Limits...
Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1.

Kadowaki M, Mizuno S, Demura Y, Ameshima S, Miyamori I, Ishizaki T - Respir. Res. (2007)

Bottom Line: To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1-21%).We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation.Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Third Department of Internal Medicine, University of Fukui, 23-3 Eiheiji-cho, Matsuoka, Yoshida-gun, Fukui, Japan. maik@u-fukui.ac.jp

ABSTRACT

Background: Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.

Methods: We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1-21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.

Results: Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.

Conclusion: Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.

Show MeSH

Related in: MedlinePlus

Effect of BPS and 8-Br-cAMP on p27kip1 mRNA stability during hypoxia. Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. The p27kip1 mRNA stability was measured after adding 400 nM of Act D using Real-time RT-PCR using LightCycler™. Degradation of p27kip1 mRNA was significantly suppressed by BPS and 8-Br-cAMP under both normoxic and moderately hypoxic conditions, and mRNA stability was slightly decreased by moderate hypoxia. Graphs show % maximal p27kip1 mRNA expression. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen; line with solid squares, 21% oxygen and BPS; line with solid triangles, 21% oxygen and 8-Br-cAMP; dotted line with open squares, 2% oxygen and BPS; dotted line with open triangle, 2% oxygen and 8-Br-cAMP. Data are expressed as means ± SE (n = 6). *P < 0.05 versus 21% oxygen. †P < 0.05 versus oxygen controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2164950&req=5

Figure 5: Effect of BPS and 8-Br-cAMP on p27kip1 mRNA stability during hypoxia. Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. The p27kip1 mRNA stability was measured after adding 400 nM of Act D using Real-time RT-PCR using LightCycler™. Degradation of p27kip1 mRNA was significantly suppressed by BPS and 8-Br-cAMP under both normoxic and moderately hypoxic conditions, and mRNA stability was slightly decreased by moderate hypoxia. Graphs show % maximal p27kip1 mRNA expression. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen; line with solid squares, 21% oxygen and BPS; line with solid triangles, 21% oxygen and 8-Br-cAMP; dotted line with open squares, 2% oxygen and BPS; dotted line with open triangle, 2% oxygen and 8-Br-cAMP. Data are expressed as means ± SE (n = 6). *P < 0.05 versus 21% oxygen. †P < 0.05 versus oxygen controls.

Mentions: To confirm the effect of BPS and hypoxia on p27kip1 mRNA expression, we assessed p27kip1 mRNA stability using Act D. Both BPS and 8-Br-cAMP significantly suppressed p27kip1 mRNA degradation in cells incubated with Act D under both normoxic and moderately hypoxic conditions. Although moderate hypoxia did not change p27kip1 mRNA expression, mRNA stability was slightly decreased under moderate hypoxia (Fig. 5).


Effect of hypoxia and Beraprost sodium on human pulmonary arterial smooth muscle cell proliferation: the role of p27kip1.

Kadowaki M, Mizuno S, Demura Y, Ameshima S, Miyamori I, Ishizaki T - Respir. Res. (2007)

Effect of BPS and 8-Br-cAMP on p27kip1 mRNA stability during hypoxia. Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. The p27kip1 mRNA stability was measured after adding 400 nM of Act D using Real-time RT-PCR using LightCycler™. Degradation of p27kip1 mRNA was significantly suppressed by BPS and 8-Br-cAMP under both normoxic and moderately hypoxic conditions, and mRNA stability was slightly decreased by moderate hypoxia. Graphs show % maximal p27kip1 mRNA expression. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen; line with solid squares, 21% oxygen and BPS; line with solid triangles, 21% oxygen and 8-Br-cAMP; dotted line with open squares, 2% oxygen and BPS; dotted line with open triangle, 2% oxygen and 8-Br-cAMP. Data are expressed as means ± SE (n = 6). *P < 0.05 versus 21% oxygen. †P < 0.05 versus oxygen controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2164950&req=5

Figure 5: Effect of BPS and 8-Br-cAMP on p27kip1 mRNA stability during hypoxia. Cultured HPASMC were exposed to 21% or 2% oxygen concentrations with or without 10 μM of BPS or 1 mM 8-Br-cAMP for indicated periods. The p27kip1 mRNA stability was measured after adding 400 nM of Act D using Real-time RT-PCR using LightCycler™. Degradation of p27kip1 mRNA was significantly suppressed by BPS and 8-Br-cAMP under both normoxic and moderately hypoxic conditions, and mRNA stability was slightly decreased by moderate hypoxia. Graphs show % maximal p27kip1 mRNA expression. Line with solid circles, 21% oxygen; dotted line with open circles, 2% oxygen; line with solid squares, 21% oxygen and BPS; line with solid triangles, 21% oxygen and 8-Br-cAMP; dotted line with open squares, 2% oxygen and BPS; dotted line with open triangle, 2% oxygen and 8-Br-cAMP. Data are expressed as means ± SE (n = 6). *P < 0.05 versus 21% oxygen. †P < 0.05 versus oxygen controls.
Mentions: To confirm the effect of BPS and hypoxia on p27kip1 mRNA expression, we assessed p27kip1 mRNA stability using Act D. Both BPS and 8-Br-cAMP significantly suppressed p27kip1 mRNA degradation in cells incubated with Act D under both normoxic and moderately hypoxic conditions. Although moderate hypoxia did not change p27kip1 mRNA expression, mRNA stability was slightly decreased under moderate hypoxia (Fig. 5).

Bottom Line: To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1-21%).We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation.Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Third Department of Internal Medicine, University of Fukui, 23-3 Eiheiji-cho, Matsuoka, Yoshida-gun, Fukui, Japan. maik@u-fukui.ac.jp

ABSTRACT

Background: Hypoxia induces the proliferation of pulmonary arterial smooth muscle cell (PASMC) in vivo and in vitro, and prostacyclin analogues are thought to inhibit the growth of PASMC. Previous studies suggest that p27kip1, a kind of cyclin-dependent kinase inhibitor, play an important role in the smooth muscle cell proliferation. However, the mechanism of hypoxia and the subcellular interactions between p27kip1 and prostacyclin analogues in human pulmonary arterial smooth muscle cell (HPASMC) are not fully understood.

Methods: We investigated the role of p27kip1 in the ability of Beraprost sodium (BPS; a stable prostacyclin analogue) to inhibit the proliferation of HPASMC during hypoxia. To clarify the biological effects of hypoxic air exposure and BPS on HPASMC, the cells were cultured in a hypoxic chamber under various oxygen concentrations (0.1-21%). Thereafter, DNA synthesis was measured as bromodeoxyuridine (BrdU) incorporation, the cell cycle was analyzed by flow cytometry with propidium iodide staining. The p27kip1 mRNA and protein expression and it's stability was measured by real-time RT-PCR and Western blotting. Further, we assessed the role of p27kip1 in HPASMC proliferation using p27kip1 gene knockdown using small interfering RNA (siRNA) transfection.

Results: Although severe hypoxia (0.1% oxygen) suppressed the proliferation of serum-stimulated HPASMC, moderate hypoxia (2% oxygen) enhanced proliferation in accordance with enhanced p27kip1 protein degradation, whereas BPS suppressed HPASMC proliferation under both hypoxic and normoxic conditions by suppressing p27kip1 degradation with intracellular cAMP-elevation. The 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), a cAMP analogue, had similar action as BPS in the regulation of p27kip1. Moderate hypoxia did not affect the stability of p27kip1 protein expression, but PDGF, known as major hypoxia-induced growth factors, significantly decreased p27kip1 protein stability. We also demonstrated that BPS and 8-Br-cAMP suppressed HPASMC proliferation under both hypoxic and normoxic conditions by blocking p27kip1 mRNA degradation. Furthermore, p27kip1 gene silencing partially attenuated the effects of BPS and partially restored hypoxia-induced proliferation.

Conclusion: Our study suggests that moderate hypoxia induces HPASMC proliferation, which is partially dependent of p27kip1 down-regulation probably via the induction of growth factors such as PDGF, and BPS inhibits both the cell proliferation and p27kip1 mRNA degradation through cAMP pathway.

Show MeSH
Related in: MedlinePlus