Regulation of osteoclast function by alpha gene tropomyosins, TM-2/3 and TM-5a/5b

Osteoclasts are large, multinucleated cells that are derived from differentiated macrophages and are mainly responsible for breakdown of bone (bone resorption). They are highly motile cells and adopt a polarized phenotype briefly during bone resorption. Because of the fluidity in cell shape associated with these processes, osteoclasts possess a dynamic cytoskeleton that contain specialized actin structures involved in motility (podosomes) and attachment during bone resorption (an F-actin ring). In our initial studies, we showed that various non-muscle tropomyosin isoforms are distributed to different intracellular locations where they regulate specific actin pools.;Results presented here describe the role that alpha gene tropomyosins, TM-2/3 and TM-5a/5b play in the regulation of mature osteoclast activity. TM-2 and -3 are closely related high molecular weight tropomyosins that differ from the low molecular weight TM-5a and -5b only by alternately spliced exons that encode the amino termini. However, this difference of 80 amino acid residues mediates markedly different patterns of expression, subcellular distribution, and function for these TMs. The high molecular weight TM-2/3 were not expressed in the monocytic and macrophage precursors of osteoclasts, but were strongly upregulated late in osteoclastogenesis. In contrast, low molecular weight TM-5a/5b were present in macrophage precursors and were modestly upregulated early during osteoclast formation. Further, TM-5a/b were strongly enriched in adhesion complexes (podosomes and actin ring) of osteoclasts, while TM-2/3 were distributed internally throughout the cells, and were not strongly associated with these actin-rich adhesion structures.;RNAi-mediated suppression of TM-2/3 caused increased spreading and flattening of the cells, accompanied by diminished motility and altered resorptive capacity, while overexpression of TM-2 caused decreased spreading and induction of actin patches or stress-fiber like actin filaments, also with effects on motility and resorption. These results demonstrated that TM-2/3 are expressed in fusing osteoclasts to regulate the cytoskeletal scaffolding of these large multinucleated cells. In contrast, suppression of TM-5a/b caused clustering and thickening of podosomes and thinning of actin ring/sealing zone. Live-cell imaging showed that the rate of appearance and disappearance of individual podosomes increased in TM-5a/5b siRNA-transfected osteoclasts, possibly explaining the increased osteopontin-mediated motility of these cells. In addition, there was overall less lateral movement of podosomes in the knockdown cells compared to the control.;On the other hand, adenovirus-mediated overexpression of TM-5a prevented formation of normal podosomes on glass and actin rings on bone. Instead TM-5a overexpressors contained an unusual looking thick ring of actin on glass, and patches and globules of actin on bone. Motility and resorption was also severely reduced in TM-5a overexpressing osteoclasts. These results indicate that TM-5a/5b plays an important role in regulating actin adhesion structures of osteoclasts, most likely by stabilizing the actin microfilaments present in podosomes and the sealing zone. Therefore, the alternately spliced exons that form the N-termini and upstream sequences in the alpha gene mediate differential patterns of expression, subcellular location, and function in osteoclasts.