Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: Bone morphogenetic protein 4 (BMP4) inhibits PDGF-stimulated cell proliferation and collagen synthesis in normal pulmonary artery smooth muscle cells (PASMCs) from conduit pulmonary arteries (Lonza). PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 24 h after which cell proliferation (A) was measured by detecting bromodeoxyuridine (BrdU) incorporation and intracellular collagen-I protein levels (B and C) were measured by Western blot analysis. B: representative immunoblots of collagen-I. C: bar graph showing the changes in protein levels of collagen-I. Results are expressed as means ± SE; n = 5. *P < 0.05 vs. control; #P < 0.05 vs. PDGF.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Incubation, BrdU Incorporation Assay, Western Blot, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: BMP4 inhibits PDGF-induced TGF-β1 signaling pathway in normal PASMCs from conduit pulmonary arteries. PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 2 h, and then p-Smad1/5, total Smad1/5, p-Smad2/3, and total Smad2/3 were measured Western blot. A: representative immunoblots of p-Smad1/5, total Smad1/5, p-Smad2/3, and total Smad2/3. B and C: bar graphs showing the changes in protein levels of p-Smad1/5 and p-Smad2/3. Results are expressed as means ± SE; n = 5. *P < 0.05 vs. control; #P < 0.05 vs. PDGF.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Incubation, Western Blot, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: BMP4 inhibits PDGF-induced calpain activation that initiates the intracrine TGF-β1 signaling pathway in normal PASMCs from conduit pulmonary arteries. A: PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5–24 h after which calpain activity was determined as described in materials and methods. B and C: PASMCs were incubated with PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 2 h after which intracellular active TGF-β1 was measured by nondenaturing electrophoresis and Western blot analysis. Results are expressed as means ± SE; n = 5. *P < 0.05 vs. control; #P < 0.05 vs. PDGF.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Activation Assay, Incubation, Activity Assay, Electrophoresis, Western Blot, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: The inhibitory effect of BMP4 on PDGF signaling pathway is not through Smad1/5 in normal PASMCs from conduit pulmonary arteries. A–D: PASMCs were transfected with siRNAs against Smad1/5 and control siRNA. After 72 h, the cells were incubated with PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 2 h. Then calpain activity was measured by using kinetic method, and p-Smad1/5, total Smad1/5, p-Smad2/3, total Smad2/3, and intracellular active TGF-β1 were measured by Western blot. E and F: after transfection with siRNAs against Smad1/5 and control siRNA, PASMCs were incubated with PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 24 h after which collagen-I protein was measured by Western blot analysis. Results are expressed as means ± SE; n = 4. *P < 0.05 vs. PDGF within control siRNA group; #P < 0.05 vs. PDGF within Smad1/5 siRNA group.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Transfection, Control, Incubation, Activity Assay, Western Blot

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: The inhibitory effect of BMP4 is not due to inhibiting PDGF-induced phosphorylation of Src or ERK in normal PASMCs from conduit pulmonary arteries. PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5 h after which p-Src, total Src, p-ERK, and total ERK were measured by Western blot analysis. A and C: representative immunoblots of p-Src, total Src, p-ERK, and total ERK. B and D: bar graph depicting the changes in p-Src and p-ERK. Results are expressed as means ± SE; n = 4. *P < 0.05 vs. control (without PDGF and BMP4); #P < 0.05 vs. BMP4.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Phospho-proteomics, Incubation, Western Blot, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: BMP4 increases PKA activity, and forskolin recapitulates and myristoylated protein kinase inhibitor (mPKI) prevents the inhibitory effects of BMP4 in normal PASMCs from conduit pulmonary arteries. A: PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5 h after which PKA activity was measured as described in materials and methods. B: PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of forsklin (10 μM) for 0.5 h after which calpain activity was measured as described in materials and methods. C: PASMCs treated with vehicle or PKI were incubated with PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5 h after which calpain activity was measured as described in materials and methods. Results are expressed as means ± SE; n = 5. *P < 0.05 vs. control (without PDGF, BMP4 or forskolin); #P < 0.05 vs. PDGF; **P < 0.05 vs. PDGF in vehicle.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Activity Assay, Incubation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: The inhibitory effect of BMP4 on PDGF-induced calpain-2 activation, cell proliferation, and collagen synthesis is abolished by knocking-down PKA in normal PASMCs from conduit pulmonary arteries. A–D: PASMCs were transfected with siRNAs against PKA and control siRNA. After 72 h, the cells were incubated with PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 2 h. Then, calpain activity was measured by using kinetic method (A and B). Some cell lysates were subjected to immunoprecipitation using specific antibodies against p-Ser369-calpain-2. The immunoprecipitates were subjected to immunoblots against calpain-2 (C and D). A, E, and F: after transfection with siRNAs against PKA and control siRNA, PASMCs were incubated with PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 24 h after which collagen-I protein (A and E) were measured by Western blot and cell proliferation (F) was measured by detecting BrdU incorporation. A and C: representative immunoblots of PKA, collagen-I, and p-Ser369-calpain-2. B, D, E, and F: bar graph depicting the changes in calpain activity, p-Ser369-calpain-2, collagen-I protein level, and cell proliferation. Results are expressed as means ± SE; n = 4. *P < 0.05 vs. PDGF within control siRNA group.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Activation Assay, Transfection, Control, Incubation, Activity Assay, Immunoprecipitation, Western Blot, BrdU Incorporation Assay

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: BMP4 attenuates the dephosphorylation of calpain-2 at serine-369 (p-Ser369) in PDGF-treated PASMCs in normal PASMCs from conduit pulmonary arteries. PASMCs were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5 h after which cells were lysed and calpain-2 proteins in cell lysates were measured by Western blot analysis. Some cell lysates were subjected to immunoprecipitation using specific antibodies against p-Ser50-calpain-2 or p-Ser369-calpain-2. The immunoprecipitates were subjected to immunoblots against calpain-2. A: representative immunoblots of calpain-2 protein. B: representative immunoblots of the immunoprecipitates for p-Ser50-calpain-2 or p-Ser369-calpain-2. C: bar graph showing the changes in the levels of p-Ser50-calpain-2 or p-Ser369-calpain-2. Results are expressed as means ± SE; n = 3. *P < 0.01 vs. control for p-Ser50-calpain-2; **P < 0.05 vs. control for p-Ser369-calpain-2.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: De-Phosphorylation Assay, Incubation, Western Blot, Immunoprecipitation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: BMP4 inhibits PDGF-stimulated cell proliferation and collagen synthesis in normal PASMCs from peripheral type-III pulmonary arteries. Normal PASMCs from peripheral type-III pulmonary arteries were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 24 h after which cell proliferation (A) was measured by detecting BrdU incorporation and intracellular collagen-I protein level (B and C) were measured by Western blot. B: representative immunoblots of collagen-I. C: bar graph showing the changes in protein levels of collagen-I. Results are expressed as means ± SE; n = 4. *P < 0.05 vs. control; #P < 0.05 vs. PDGF.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Incubation, BrdU Incorporation Assay, Western Blot, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: BMP4 does not attenuate PDGF-induced increases in cell proliferation, collagen-I protein levels, and calpain activation in PASMCs from peripheral type-III pulmonary arteries of idiopathic pulmonary arterial hypertension (PAH) patients. PASMCs from peripheral type-III pulmonary arteries of PAH patients were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 24 h after which cell proliferation (A) was measured by detecting BrdU incorporation and intracellular collagen-I protein levels (B and C) and calpain activity (D; 30-min incubation) were measured. B: representative immunoblots of collagen-I. C: bar graph showing the changes in protein levels of collagen-I. Results are expressed as means ± SE; n = 4. *P < 0.05 vs. control; **P < 0.05 vs. BMP4.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Activation Assay, Incubation, BrdU Incorporation Assay, Activity Assay, Western Blot, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: BMP4 does not induce PKA activation and does not prevent PDGF-induced decrease in calpain-2 phosphorylation at serine-369 in PASMCs from peripheral type-III pulmonary arteries of IPAH patients. PASMCs from peripheral type-III pulmonary arteries of IPAH patients were incubated with and without PDGF-BB (10 ng/ml) in the presence and absence of BMP4 (30 ng/ml) for 0.5 h after which after which PKA activity was measured as described in materials and methods (A). Some cells were lysed and calpain-2 proteins in cell lysates were measured by Western blot analysis (B). Some cell lysates were subjected to immunoprecipitation using specific antibodies against p-Ser50-calpain-2 or p-Ser369-calpain-2. The immunoprecipitates were subjected to immunoblots against calpain-2 (C and D). B: representative immunoblots of calpain-2 protein. C: representative immunoblots of the immunoprecipitates for p-Ser50-calpain-2 or p-Ser369-calpain-2. D: bar graph showing the changes in the levels of p-Ser50-calpain-2 or p-Ser369-calpain-2. Results are expressed as means ± SE; n = 4. *P < 0.01 vs. control; **P < 0.01 vs. BMP4.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Activation Assay, Phospho-proteomics, Incubation, Activity Assay, Western Blot, Immunoprecipitation, Control

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: BMP4 inhibits PDGF-induced proliferation and collagen synthesis via PKA-mediated inhibition of calpain-2 in pulmonary artery smooth muscle cells

doi: 10.1152/ajplung.00260.2016

Figure Lengend Snippet: A schematic pathway illustrating the inhibitory effects of BMP4 on PDGF-induced calpain-2 activation, cell proliferation, and collagen synthesis in normal and PAH PASMCs.

Article Snippet: To determine the effect of BMP4 on PDGF signaling pathway, normal human PASMCs from conduit pulmonary arteries (Lonza) were incubated with PDGF-BB (10 ng/ml) in the absence and presence of BMP4 (30 ng/ml) for 24 h, and cell proliferation and intracellular collagen were then determined.

Techniques: Activation Assay