Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Dec;299(6):C1370-8.
doi: 10.1152/ajpcell.00040.2010. Epub 2010 Sep 15.

Bone morphogenetic protein 4 enhances canonical transient receptor potential expression, store-operated Ca2+ entry, and basal [Ca2+]i in rat distal pulmonary arterial smooth muscle cells

Wenju Lu  1 , Pixin Ran, Dandan Zhang, Ning Lai, Nanshan Zhong, Jian Wang
Affiliations

Bone morphogenetic protein 4 enhances canonical transient receptor potential expression, store-operated Ca2+ entry, and basal [Ca2+]i in rat distal pulmonary arterial smooth muscle cells

Wenju Lu et al. Am J Physiol Cell Physiol. 2010 Dec.

Abstract

Recent advances have identified an important role of bone morphogenetic protein 4 (BMP4) in pulmonary vascular remodeling, yet the underlying mechanisms remain largely unexplored. We have previously found that Ca(2+) influx through store-operated calcium channels (SOCC), which are mainly thought to be composed of canonical transient receptor potential (TRPC) proteins, likely contribute to the pathogenic development of chronic hypoxic pulmonary hypertension. In this study, we investigated the effect of BMP4 on expression of TRPC and store-operated Ca(2+) entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs). Real-time quantitative PCR and Western blotting revealed that treatment with BMP4 (50 ng/ml, 60 h) increased TRPC1, TRPC4, and TRPC6 mRNA and protein expression in growth-arrested rat distal PASMCs. Moreover, in comparison to vehicle control, cells treated with BMP4 also exhibited enhanced SOCE, and elevated basal intracellular calcium concentration ([Ca(2+)](i)) as determined by fluorescent microscopy using the Ca(2+) indicator Fura-2 AM. Perfusing cells with Ca(2+)-free Krebs-Ringer bicarbonate solution (KRBS) or KRBS containing SOCC antagonists SKF-96365 or NiCl(2) attenuated the increases in basal [Ca(2+)](i) caused by BMP4. Specific knockdown of BMP4 by small interference RNA significantly decreased the mRNA and protein expression of TRPC1, TRPC4, and TRPC6 and reduced SOCE and basal [Ca(2+)](i) in serum-stimulated PASMCs. We conclude that BMP4 regulates calcium signaling in PASMCs likely via upregulation of TRPC expression, leading to enhanced SOCE and basal [Ca(2+)](i) in PASMCs, and by this mechanism contributes to pulmonary vascular remodeling during pulmonary arterial hypertension.

PubMed Disclaimer

Figures

Fig. 1.
Fig. 1.
A: expression of canonical transient receptor potential (TRPC) mRNA relative to cyclophilin B in pulmonary arterial smooth muscle cells (PASMCs) treated with bone morphogenetic protein 4 (BMP4) at 5 ng/ml, 50 ng/ml, 250 ng/ml, or vehicle (control) for 60 h as determined by real-time quantitative PCR (n = 4 in each group). B: expression of TRPC mRNA relative to cyclophilin B in PASMCs treated with 50 ng/ml BMP4 or vehicle control for 24 h, 48 h, or 60 h as determined by real-time quantitative PCR (n = 4 in each group). Data are presented as percent change from control. *P < 0.05 vs. vehicle control cells.
Fig. 2.
Fig. 2.
Representative Western blots for TRPC1, TRPC4, TRPC6, and α-actin in PASMCs treated with 50 ng/ml BMP4 or vehicle (control) for 60 h. Bar graph shows mean protein expression for TRPC1, TRPC4, and TRPC6 relative to α-actin (n = 4 on samples from 4 animals; *P < 0.05 vs. vehicle control cells). Bar values are means ± SE.
Fig. 3.
Fig. 3.
A: representative traces of intracellular Ca2+ concentration ([Ca2+]i) responses to restoration of extracellular [Ca2+] to 2.5 mM after perfusion with Ca2+-free Krebs-Ringer bicarbonate solution (KRBS; 0 Ca2+) containing cyclopiazonic acid (CPA; 10 μM), nifedipine (Nif, 5 μM), and EGTA (1 mM) in rat distal PASMCs treated with BMP4 (n = 4 experiments in 109 cells), or vehicle for 60 h (n = 4 experiments in 105 cells). Bar graph indicates that maximum increase in [Ca2+]i after restoration of extracellular [Ca2+] was greater in BMP4-treated PASMCs than that in control cells (*P < 0.001). B: time course of quenching of Fura-2 AM fluorescence at 360 nm by 200 μM Mn2+ after perfusion with Ca2+-free KRBS (0 Ca2+) containing nifedipine (5 μM) and CPA (10 μM) in rat distal PASMCs treated with 50 ng/ml BMP4 (n = 3 experiments in 88 cells), or vehicle for 60 h, normalized to fluorescence at time 0 (F/F0). Bar graph shows that Mn2+ quenching, expressed as percent decrease in fluorescence at time 10 min from time 0, was greater in BMP4-treated PASMCs compared with that in control cells (*P < 0.001). Bar values are means ± SE.
Fig. 4.
Fig. 4.
A: changes in basal [Ca2+]i in rat distal PASMCs treated with 50 ng/ml BMP4 (n = 16 from 365 cells) for 60 h. Bar values are means ± SE. *P < 0.001 vs. vehicle control cells (n = 16 from 358 cells). B: removal of extracellular Ca2+ reduced basal [Ca2+]i in rat distal PASMCs treated with BMP4 (50 ng/ml, 60 h) (n = 4 in 115 cells), but not in control cells treated with vehicle (n = 4 in 136 cells). C: store-operated calcium channel (SOCC) antagonist SKF-96365 and NiCl2 attenuated basal [Ca2+]i in rat distal PASMCs treated with BMP4 (50 ng/ml, 60 h) (n = 4 in 109 cells), but not in control cells treated with vehicle (n = 4 in 121 cells). D: SOCC antagonist attenuated basal [Ca2+]i in PASMCs treated with BMP4 (50 ng/m, 60 h) (n = 4 in 112 cells), but not in control cells treated with vehicle (n = 4 in 119 cells). Values are means ± SE. *Significantly different from vehicle control: P < 0.05.
Fig. 5.
Fig. 5.
A: BMP4 and BMP2 mRNA relative to cyclophilin B as determined by real-time quantitative (q)PCR in PASMCs treated with BMP4 small interference (si)RNA or nontargeting (NT) control siRNA (n = 3 for each group). *P < 0.05 vs. NT siRNA-treated cells. B: representative Western blots of the secreted BMP4 and BMP2 protein in conditioned media from PASMC cultures treated with BMP4 siRNA or NT control siRNA. C: TRPC1, TRPC4, and TRPC6 mRNA relative to cyclophilin B as determined by real-time qPCR in PASMCs treated with BMP4 siRNA or NT control siRNA (n = 3 for each group; *P < 0.05 vs. NT siRNA-treated cells). D: representative Western blots of TRPC1, TRPC4, and TRPC6 protein in PASMCs treated with BMP4 siRNA or NT control siRNA. Bar graph shows mean protein expression for TRPC1, TRPC4, and TRPC6 relative to α-actin (n = 3 for each group; *P < 0.05 vs. NT siRNA treated cells). Bar values are means ± SE.
Fig. 6.
Fig. 6.
A: representative traces of [Ca2+]i responses to restoration of extracellular [Ca2+] to 2.5 mM after perfusion with Ca2+-free KRBS (0 Ca2+) containing CPA (10 μM), nifedipine (Nifed; 5 μM), and EGTA (1 mM) in rat distal PASMCs treated with BMP4 siRNA (n = 3 experiments in 86 cells) or NT siRNA (n = 3 experiments in 87 cells). Bar graph indicates that maximum increase in [Ca2+]i after restoration of extracellular [Ca2+] was reduced in BMP4 siRNA-treated PASMCs compared with that in NT siRNA-treated cells (*P < 0.001). B: time course of quenching of Fura-2 AM fluorescence at 360 nm by 200 μM Mn2+ after perfusion with Ca2+-free KRBS (0 Ca2+) containing nifedipine (5 μM) and CPA (10 μM) in rat distal PASMCs treated with BMP4 siRNA (n = 3 experiments in 83 cells), or NT siRNA, normalized to fluorescence at time 0 (F/F0). Bar graph shows that Mn2+ quenching, expressed as percent decrease in fluorescence from time 0, was reduced in BMP4 siRNA-treated PASMCs compared with that in NT siRNA-treated cells (*P < 0.05). Bar values are means ± SE.
Fig. 7.
Fig. 7.
Changes in basal [Ca2+]i in rat distal PASMCs treated with BMP4 siRNA (n = 3 from 88 cells) or NT siRNA. Bar values are means ± SE. *P < 0.01 vs. NT siRNA-treated cells (n = 3 from 89 cells).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC, Morrell NW. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 105: 1672–1678, 2002 - PubMed
    1. Berridge MJ. Calcium signalling and cell proliferation. Bioessays 17: 491–500, 1995 - PubMed
    1. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 4: 517–529, 2003 - PubMed
    1. Boulay G, Zhu X, Peyton M, Jiang M, Hurst R, Stefani E, Birnbaumer L. Cloning and expression of a novel mammalian homolog of Drosophila transient receptor potential (Trp) involved in calcium entry secondary to activation of receptors coupled by the Gq class of G protein. J Biol Chem 272: 29672–29680, 1997 - PubMed
    1. Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE, Knowles JA. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet 67: 737–744, 2000 - PMC - PubMed

Publication types

MeSH terms

LinkOut - more resources