Expression Profile of RhoGTPases and RhoGEFs During RANKL‐Stimulated Osteoclastogenesis: Identification of Essential Genes in Osteoclasts

• Published in: Journal of bone and mineral research 2006-09, Vol.21 (9), p.1387-1398
• Main Authors: Brazier, Hélène, Stephens, Sébastien, Ory, Stéphane, Fort, Philippe, Morrison, Nigel, Blangy, Anne
• Format: Article
• Language: English
• Subjects: actin; Adaptor Proteins, Signal Transducing; Adaptor Proteins, Signal Transducing - metabolism; Animals; Biochemistry, Molecular Biology; Biodiversity; Biological and medical sciences; bone; Cancer; Carrier Proteins; Carrier Proteins - pharmacology; Cell Differentiation; Cell Differentiation - drug effects; Cellular Biology; Development Biology; Embryology and Organogenesis; Fundamental and applied biological sciences. Psychology; gene array; Gene Expression Profiling; Gene Expression Profiling - methods; Guanine Nucleotide Exchange Factors; Guanine Nucleotide Exchange Factors - metabolism; Humans; Life Sciences; Membrane Glycoproteins; Membrane Glycoproteins - pharmacology; Mice; Mice, Inbred C57BL; Molecular biology; NIH 3T3 Cells; osteoclast; Osteoclasts; Osteoclasts - metabolism; Phosphoproteins; Phosphoproteins - metabolism; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; rho GTP-Binding Proteins; rho GTP-Binding Proteins - metabolism; Skeleton and joints; Subcellular Processes; Systematics, Phylogenetics and taxonomy; transcription; Vertebrates: osteoarticular system, musculoskeletal system
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1359/jbmr.060613

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. Introduction: 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. Materials and 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.