Abstract
Determining the underlying mechanisms of macrophage colony-stimulating factor (M-CSF)-mediated osteoclast survival may be important in identifying novel approaches for treating excessive bone loss. This study investigates M-CSF-mediated MEK/ERK activation and identifies a downstream effector of this pathway. M-CSF activates MEK/ERK and induces MEK-dependent expression of the immediate early gene Egr2. Inhibition of either MEK1/2 or inhibition of Egr2 increases osteoclast apoptosis. In contrast, wild-type Egr2 or an Egr2 point mutant unable to bind the endogenous repressors Nab1/2 (caEgr2) suppresses basal osteoclast apoptosis and rescues osteoclasts from apoptosis induced by MEK1/2 or Egr2 inhibition. Mechanistically, Egr2 induces pro-survival Blc2 family member Mcl1 while stimulating proteasome-mediated degradation of pro-apoptotic Bim. In addition, Egr2 increased the expression of c-Cbl, the E3 ubiquitin ligase that catalyzes Bim ubiquitination. M-CSF, therefore, promotes osteoclast survival through MEK/ERK-dependent induction of Egr2 to control the Mcl1/Bim ratio, documenting a novel function of Egr2 in promoting survival.
Highlights
MARCH 21, 2008 VOLUME 283 NUMBER 12 oclast differentiation from hematopoietic cells within the monocyte/macrophage lineage [2, 3]
macrophage colony-stimulating factor (M-CSF) promotes the survival of cells within the monocyte/macrophage cell lineage, including boneresorbing osteoclasts
M-CSF-induced osteoclast survival may contribute toward the treatment of conditions resulting in increased bone loss such as osteoporosis and tumor-induced bone loss
Summary
All chemical were purchased form Sigma-Aldrich. Osteoclast Differentiation—Mouse marrow osteoclast precursors were obtained from female Balb/c mice (The Jackson Laboratory, Bar Harbor, ME) as previously described [23]. Osteoclasts were harvested immediately for total RNA or cultured with 25 ng/ml M-CSF for the indicated period, rinsed with phosphate-buffered saline, and harvested for total RNA. Osteoclasts were either harvested immediately for Western blotting or cultured with 25 ng/ml M-CSF for the indicated time period, rinsed with phosphate-buffered saline, and harvested for Western blotting. The resulting supernatant was used in a Western blot for ubiquitin and stripped and reprobed for total Bim. Apoptosis Detection—Mature osteoclasts were serum-starved for 60 min and treated with 25 ng/ml. Western blotting for phospho- and total ERK in osteoclasts treated with M-CSF in the presence of UO126 or vehicle control. Osteoclasts were serum-starved and cultured with M-CSF as indicated in the presence of the MEK1/2 inhibitor UO126 or vehicle control or no treatment.
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