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. 2014 Sep 9;9(9):e107135.
doi: 10.1371/journal.pone.0107135. eCollection 2014.

NOX4 mediates BMP4-induced upregulation of TRPC1 and 6 protein expressions in distal pulmonary arterial smooth muscle cells

Qian Jiang  1 , Xin Fu  1 , Lichun Tian  1 , Yuqin Chen  1 , Kai Yang  2 , Xiuqing Chen  1 , Jie Zhang  1 , Wenju Lu  1 , Jian Wang  3
Affiliations

NOX4 mediates BMP4-induced upregulation of TRPC1 and 6 protein expressions in distal pulmonary arterial smooth muscle cells

Qian Jiang et al. PLoS One. .

Abstract

Rationale: Our previous studies demonstrated that bone morphogenetic protein 4 (BMP4) mediated, elevated expression of canonical transient receptor potential (TRPC) largely accounts for the enhanced proliferation in pulmonary arterial smooth muscle cells (PASMCs). In the present study, we sought to determine the signaling pathway through which BMP4 up-regulates TRPC expression.

Methods: We employed recombinant human BMP4 (rhBMP4) to determine the effects of BMP4 on NADPH oxidase 4 (NOX4) and reactive oxygen species (ROS) production in rat distal PASMCs. We also designed small interfering RNA targeting NOX4 (siNOX4) and detected whether NOX4 knockdown affects rhBMP4-induced ROS, TRPC1 and 6 expression, cell proliferation and intracellular Ca2+ determination in PASMCs.

Results: In rhBMP4 treated rat distal PASMCs, NOX4 expression was (226.73±11.13) %, and the mean ROS level was (123.65±1.62) % of that in untreated control cell. siNOX4 transfection significantly reduced rhBMP4-induced elevation of the mean ROS level in PASMCs. Moreover, siNOX4 transfection markedly reduced rhBMP4-induced elevation of TRPC1 and 6 proteins, basal [Ca2+]i and SOCE. Furthermore, compared with control group (0.21±0.001), the proliferation of rhBMP4 treated cells was significantly enhanced (0.41±0.001) (P<0.01). However, such increase was attenuated by knockdown of NOX4. Moreover, external ROS (H2O2 100 µM, 24 h) rescued the effects of NOX4 knockdown, which included the declining of TRPC1 and 6 expression, basal intracellular calcium concentration ([Ca2+]i) and store-operated calcium entry (SOCE), suggesting that NOX4 plays as an important mediator in BMP4-induced proliferation and intracellular calcium homeostasis.

Conclusion: These results suggest that BMP4 may increase ROS level, enhance TRPC1 and 6 expression and proliferation by up-regulating NOX4 expression in PASMCs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Bone morphogenetic protein 4 (BMP4) increased NOX4 NADPH oxidase 4 (NOX4) expression and ROS mean level in PASMCs.
A: Western blot analysis of BMP4 protein impacting NOX4 in rat distal PASMCs, of which the upper band is NOX4, the following band is α-actin. Control group and BMP4 group indicate blank control group and recombinant BMP4 (50 ng/ml for 24 hours) group respectively. B: BMP4 effects on NOX4 protein in rat distal PASMCs (X±S, n = 3) #P<0.01 vs. control group, the results have significant difference. C: BMP4 induced reactive oxygen species (ROS) generation in rat distal PASMCs. Control group and BMP4 group indicate blank control group and recombinant BMP4 (50 ng/ml for 24 hours) group respectively. (X±S, n = 3) #P<0.01 vs. control group, the results have significant difference.
Figure 2
Figure 2. NOX4 knockdown downregulated BMP4 induced ROS generation in rat distal PASMCs.
A: Western blot analysis of NOX4 siRNA affecting NOX4 protein in rat distal PASMCs, of which the upper band is NOX4, the following band is α-actin. NC group, NOX4-205 siRNA group and NOX4-1048 siRNA group indicate negative control group, NOX4 siRNA group respectively. B: NOX4 siRNA affects NOX4 protein expression in rat distal PASMCs (formula image±S, n = 4). #indicates P<0.01 vs. negative control group, the results have significant difference. C: From left to right are negative control group, negative control+BMP4 group, NOX4 siRNA group, and NOX4 siRNA+BMP4 group respectively. (X±S, n = 3), #indicates P<0.01 vs. NC-siRNA group, *p<0.01 vs. NC-siRNA plus BMP4 group.
Figure 3
Figure 3. NOX4 knockdown decreased TRPC1 and TRPC6 expression under both control and BMP4 treatment circumstances.
A: Western blot analysis of TRPC1 protein after knockout of NOX4 in rat distal PASMCs, of which the upper band is TRPC1, the following band is α-actin; NC and NOX4 siRNA group indicate negative control group and NOX4 knockout group respectively. B: results of knockout of NOX4 affecting TRPC1 protein in rat distal PASMCs. (X±S, n = 3), #indicates P<0.01 vs. Negative control group, the results have significant difference. C: Western blot analysis of BMP4 affecting TRPC1 in rat distal PASMCs after knockout of NOX4, of which the upper band is TRPC1, the following band is α-actin. NC+BMP4 group, NOX4 siRNA+BMP4 group indicate negative control group and NOX4-knockout with recombinant BMP4 treatment (for 24 hours) group, respectively. D: Results of BMP4 affecting TRPC1 expression in rat distal PASMCs after knockout of NOX4. (X±S, n = 3), #indicates P<0.01 vs. Negative control+BMP4 group, the results have significant difference. E: Western blot analysis of TRPC6 protein after knockout of NOX4 in rat distal PASMCs, of which the upper band is TRPC1, the following band is α-actin; NC and NOX4 siRNA group indicate negative control group and NOX4 knockout group respectively. F: results of knockout of NOX4 affecting TRPC1 protein in rat distal PASMCs. (X±S, n = 3), #indicates P<0.01 vs. Negative control group, the results have significant difference. G: Western blot analysis of BMP4 affecting TRPC6 in rat distal PASMCs after knockout of NOX4, of which the upper band is TRPC6, the following band is α-actin; NC+BMP4 NOX4 siRNA+BMP4 group indicate negative control group and NOX4-knockout with recombinant BMP4 treatment (for 24 hours) group, respectively. H: Results of BMP4 affecting TRPC6 expression in rat distal PASMCs after knockout of NOX4. (X±S, n = 3), #indicates P<0.01 vs. Negative control+BMP4 group, the results have significant difference.
Figure 4
Figure 4. NOX4 knockdown inhibits proliferation of PASMCs under both control and BMP4 treatment circumstances A: control group was the control group absorbance at 490 nm, BMP4 group was absorbance of recombinant BMP4 protein intervention for 24 h group at 490 nm, #indicates P<0.01 vs. control group, the results are statistically significant.
B: absorbance of rat distal PASMCS at 490 nm after transfection with NOX4 siRNA and knockout of NOX4, NC indicates negative control group; NOX4 siRNA indicates NOX4 gene silencing group; #indicates P<0.01 vs. negative control group, results have statistically significance. C: absorbance of rat distal PASMCS at 490 nm after transfection with NOX4 siRNA and knockout of NOX4, and treatment with recombinant BMP4 for 24 hours. NC+BMP4 group indicates negative control+BMP4 treatment group; NOX4 siRNA+BMP4 group was BMP4 with silent NOX4 group, #indicates P<0.01 vs. negative control+ BMP4 group, the results are statistically significant.
Figure 5
Figure 5. NOX4 knockdown downregulate basal [Ca2+]i and SOCE of rat distal PASMCs in the condition with or without BMP4 treatment.
A and C: changes of SOCE in PASMCs from NC-siRNA group, NC-siRNA+rhBMP4 group, NOX4-siRNA group, NOX4-siRNA+rhBMP4 group (X±S, n = 5, #indicates P<0.01 vs. NC-siRNA group, the results are statistically significant. *indicates P<0.01 vs. NC-siRNA group, the results are statistically significant. **indicates P<0.01 vs. NC-siRNA group and NC-siRNA+rhBMP4 group, the results are statistically significant). B: changes of basal [Ca2+]i in four group (X±S, n = 5, #indicates P<0.01 vs. NC-siRNA group, the results are statistically significant. *indicates P<0.01 vs. NC-siRNA group, the results are statistically significant. **indicates P<0.01 vs. NC-siRNA group and NC-siRNA+rhBMP4 group, the results are statistically significant.).
Figure 6
Figure 6. H2O2 reversed the effect of knockdown NOX4 upregulated TRPC1 and TRPC6 expression, basal [Ca2+]i and SOCE in rat distal PASMCs.
A–D: expression levels of TRPC1 and TRPC6 protein were detected by western blot (A, C: representative blots, B, D: mean intensity for TRPC1 and TRPC6) (X±S, n = 4, #indicates P<0.01 vs. NC-siRNA group, the results are statistically significant. *indicates P<0.01 vs. NOX4-siRNA group, the results are statistically significant.) F: basal [Ca2+]i in PASMCs of three group (NC-siRNA group, NOX4-siRNA group, NOX4-siRNA+ H2O2 group) (X±S, n = 4, #indicates P<0.01 vs. NC-siRNA group, the results are statistically significant. *indicates P<0.01 vs. NC-siRNA group and NOX4-siRNA group, the results are statistically significant.) E, G: changes of SOCE in NC-siRNA group, NOX4-siRNA group and NOX4-siRNA+ H2O2 group. (X±S, n = 5, #indicates P<0.01 vs. NC-siRNA group, the results are statistically significant. *indicates P<0.01 vs. NC-siRNA group and NOX4-siRNA group, the results are statistically significant.).
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