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. 2013 Aug;49(2):212-20.
doi: 10.1165/rcmb.2012-0051OC.

BMP4 increases canonical transient receptor potential protein expression by activating p38 MAPK and ERK1/2 signaling pathways in pulmonary arterial smooth muscle cells

Xiaoyan Li  1 , Wenju Lu, Xin Fu, Yi Zhang, Kai Yang, Nanshan Zhong, Pixin Ran, Jian Wang
Affiliations

BMP4 increases canonical transient receptor potential protein expression by activating p38 MAPK and ERK1/2 signaling pathways in pulmonary arterial smooth muscle cells

Xiaoyan Li et al. Am J Respir Cell Mol Biol. 2013 Aug.

Abstract

Abnormal bone morphogenetic protein (BMP) signaling has been implicated in the pathogenesis of pulmonary hypertension. We previously found that BMP4 elevated basal intracellular Ca(2+) ([Ca(2+)]i) concentrations in distal pulmonary arterial smooth muscle cells (PASMCs), attributable in large part to enhanced store-operated Ca(2+) entry through store-operated Ca(2+) channels (SOCCs). Moreover, BMP4 up-regulated the expression of canonical transient receptor potential (TRPC) proteins thought to compose SOCCs. The present study investigated the signaling pathways through which BMP4 regulates TRPC expression and basal [Ca(2+)]i in distal PASMCs. Real-time quantitative PCR was used for the measurement of mRNA, Western blotting was used for the measurement of protein, and fluorescent microscopic for [Ca(2+)]i was used to determine the involvement of p38 and extracellular regulated kinase (ERK)-1/2 mitogen-activated protein kinase (MAPK) signaling in BMP4-induced TRPC expression and the elevation of [Ca(2+)]i in PASMCs. We found that the treatment of BMP4 led to the activation of both p38 MAPK and ERK1/2 in rat distal PASMCs. The induction of TRPC1, TRPC4, and TRPC6 expression, and the increases of [Ca(2+)]i caused by BMP4 in distal PASMCs, were inhibited by treatment with either SB203580 (10 μM), the selective inhibitor for p38 activation, or the specific p38 small interfering RNA (siRNA). Similarly, those responses induced by BMP4 were also abolished by treatment with PD98059 (5 μM), the selective inhibitor of ERK1/2, or by the knockdown of ERK1/2 using its specific siRNA. These results indicate that BMP4 participates in the regulation of Ca(2+) signaling in PASMCs by modulating TRPC channel expression via activating p38 and ERK1/2 MAPK pathways.

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Figures

<i>Figure 1.</i>
Figure 1.
Bone morphogenetic protein (BMP)–4 increased p38 mitogen-activated protein kinase (MAPK) and extracellular signal–regulated kinase–1/2 (ERK1/2) phosphorylation in rat distal pulmonary arterial smooth muscle cells (PASMCs). Cells were treated with BMP4 (50 ng/ml) for various times, from 0.25 hour up to 60 hours. (A and C) Representative Western blots indicate changes in the amount of phospho-p38 (P-p38; A) and phospho-ERK1/2 (P-ERK1/2; B) proteins in cell lysates. Cells treated with vehicle served as control samples (Cont). Total p38 (T-p38) and ERK1/2 (T-ERK1/2) proteins were also blotted to verify equal protein loading among the samples. (B and D) The intensity of P-p38 and P-ERK1/2 bands was standardized according to that of their total proteins in each sample. Values are presented as percentages of Cont. Bars represent means ± SEMs (n = 5 in each group). *P < 0.05, versus respective Cont.
<i>Figure 2.</i>
Figure 2.
SB203580 (SB) and PD98059 (PD) inhibited BMP4-induced p38 and ERK1/2 phosphorylation in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then incubated with BMP4 (50 ng/ml) or its vehicle (Vehi) for 15 minutes. (A and C) Representative Western blots indicate the changes in the amount of phospho-p38 (P-p38; A) and phospho-ERK1/2 (P-ERK1/2; C) proteins in cell lysates. Total p38 (T-p38) and ERK1/2 (T-ERK1/2) proteins were also blotted to verify equal protein loading among the samples. (B and D) The intensity of the P-p38 and P-ERK1/2 bands was standardized according to that of their total proteins in each sample. Values are presented as percentages of DMSO + Vehi. Bars represent means ± SEMs (n = 3 in each group). *P < 0.05, compared with respective vehicle control. **P < 0.05, compared with DMSO + BMP4.
<i>Figure 3.</i>
Figure 3.
SB203580 and PD98059 inhibited BMP4-induced transient receptor potential (TRPC)–1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, and were then incubated with BMP4 (50 ng/ml) or an equal amount of BMP4 vehicle (Vehi) for 60 hours. (A–C) Real-time PCR demonstrated that the induction of TRPC1 (A), TRPC4 (B), and TRPC6 (C) mRNA in response to BMP4 was inhibited by SB203580 and PD98059. The data are presented as percentages of DMSO + Vehi. (D) Representative Western blots show the effects of SB203580 and PD98059 on the protein expression of TRPC1, TRPC4, and TRPC6 induced by BMP4. (E–G) Intensity of the TRPC1 (E), TRPC4 (F), and TRPC6 (G) bands was standardized by α-actin in each sample. Bar values indicate means ± SEMs (n = 3 in each group). *P < 0.05, compared with the respective DMSO + Vehi. **P < 0.05, compared with the respective DMSO + BMP4.
<i>Figure 4.</i>
Figure 4.
Effects of p38 small interfering RNA (siRNA) on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with p38 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4 (50 ng/ml) for 60 hours. (A) p38 and ERK1/2 mRNA relative to cyclophilin B (CpB) was determined by real-time quantitative PCR. (B) TRPC1, TRPC4, and TRPC6 mRNA relative to CpB was determined by real-time quantitative PCR. (C) Representative Western blots of TRPC1, TRPC4, TRPC6, and α-actin protein. (D) Bar graph shows protein expression levels for TRPC1, TRPC4, and TRPC6 relative to α-actin. Data are presented as percentages of the respective NT siRNA. Bar values indicate means ± SEMs (n = 3 in each group). *P < 0.05, versus respective NT siRNA–treated cells.
<i>Figure 5.</i>
Figure 5.
Effects of ERK1/2 siRNA on BMP4-induced TRPC1, TRPC4, and TRPC6 expression in rat distal PASMCs. Cells were pretreated with ERK1/2 siRNA (50 nM) or an equal amount of nontargeting control siRNA (NT siRNA) for 24 hours, and were then incubated with BMP4 (50 ng/ml) for 60 hours. (A) ERK1/2 and p38 mRNA relative to cyclophilin B (CpB) was determined by real-time quantitative PCR. (B) TRPC1, TRPC4, and TRPC6 mRNA relative to CpB was determined by real-time quantitative PCR. (C) Representative Western blots of TRPC1, TRPC4, TRPC6, and α-actin protein. (D) Bar graph shows protein expression levels for TRPC1, TRPC4, and TRPC6 relative to α-actin. Data are presented as percentages of the respective NT siRNA. Bar values indicate means ± SEMs (n = 3 for each group). *P < 0.05, versus respective NT siRNA–treated cells.
<i>Figure 6.</i>
Figure 6.
Inhibition of p38 and ERK1/2 activation and expression prevented BMP4-induced increases of basal intracellular Ca2+ ([Ca2+]i) in rat distal PASMCs. Cells were pretreated with DMSO, p38 inhibitor SB203580 (10 μM), or ERK1/2 inhibitor PD98059 (5 μM) for 30 minutes, or with p38, ERK1/2, or nontargeting control (NT) siRNA (50 nM) for 24 hours, and cells were then incubated with BMP4 (50 ng/ml) or an equal amount of BMP4 vehicle (Vehi) for 60 hours. (A) Changes in basal [Ca2+]i in cells treated with DMSO + Vehi (n = 4 from 112 cells), DMSO + BMP4 (n = 4 in 135 cells), SB203580 + BMP4 (n = 3 from 115 cells), or PD98059 + BMP4 (n = 3 from 116 cells) were measured by Fura-2–based fluorescent microcopy. Bar values indicate means ± SEMs. *P < 0.05, versus Vehi control cells. **P < 0.05, versus Vehi + BMP4–treated cells. (B) Changes in basal [Ca2+]i in cells treated with NT siRNA + Vehi (n = 6 from 177 cells), NT siRNA + BMP4 (n = 3 from 103 cells), p38 siRNA + BMP4 (n = 3 from 89 cells), and ERK1/2 siRNA + BMP4 (n = 3 from 97 cells). Bar values indicate means ± SEMs. *P < 0.01, versus NT siRNA + Vehi–treated cells. ** P < 0.05, versus NT siRNA + BMP4–treated cells.
<i>Figure 7.</i>
Figure 7.
Schematic graph illustrates the hypothesized regulation-signaling axis of BMP4 on TRPC expression in PASMCs. BMP4 stimulation induces the activation of ERK1/2 and p38 MAPK through binding with the specific BMP receptors, which subsequently results in the up-regulation of TRPC (i.e., TRPC1, TRPC4, and TRPC6) expression. Increased TRPC concentrations could cause enhanced basal [Ca2+]i through triggered store-operated calcium entry (SOCE), eventually leading to PASMC contraction, proliferation, and migration, which relates to the pathogenesis of chronic hypoxic pulmonary hypertension (CHPH). SOCCs, store-operated calcium channels.
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