The Rho GTPase Wrch1 regulates osteoclast precursor adhesion and migration

Brazier, Hélène; Pawlak, Geraldine; Vives, Virginie; Blangy, Anne

doi:10.1016/j.biocel.2008.12.007

Cited by 72 publications

(47 citation statements)

References 50 publications

(94 reference statements)

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“…(18) Microscopy, immunofluorescence, and TRACP labeling OCs were fixed and stained for DNA, actin, or vinculin or TRAP as described previously. (16,19) Anti-vinculin antibody (Sigma, St Louis, MO, USA) was revealed with Alexa Fluor 546-conjugated secondary antibody and actin stained with Alexa Fluor 360-or 488-conjugated phalloidin (Invitrogen, Carlsbad, CA, USA). Preparations were mounted in Mowiol 40-88 (Sigma) and imaged with a Zeiss Axioimager Z2 microscope with Coolsnap HQ2 camera for fluorescence and Coolsnap color Cf camera using a Zeiss 40Â PLAN-NEOFLUAR 1.3 oil DIC or Zeiss 20 Â PLAN-APOCHROMAT 0.8 or Zeiss 10 Â EC PLAN-NEOFLUAR 0.3 (Zeiss, Inc., Thornwood, NY, USA).…”

Section: Production Of Ocs and Osteoblastsmentioning

confidence: 99%

“…Wells then were rinsed with PBS, fixed, stained with 0.1% crystal violet, and lysed, and OD595 was measured as described previously. (19) Each condition was performed in triplicate wells.…”

Section: Adhesion Assaysmentioning

confidence: 99%

“…(16,19) RT-PCR and primers Reverse-transcriptase polymerase chain reaction (RT-PCR) analyses were performed as described previously. (19) Amplification primers were 5'-GGC TGT GTT TAC CGA CGA GC-3' and 5'-CAA GCA CGC GGA CAA TGT TG-3' for calcitonin receptor, 5'-TCA GCT TCA GCA TTC AGC CC-3' and 5'-ACT GCA CGA TTC CAG AGT CC-3' for Dock1 and 5'-AGC CTT GCA TCT CCT GTG GC-3' and 5'-CAT GCG TCC CTT GGA TGC TG-3' for Dock2, 5'-GCG CTC TGT CTC TCT GAC CT-3' and 5'-GCC GGA GTC TGT TCA CTA CC-3' for osteocalcin, 5'-AAT GCC CTG AAA CTC CAA AA-3' and 5'-AGG GGA ATT TGT CCA TCT CC-3' for alkaline phosphatase, and 5'-TGT TCA GCT TTG TGG ACC TC-3' and 5'-TCA AGC ATA CCT CGG GTT TC-3' for collagen 1. Other primers were described: Gapdh, Dock5, Vav3, Src, TRACP, NFATc1, CtsK, integrin b3, DC-STAMP, ATP6v0d2, (16,19) OSCAR, CD44, (6) and ADAM8.…”

Section: Plasmid Dnasmentioning

confidence: 99%

“…(19) Amplification primers were 5'-GGC TGT GTT TAC CGA CGA GC-3' and 5'-CAA GCA CGC GGA CAA TGT TG-3' for calcitonin receptor, 5'-TCA GCT TCA GCA TTC AGC CC-3' and 5'-ACT GCA CGA TTC CAG AGT CC-3' for Dock1 and 5'-AGC CTT GCA TCT CCT GTG GC-3' and 5'-CAT GCG TCC CTT GGA TGC TG-3' for Dock2, 5'-GCG CTC TGT CTC TCT GAC CT-3' and 5'-GCC GGA GTC TGT TCA CTA CC-3' for osteocalcin, 5'-AAT GCC CTG AAA CTC CAA AA-3' and 5'-AGG GGA ATT TGT CCA TCT CC-3' for alkaline phosphatase, and 5'-TGT TCA GCT TTG TGG ACC TC-3' and 5'-TCA AGC ATA CCT CGG GTT TC-3' for collagen 1. Other primers were described: Gapdh, Dock5, Vav3, Src, TRACP, NFATc1, CtsK, integrin b3, DC-STAMP, ATP6v0d2, (16,19) OSCAR, CD44, (6) and ADAM8. (24) Active Rac1 quantification GTP-bound Rac1 was pulled down from OC or 293T cell extracts using GST-fused PAK1 CRIB domain as described previously (22) and revealed by Western blot with monoclonal anti-Rac1 antibodies (Transduction Laboratories, Le Pont de Claix, France) and anti-GFP polyclonal antibodies (Torrey Pines Biolabs, Houston, TX, USA).…”

Section: Plasmid Dnasmentioning

confidence: 99%

“…(19) The entire well surface was imaged using a Zeiss AxioimagerZ1 microscope. The well image was reconstituted, and resorbed areas were measured using MetaMorph 7.0 software (Molecular Devices).…”

Section: Resorption Assaysmentioning

confidence: 99%

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The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts

Vives

Laurin

Crès

et al. 2010

Journal of Bone and Mineral Research

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106

148

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Osteoporosis, which results from excessive bone resorption by osteoclasts, is the major cause of morbidity for elder people. Identification of clinically relevant regulators is needed to develop novel therapeutic strategies. Rho GTPases have essential functions in osteoclasts by regulating actin dynamics. This is of particular importance because actin cytoskeleton is essential to generate the sealing zone, an osteoclast-specific structure ultimately mediating bone resorption. Here we report that the atypical Rac1 exchange factor Dock5 is necessary for osteoclast function both in vitro and in vivo. We discovered that establishment of the sealing zone and consequently osteoclast resorbing activity in vitro require Dock5. Mechanistically, our results suggest that osteoclasts lacking Dock5 have impaired adhesion that can be explained by perturbed Rac1 and p130Cas activities. Consistent with these functional assays, we identified a novel small-molecule inhibitor of Dock5 capable of hindering osteoclast resorbing activity. To investigate the in vivo relevance of these findings, we studied Dock5 -/-mice and found that they have increased trabecular bone mass with normal osteoclast numbers, confirming that Dock5 is essential for bone resorption but not for osteoclast differentiation. Taken together, our findings characterize Dock5 as a regulator of osteoclast function and as a potential novel target to develop antiosteoporotic treatments. ß

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Section: Production Of Ocs and Osteoblastsmentioning

confidence: 99%

“…Wells then were rinsed with PBS, fixed, stained with 0.1% crystal violet, and lysed, and OD595 was measured as described previously. (19) Each condition was performed in triplicate wells.…”

Section: Adhesion Assaysmentioning

confidence: 99%