC ( rearranged in transformation/papillary thyroid carcinomas) gene rearrangements are the most frequent genetic alterations identified in papillary thyroid carcinoma. Although it has been established that RET/PTC kinase plays a crucial role in intracellular signaling pathways that regulate cellular transformation, growth, and proliferation in thyroid epithelial cells, the upstream signaling that leads to the activation of RET/PTC is largely unknown. Based on the observation of high levels of PLD expression in human papillary thyroid cancer tissues, we investigated whether PLD plays a role in the regulating the RET/ PTC-induced STAT3 activation. Methods: Cancer tissue samples were obtained from papillary thyroid cancer patients ( n = 6). The expression level of PLD was examined using immunohistochemistry and western blotting. Direct interaction between RET/ PTC and PLD was analyzed by co-immunoprecipitation assay. PLD activity was assessed by measuring the formation of [H-3] phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of n-butanol. The transcriptional activity of STAT3 was assessed by m67 luciferase reporter assay. Results: In human papillary thyroid cancer, the expression levels of PLD2 protein were higher than those in the corresponding paired normal tissues. PLD and RET/ PTC could be coimmunoprecipitated from cells where each protein was over-expressed. In addition, the activation of PLD by pervanadate triggered phosphorylation of tyrosine 705 residue on STAT-3, and its phosphorylation was dramatically higher in TPC-1 cells ( from papillary carcinoma) that have an endogenous RET/PTC1 than in ARO cells ( from anaplastic carcinoma) without alteration of total STAT-3 expression. Moreover, the RET/PTC- mediated transcriptional activation of STAT-3 was synergistically increased by over-expression of PLD, whereas the PLD activity as a lipid hydrolyzing enzyme was not affected by RET/PTC. Conclusion: These findings led us to suggest that the PLD synergistically functions to activate the STAT3 signaling by interacting directly with the thyroid oncogenic kinase RET/PTC.