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

Abstract

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.

The zinc transporter ZIP12 regulates monocrotaline-induced proliferation and migration of pulmonary arterial smooth muscle cells via the AKT/ERK signaling pathways

“…Moreover, it has recently been shown that ZIP12 plays an important role in the hypoxia-induced phenotypic transition of PASMCs [ 32 ]. In our recently published work, we demonstrated that elevated intracellular labile zinc possibly from ZIP12 was associated with increased CREB-mediated transcriptional activity and PASMCs proliferation [ 16 ]. However, functional characterization is further required for ZIP12 via the transgenic approaches including the gene overexpression and silencing analysis.…”

“…ZIP12 knockdown was shown to inhibit hypoxia-induced influx of zinc ions and subsequent proliferation of the cells, ameliorate pulmonary arterial remodeling, and suppress elevated pulmonary arterial pressure [ 15 ]. Our previous study [ 16 ] and another recent study [ 17 ] have demonstrated that ZIP12 expression is upregulated in MCT-induced PAH. However, the role of ZIP12 in mechanisms of MCT-induced PAH rats have not been described previously.…”

The Role of Zinc in the Pathogenesis of Lung Disease

“…Emerging evidence suggests that altered zinc and zinc transporter protein levels are associated with the occurrence and progression of PH [ 34 , 35 , 36 , 37 ]. A previous study by Zhao et al identified that plasmalemma zinc transporter ZIP12, encoded by the gene Slc39a12, plays a critical role in hypoxia-induced pulmonary vascular remodeling [ 34 ].…”

“…While the results of these studies point to ZIP12 having a fundamental role in regulating pulmonary vascular homeostasis and remodeling under hypoxic conditions, the underlying mechanisms of how ZIP12 affects hypoxic responses remain unclear. Another study by Xiao et al demonstrated that increased intracellular labile zinc, possibly from ZIP12, was linked with reduced phosphatases, increased transcription factor CREB-mediated activity, and PASMC proliferation [ 35 ]. Zhu et al showed that ZIP12 contributed to phenotypic switching in hypoxia-induced PASMCs and promoted PH.…”

Alternative Splicing Isoforms of Porcine CREB Are Differentially Involved in Transcriptional Transactivation

“…CREB gene knockout mice had embryonal neuronal deficits and a reduced lifespan [ 2 , 3 ]. The dysregulation of the CREB gene results in aberrant signal transduction affecting apoptosis, proliferation and differentiation, immune surveillance, and metabolism [ 4 , 5 , 6 , 7 ].…”