Expression profile of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis: identification of essential genes in osteoclasts.
RhoGTPases regulate actin cytoskeleton dynamics, a key element in osteoclast biology. We identified three novel genes induced during RANKL-stimulated osteoclastogenesis among RhoGTPases and their exchange factors that are essential in osteoclast biology.


BACKGROUND
During the process of differentiation, adhesion to the bone matrix or osteolysis, the actin cytoskeleton of osteoclasts undergoes profound reorganization. RhoGTPases are key regulators of actin dynamics. They control cell adhesion, migration, and morphology through their action on actin cytoskeleton. In mice, there are 18 low molecular weight RhoGTPases. They are activated by guanine nucleotide exchange factors: the RhoGEFs. There are 76 RhoGEFs in mice: 65 belong to the Dbl family and 11 to the CZH family. To identify novel genes among RhoGTPases and RhoGEFs important in osteoclasts, we established the expression profiles of the complete families of RhoGTPases and RhoGEFs during RANKL-stimulated osteoclastogenesis.


METHODS
The RAW264.7 cell line, mouse bone marrow macrophages, and hematopoietic stem cells were used as precursors for RANKL-induced osteoclastogenesis. Gene arrays and real-time quantitative PCR analyses were performed to establish the transcription profiles of RhoGTPase and RhoGEF genes during differentiation. Small hairpin RNA was used to knock down genes of interest.


RESULTS
Of the 18 RhoGTPases and 76 RhoGEFs, the expression of three genes was upregulated by RANKL: the RhoGTPase RhoU/Wrch1, the Dbl family exchange factor Arhgef8/Net1, and the CZH family exchange factor Dock5. The inductions were observed in gene array and real-time quantitative PCR experiments performed in RAW264.7 cells. They were further confirmed in bone marrow macrophages and hematopoietic stem cells. Silencing of Wrch1 and Arhgef8 expression severely inhibited differentiation and affected osteoclast morphology. Dock5 suppression was lethal in osteoclast precursors while having no effect in fibroblasts.


CONCLUSIONS
We identified three genes among RhoGTPase signaling pathways that are upregulated during RANKL-induced osteoclastogenesis. These genes are novel essential actors in osteoclasts, most likely through the control of actin cytoskeleton dynamics.
Brazier
Hélène
H
Centre de Recherches en Biochimie Macromoléculaire, CNRS FRE 2593, Montpellier, France.
Stephens
Sébastien
S
Ory
Stéphane
S
Fort
Philippe
P
Morrison
Nigel
N
Blangy
Anne
A
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000494:pharmacology*; D002454:Cell Differentiation / Q000187:drug effects*; D020869:Gene Expression Profiling / Q000379:methods; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D010010:Osteoclasts / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C432912:DOK5 protein, human; D020662:Guanine Nucleotide Exchange Factors; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; C432668:RHOU protein, human; D020741:rho GTP-Binding Proteins