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. Video abstract.

Keywords: CREB; Intracellular labile zinc; Protein phosphatases; Pulmonary artery smooth muscle cells proliferation; Pulmonary hypertension.

Fig. 1

CRE-containing genes among DEGs induced by MCT and hypoxia. a Volcano plot showing overrepresention of CRE-containing DEGs in MCT-induced PH. b The percentage of CRE-containing DEGs in hypoxia-induced PH. The DEGs induced by hypoxia were derived from Kwap iszewska et al. Respir Res, 2005. 6: p.109. CRE-containing genes were identified by searching CREB target gene database (http://natural.salk.edu/CREB)

Fig. 2

The effect of MCT on the expression and phosphorylation of CREB. a The elevated CREB phosphorylation in lung tissues. The lung tissues were isolated from control and rats treated with MCT for 1, 2, 3 and 4 weeks. n = 7. b The CREB phosphorylation and expression in PASMCs derived from control and rats treated with MCT for 1, 2, 3 and 4 weeks. n = 7. c Immunofluorescence staining showed the increased phosphorylation of CREB (red staining) in the distal pulmonary arteries. n = 5. d The increased entry of phosphorylated-CREB (green staining) in PH-PASMCs, revealed by immunofluorescence staining. n = 3. *p < 0.05 versu Ctr

Fig. 3

Identification of potential protein kinases involved in CREB phosphorylation. a The effect of inhibition of ERK1/2, P38MAPK and PI3K on CREB phosphorylation in PH-PASMCs. The PH-PASMCs were starved with serum-free DMEM/F12 for 24 h, and then the cells were pretreated with ERK1/2 inhibitor PD98059 (20 μM), P38MAPK inhibitor SB203580 (20 μM) and PI3K inhibitor LY294002 (50 μM) for 24 h, respectively. n = 4, 7 and 5. b The effects of inhibition of PKA, CAMKII and PKC on CREB phosphorylation in PH-PASMCs. The PH-PASMCs were starved with serum-free DMEM/F12 for 24 h, and then the cells were pretreated with PKA inhibitor H89 (10 μM), CaMK inhibitor KN62 (10 μM) and PKC inhibitor chelerythrine chloride (CHE, 10 µM) for 24 h, respectively. n = 5, 7 and 5. c The effect of staurosporine (Sto, 10 nM) on CREB phosphorylation in PH-PASMCs. The PH-PASMCs were starved with serum-free DMEM/F12 for 24 h, and then were pretreated with Sto for 1 h. n = 4. Ctr, control; MCT, MCT treatment for 4 weeks. *p < 0.05 vs. Ctr, #p < 0.05 vs. MCT group

Fig. 4

Profiling of protein kinases and phosphatases associated with CREB phosphorylation in MCT-induced PH. a, c Hierarchical clustering of the protein kinases and phosphatases related to CREB phosphorylation by using ClustVis. Rows in the heatmap represent averaged gene expression of replicates and columns represent time point. n = 3 MCT-treated rats at each time point and n = 5 control. b, d Heatmap of differentially expressed protein kinases and phosphatases induced by MCT. Rows in the heatmap represent gene expression levels and columns represent each sample. The DEGs between control and MCTW4 were identified by using a threshold of fold change ≥ 2 and p ≤ 0.05. Ctr, control; MCTW1, MCTW2, MCTW3 and MCTW4 represent MCT treatment for 1, 2, 3 and 4 weeks, respectively

Fig. 5

The effect of CREB-mediated transcriptional activity on PASMCs proliferation. a, d The elevated cell proliferation in PH-PASMCs. The PASMCs were isolated from control and MCT-treated rats. n = 6, 4. b, e The inhibitory effect of FK506 on cell proliferation. Cells were starved with serum-free DMEM/F12 for 24 h, and then treated with FK506 (1 nM) for 24 h. n = 6, 5. c, f The inhibitory effect of CREM on cell proliferation. Cells were starved with serum-free DMEM/F12 for 24 h and transfected with Ad-CREM and Ad-GFP for 24 h. n = 6, 5. Cell proliferation was determined by MTT assay (a-c) and measurement of PCNA expression (d–f). Ad-CREM, ICER-expressing adenovirus vector; Ad-GFP, green fluorescent protein-empty adenovirus vector. Ctr, control; MCT, MCT treatment for 4 weeks. *p < 0.05 vs. Ctr, ^#p < 0.05 vs MCT

Fig. 6

Role of zinc in regulation of CREB-mediated transcription and cell proliferation. a Representative zinc probe fluorescence images in PASMCs isolated from control and MCT-treated rats. The intracellular labile zinc levels in PASMCs were stained by fluozin-3. b Summarizing the relative intensities of fluorescence in PASMCs. n = 4. c The effect of zinc on CRE-luciferase reporter activity in PH-PASMCs. PASMCs were infected with pGL4.20-TH reporter, an adenoviral vector expressing a CRE-luciferase. n = 4. d The effect of zinc on PASMCs proliferation determined by MTT. Serum starved cells were pretreated with TPEN (10 μM) for 24 h. n = 6. TPEN, N,N,N′,N′-Tetrakis (2-pyridylmethyl) ethylenediamine; Ctr, control; MCT, MCT treatment for 4 weeks. *p < 0.05 vs. Ctr, ^#p < 0.05 vs. MCT. e Elevated expression of ZIP12 protein in rat lung homogenate. n = 8. f Elevated expression of ZIP12 protein in isolated PH-PASMCs. n = 4. Ctr, control; MCT, MCT treatment for 4 weeks. *p < 0.05 vs. Ctr

Fig. 7

A proposed model for the role and regulation of CREB in the proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension