title: Proteomic analysis of lysosomal acid hydrolases secreted by osteoclasts: implications for lytic enzyme transport and bone metabolism.
abstract: Osteoclasts, the bone-digesting cells, are polarized cells that secrete acid hydrolases into a resorption lacuna where bone degradation takes place. The molecular mechanisms underlying this process are poorly understood. To analyze the nature of acid hydrolases secreted by osteoclasts, we used the mouse myeloid Raw 264.7 cell line that differentiates in vitro into mature osteoclasts in the presence of the receptor activator of NF-kappaB ligand. Upon differentiation, we observed a strong increase in the secretion of mannose 6-phosphate-containing acid hydrolases. A proteomic analysis of the secreted proteins captured on a mannose 6-phosphate receptor affinity column revealed 58 different proteins belonging to several families of acid hydrolases of which 16 are clearly involved in bone homeostasis. Moreover these acid hydrolases were secreted as proproteins. The expression of most of the identified acid hydrolases is unchanged during osteoclastogenesis. Thus, our data strongly support the notion that the polarized secretion of acid hydrolases by osteoclasts results from a reorganization of key steps of membrane traffic along the lysosomal pathway rather than from a fusion of lysosomes with the membrane facing the resorption lacuna.
authors.affiliation: Technical University of Dresden, 01307 Dresden, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Czupalla
authors.forename: Hannu
authors.initials: H
authors.lastname: Mansukoski
authors.forename: Thilo
authors.initials: T
authors.lastname: Riedl
authors.forename: Dorothee
authors.initials: D
authors.lastname: Thiel
authors.forename: Eberhard
authors.initials: E
authors.lastname: Krause
authors.forename: Bernard
authors.initials: B
authors.lastname: Hoflack
mesh_terms: D000818:Animals; D001842:Bone and Bones / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism; D002454:Cell Differentiation; D002846:Chromatography, Affinity; D020869:Gene Expression Profiling; D006867:Hydrolases / Q000032:analysis*; D008247:Lysosomes / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis; D010010:Osteoclasts / Q000201:enzymology*; D021381:Protein Transport; D040901:Proteomics; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D017527:Receptor, IGF Type 2; D011994:Recombinant Proteins; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D006867:Hydrolases; D002402:Cathepsin D