Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal.
METHODS
To examine the impact of hypoxia, rat nucleus pulposus cells were maintained in monolayer culture in 2% O2 and survival and signal transduction pathways identified.


OBJECTIVE
To elucidate the signaling pathways that allow nucleus pulposus cells to adapt to low oxygen environment.


BACKGROUND
Mammalian cell function is critically dependent on a continuous supply of oxygen. Interestingly, some specialized cell types that include nucleus pulposus cells of the intervertebral disc reside in a hypoxic environment. However, the mechanism of their adaptation to this low oxygen environment is not known.


METHODS
Rat nucleus pulposus cells were harvested from explant cultures and grown to confluence in monolayer. Cells from passage 3-7 were maintained under hypoxia (2% O2) and normoxia (20% O2) for various time periods in complete or serum-free medium. Cells were also treated with pharmacologic agents that block PI3K and MAPK signaling pathways. Cell survival was assessed by MTT assay, annexinV-PI dual-color flow cytometry, and the TUNEL procedure. Expression of signaling proteins was evaluated by Western blot analysis. Cell phenotype was studied by semiquantitative RT-PCR.


RESULTS
Under hypoxic conditions, rat nucleus pulposus cells were resistant to apoptosis induced by serum starvation. Protection was also observed after treatment of the nucleus cells by desferrioxamine, a compound that mimics many of the effects of hypoxia. Cell survival in hypoxia was related to activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways. Induction of Akt activation and ERK1/2 activationunder hypoxic condition was detected at 12 hours and correlated with inactivation of glycogen synthase kinase-3beta (GSK-3beta), an effector protein involved in regulation of apoptosis. Finally, inhibition of PI3K/Akt and MEK/ERK pathway using the inhibitors LY294002 and PD98059, respectively, impaired cell survival.


CONCLUSIONS
It is concluded that under hypoxic conditions, rat nucleus pulposus cells are adapted for survival by regulation of expression of critical genes, downregulation of apoptosis through activation of the PI3K/Akt and MAPK survival pathways.
Risbud
Makarand V
MV
Division of Orthopaedic Research, Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA. makarand.risbud@jefferson.edu
Fertala
Jolanta
J
Vresilovic
Edward J
EJ
Albert
Todd J
TJ
Shapiro
Irving M
IM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D015687:Cell Hypoxia / Q000502:physiology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D020935:MAP Kinase Signaling System / Q000502:physiology*; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000502:physiology*; D015854:Up-Regulation
D015415:Biomarkers; D016895:Culture Media, Serum-Free; D019869:Phosphatidylinositol 3-Kinases; D000071679:Glycogen Synthase Kinase 3 beta; C000605508:Gsk3b protein, rat; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D038362:Glycogen Synthase Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases