Korean J Orthod.  2003 Jun;33(3):169-183.

Role of p-38 MAP Kinase in apoptosis of hypoxia-induced osteoblasts

Affiliations
  • 1Department of Orthodontics, School of Dentistry, Wonkwang University, Korea. sangkim@wonkwang.ac.kr

Abstract

Tooth movement by orthodontic force effects great tissue changes within the periodontium, especially by shifting the blood flow in the pressure side and resulting in a hypoxic state of low oxygen tension. The aim of this study is to elucidate the possible mechanism of apoptosis in response to hypoxia in MC3T3E1 osteoblasts, the main cells in bone remodeling during orthodontic tooth movement. MC3T3E1 osteoblasts under hypoxic conditions (2% oxygen) resulted in apoptosis in a time-dependent manner as estimated by DNA fragmentation assay and nuclear morphology stained with fluorescent dye, Hoechst 33258. Pretreatment with Z-VAD-FMK, a pan-caspase inhibitor, or Z-DEVD-CHO, a specific caspase-3 inhibitor, completely suppressed the DNA ladder in response to hypoxia. An increase in caspase-3-like protease (DEVDase) activity was observed during apoptosis, but no caspase-1 activity (YVADase) was detected. To confirm what caspases are involved in apoptosis, Western blot analysis was performed using anti-caspase-3 or -6 antibodies. The 10-kDa protein, corresponding to the active products of caspase-3, and the 10-kDa protein of the active protein of caspase-6 were generated in hypoxia-challenged cells in which the processing of the full length form of caspase-3 and -6 was evident. While a time course similar to this caspase-3 and -6 activation was evident, hypoxic stress caused the cleavage of lamin A, which was typical of caspase-6 activity. In addition, the stress elicited the release of cytochrome c into the cytosol during apoptosis. Furthermore, we observed that pre-treatment with SB203580, a selective p38 mitogen activated protein kinase inhibitor, attenuated the hypoxia-induced apoptosis. The addition of SB203580 suppressed caspase-3 and -6-like protease activity by hypoxia up to 50%. In contrast, PD98059 had no effect on the hypoxia-induced apoptosis. To confirm the involvement of MAP kinase, JNK/SAPK, ERK, or p38 kinase assay was performed. Although p38 MAPK was activated in response to hypoxic treatment, the other MAPK -JNK/SAPK or ERK- was either only modestly activated or not at all. These results suggest that p38 MAPK is involved in hypoxia-induced apoptosis in MC3T3E1 osteoblasts.

Keyword

Hypoxia; Apoptosis; Osteoblast; P-38 MAP kinase

MeSH Terms

Anoxia
Antibodies
Apoptosis*
Bisbenzimidazole
Blotting, Western
Bone Remodeling
Caspase 3
Caspase 6
Caspases
Cytochromes c
Cytosol
DNA
DNA Fragmentation
Lamin Type A
Osteoblasts*
Oxygen
p38 Mitogen-Activated Protein Kinases
Periodontium
Phosphotransferases*
Protein Kinases
Tooth Movement
Antibodies
Bisbenzimidazole
Caspase 3
Caspase 6
Caspases
Cytochromes c
DNA
Lamin Type A
Oxygen
Phosphotransferases
Protein Kinases
p38 Mitogen-Activated Protein Kinases
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