Zinc-mediated activation of CREB pathway in proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension

Background Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH. Methods A series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. Results Using bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Conclusions CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH. Plain English summary Transcription factor CREB plays an important role in the development of pulmonary hypertension (PH). However, paradoxical roles have been reported in the pathogenesis of PH, and the regulatory mechanisms of CREB activation in pulmonary artery smooth muscle cells (PASMCs) proliferation remained unknown. In this study, we showed that CRE-containing genes were overrepresented among the differentially expressed genes in experimental PH, which resulted from the sustained activation of CREB pathway. The sustained activation of CREB pathway may be associated with the activation of multiple protein kinases that positively regulate CREB and down-regulation of numerous phosphatases involved in CREB dephosphorylation. Additionally, we found that the proliferation of PAMSCs was dependent on the CREB-mediated transcriptional activity in experimental PH. Moreover, the raised intracellular labile zinc possibly from ZIP12 may be associated with reduced protein phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Collectively, we found CREB-mediated transcriptional activity in the proliferation of PASMCs in PH, which may be associated with multiple protein kinases and/or reduced phosphatases and elevated intracellular zinc. This study may reveal a critical role of zinc-mediated activation of CREB pathway in the proliferation of PASMCs, thus providing a more comprehensive understanding of CREB pathway in the pathogenesis of PH.

Automated summary

In experimental pulmonary hypertension (PH), the CREB pathway is overactivated and contributes to PASMCs proliferation, possibly involving multiple protein kinases, reduced CREB phosphatases, and elevated intracellular zinc.