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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.

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Effects of BPS and 8-Br-cAMPon p27kip1 mRNA expression 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. Expression of p27kip1 mRNA was measured using Real-time RT-PCR using LightCycler™. (a) BPS suppressed p27kip1 mRNA reduction under both normoxic and hypoxic conditions. Expression of p27kip1 mRNA between normoxic and hypoxic conditions did not significantly change. Graph shows ratio of p27kip1 to β-actin mRNA expression. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 6). *P < 0.05 versus control. (b) Agarose gel electrophoresis with EB staining revealed single amplification of predicted PCR products (lane 1, DNA molecular weight markers; lane 2, p27kip1; 109 bp; lane 3, β-actin 144 bp).
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Figure 4: Effects of BPS and 8-Br-cAMPon p27kip1 mRNA expression 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. Expression of p27kip1 mRNA was measured using Real-time RT-PCR using LightCycler™. (a) BPS suppressed p27kip1 mRNA reduction under both normoxic and hypoxic conditions. Expression of p27kip1 mRNA between normoxic and hypoxic conditions did not significantly change. Graph shows ratio of p27kip1 to β-actin mRNA expression. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 6). *P < 0.05 versus control. (b) Agarose gel electrophoresis with EB staining revealed single amplification of predicted PCR products (lane 1, DNA molecular weight markers; lane 2, p27kip1; 109 bp; lane 3, β-actin 144 bp).

Mentions: After incubation in serum-depleted medium (quiescent state), p27kip1 mRNA expression was obviously up-regulated, and decreased by 24 h of serum stimulation. On the other hand, BPS and 8-Br-cAMP significantly attenuated the suppression induced by 24 h of serum stimulation. However, p27kip1 mRNA expression did not significantly differ between normoxia and moderate hypoxia (Fig. 4a).


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)

Effects of BPS and 8-Br-cAMPon p27kip1 mRNA expression 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. Expression of p27kip1 mRNA was measured using Real-time RT-PCR using LightCycler™. (a) BPS suppressed p27kip1 mRNA reduction under both normoxic and hypoxic conditions. Expression of p27kip1 mRNA between normoxic and hypoxic conditions did not significantly change. Graph shows ratio of p27kip1 to β-actin mRNA expression. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 6). *P < 0.05 versus control. (b) Agarose gel electrophoresis with EB staining revealed single amplification of predicted PCR products (lane 1, DNA molecular weight markers; lane 2, p27kip1; 109 bp; lane 3, β-actin 144 bp).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effects of BPS and 8-Br-cAMPon p27kip1 mRNA expression 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. Expression of p27kip1 mRNA was measured using Real-time RT-PCR using LightCycler™. (a) BPS suppressed p27kip1 mRNA reduction under both normoxic and hypoxic conditions. Expression of p27kip1 mRNA between normoxic and hypoxic conditions did not significantly change. Graph shows ratio of p27kip1 to β-actin mRNA expression. Open and solid bars, 21% and 2% oxygen, respectively. Data are expressed as means ± SE (n = 6). *P < 0.05 versus control. (b) Agarose gel electrophoresis with EB staining revealed single amplification of predicted PCR products (lane 1, DNA molecular weight markers; lane 2, p27kip1; 109 bp; lane 3, β-actin 144 bp).
Mentions: After incubation in serum-depleted medium (quiescent state), p27kip1 mRNA expression was obviously up-regulated, and decreased by 24 h of serum stimulation. On the other hand, BPS and 8-Br-cAMP significantly attenuated the suppression induced by 24 h of serum stimulation. However, p27kip1 mRNA expression did not significantly differ between normoxia and moderate hypoxia (Fig. 4a).

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