The Critical Role and Regulation of CREB Pathway in Pulmonary Hypertension

Background: Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, the regulatory mechanisms of CREB activation in PH remain unknown. Methods: A series of techniques, including bioinformatics methods, western blot and luciferase reporter assay were used to perform a comprehensive analysis of expression and regulation of CREB in experimental PH. Findings: 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 pulmonary artery smooth muscle cells (PASMCs) 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 MCT-induced PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation assay showed increased proliferation of PASMCs derived from rats with 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. Interpretation: CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which may be associated with multiple protein kinases, reduced CREB phosphatases and raised intracellular labile zinc. Funding: This work was supported by a grant from the National Natural Science Foundation of China (Grant No. 81873537). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: The procedures were approved by the Laboratory Animal Welfare and Ethics Committee of Fujian Medical University (Approval No. 2017–070, Fuzhou, China)