Abstract
Bone formation in response to exogenous mechanical loading is dependent on prostaglandin synthesis by the inducible isoform of cyclooxygenase, COX-2. While several transcription factors target the COX-2 gene, we examined the role of nuclear factor kappa B (NFκB) on COX-2 upregulation in osteoblasts in response to fluid shear due to its involvement in immune and inflammatory responses in other cell types. Application of 12 dyn/cm2 laminar flow to MC3T3-E1 osteoblast-like cells resulted in translocation of NFκB to the nucleus within 1 h of the onset of shear, with NFκB returning to the cytoplasm after 2 h of continuous flow. NFκB translocation in response to shear was inhibited by the protease inhibitor, Nα -p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), or a cell-permeant peptide that blocks the nuclear localization sequence (NLS) on NFκB. Block of NFκB translocation with these inhibitors blocked the shear-induced upregulation of COX-2. We found that disruption of the actin cytoskeleton with cytochalasin D or microtubules with nocodozol did not alter NFκB translocation in response to shear. However, addition of the intracellular Ca2+ chelator BAPTA completely blocked NFκB translocation. While block of Ca2+ entry with channel blockers failed to inhibit NFκB translocation, inhibition of phospholipase C (PLC)-induced intracellular Ca2+ release with the PLC inhibitor U73122 completely abrogated the NFκB response to shear. These data indicate that NFκB translocation to the nucleus is essential for the fluid shear-induced increase in COX-2. Further, these studies suggest that intracellular Ca2+ release, but not the cytoskeletal architecture, is important to NFκB translocation.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 399-410 |
| Number of pages | 12 |
| Journal | Bone |
| Volume | 33 |
| Issue number | 3 |
| DOIs | |
| State | Published - Sep 1 2003 |
| Externally published | Yes |
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Keywords
- Intracellular Ca release
- Mechanotransduction
- NFκB
- Osteoblasts
- Phospholipase C
ASJC Scopus subject areas
- Physiology
- Hematology
Cite this
Fluid shear-induced NFκB translocation in osteoblasts is mediated by intracellular calcium release. / Chen, Neal X.; Geist, Derik J.; Genetos, Damian C; Pavalko, Fredrick M.; Duncan, Randall L.
In: Bone, Vol. 33, No. 3, 01.09.2003, p. 399-410.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Fluid shear-induced NFκB translocation in osteoblasts is mediated by intracellular calcium release
AU - Chen, Neal X.
AU - Geist, Derik J.
AU - Genetos, Damian C
AU - Pavalko, Fredrick M.
AU - Duncan, Randall L.
PY - 2003/9/1
Y1 - 2003/9/1
N2 - Bone formation in response to exogenous mechanical loading is dependent on prostaglandin synthesis by the inducible isoform of cyclooxygenase, COX-2. While several transcription factors target the COX-2 gene, we examined the role of nuclear factor kappa B (NFκB) on COX-2 upregulation in osteoblasts in response to fluid shear due to its involvement in immune and inflammatory responses in other cell types. Application of 12 dyn/cm2 laminar flow to MC3T3-E1 osteoblast-like cells resulted in translocation of NFκB to the nucleus within 1 h of the onset of shear, with NFκB returning to the cytoplasm after 2 h of continuous flow. NFκB translocation in response to shear was inhibited by the protease inhibitor, Nα -p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), or a cell-permeant peptide that blocks the nuclear localization sequence (NLS) on NFκB. Block of NFκB translocation with these inhibitors blocked the shear-induced upregulation of COX-2. We found that disruption of the actin cytoskeleton with cytochalasin D or microtubules with nocodozol did not alter NFκB translocation in response to shear. However, addition of the intracellular Ca2+ chelator BAPTA completely blocked NFκB translocation. While block of Ca2+ entry with channel blockers failed to inhibit NFκB translocation, inhibition of phospholipase C (PLC)-induced intracellular Ca2+ release with the PLC inhibitor U73122 completely abrogated the NFκB response to shear. These data indicate that NFκB translocation to the nucleus is essential for the fluid shear-induced increase in COX-2. Further, these studies suggest that intracellular Ca2+ release, but not the cytoskeletal architecture, is important to NFκB translocation.
AB - Bone formation in response to exogenous mechanical loading is dependent on prostaglandin synthesis by the inducible isoform of cyclooxygenase, COX-2. While several transcription factors target the COX-2 gene, we examined the role of nuclear factor kappa B (NFκB) on COX-2 upregulation in osteoblasts in response to fluid shear due to its involvement in immune and inflammatory responses in other cell types. Application of 12 dyn/cm2 laminar flow to MC3T3-E1 osteoblast-like cells resulted in translocation of NFκB to the nucleus within 1 h of the onset of shear, with NFκB returning to the cytoplasm after 2 h of continuous flow. NFκB translocation in response to shear was inhibited by the protease inhibitor, Nα -p-tosyl-L-lysine chloromethylketone hydrochloride (TLCK), or a cell-permeant peptide that blocks the nuclear localization sequence (NLS) on NFκB. Block of NFκB translocation with these inhibitors blocked the shear-induced upregulation of COX-2. We found that disruption of the actin cytoskeleton with cytochalasin D or microtubules with nocodozol did not alter NFκB translocation in response to shear. However, addition of the intracellular Ca2+ chelator BAPTA completely blocked NFκB translocation. While block of Ca2+ entry with channel blockers failed to inhibit NFκB translocation, inhibition of phospholipase C (PLC)-induced intracellular Ca2+ release with the PLC inhibitor U73122 completely abrogated the NFκB response to shear. These data indicate that NFκB translocation to the nucleus is essential for the fluid shear-induced increase in COX-2. Further, these studies suggest that intracellular Ca2+ release, but not the cytoskeletal architecture, is important to NFκB translocation.
KW - Intracellular Ca release
KW - Mechanotransduction
KW - NFκB
KW - Osteoblasts
KW - Phospholipase C
UR - http://www.scopus.com/inward/record.url?scp=0042834331&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0042834331&partnerID=8YFLogxK
U2 - 10.1016/S8756-3282(03)00159-5
DO - 10.1016/S8756-3282(03)00159-5
M3 - Article
C2 - 13678782
AN - SCOPUS:0042834331
VL - 33
SP - 399
EP - 410
JO - Bone
JF - Bone
SN - 8756-3282
IS - 3
ER -