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Spine (Phila Pa 1976). 2005 Apr 15;30(8):882-9.

Nucleus pulposus cells upregulate PI3K/Akt and MEK/ERK signaling pathways under hypoxic conditions and resist apoptosis induced by serum withdrawal.

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1
Division of Orthopaedic Research, Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA. makarand.risbud@jefferson.edu

Abstract

STUDY DESIGN:

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.

SUMMARY OF BACKGROUND DATA:

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.

CONCLUSION:

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.

PMID:
15834331
[Indexed for MEDLINE]
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