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


CONCLUSIONS
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.
Kadowaki
Maiko
M
Third Department of Internal Medicine, University of Fukui, 23-3 Eiheiji-cho, Matsuoka, Yoshida-gun, Fukui, Japan. maik@u-fukui.ac.jp
Mizuno
Shiro
S
Demura
Yoshiki
Y
Ameshima
Shingo
S
Miyamori
Isamu
I
Ishizaki
Takeshi
T
D015687:Cell Hypoxia / Q000502:physiology; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004305:Dose-Response Relationship, Drug; D011464:Epoprostenol / Q000008:administration & dosage / Q000031:analogs & derivatives*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D010100:Oxygen / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D014665:Vasodilator Agents / Q000008:administration & dosage
C436291:CDKN1B protein, human; D047908:Intracellular Signaling Peptides and Proteins; D014665:Vasodilator Agents; D050760:Cyclin-Dependent Kinase Inhibitor p27; C048081:beraprost; D011464:Epoprostenol; D010100:Oxygen