Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function.
The uPAR and its ligand uPA are expressed by both osteoblasts and osteoclasts. Their function in bone remodeling is unknown. We report that uPAR-lacking mice display increased BMD, increased osteogenic potential of osteoblasts, decreased osteoclasts formation, and altered cytoskeletal reorganization in mature osteoclasts.


BACKGROUND
Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express uPAR and produce urokinase (uPA). The purpose of this study was to investigate the role of uPAR in bone remodeling.


METHODS
In vivo studies were performed in uPAR knockout (KO) and wildtype (WT) mice on a C57Bl6/SV129 (75:25) background. Bone mass was analyzed by pQCT. Excised tibias were subjected to mechanical tests. UPAR KO calvaria osteoblasts were characterized by proliferation assays, RT-PCR for important proteins secreted during differentiation, and immunoblot for activator protein 1 (AP-1) family members. In vitro osteoclast formation was tested with uPAR KO bone marrow monocytes in the presence of macrophage-colony stimulating factor (M-CSF) and RANKL. Phalloidin staining in osteoclasts served to study actin ring and podosome formation.


RESULTS
pQCT revealed increased bone mass in uPAR-null mice. Mechanical tests showed reduced load-sustaining capability in uPAR KO tibias. uPAR KO osteoblasts showed a proliferative advantage with no difference in apoptosis, higher matrix mineralization, and earlier appearance of alkaline phosphatase (ALP). Surface RANKL expression at different stages of differentiation was not altered. AP-1 components, such as JunB and Fra-1, were upregulated in uPAR KO osteoblasts, along with other osteoblasts markers. On the resorptive side, the number of osteoclasts formed in vitro from uPAR KO monocytes was decreased. Podosome imaging in uPAR KO osteoclasts revealed a defect in actin ring formation.


CONCLUSIONS
The defective proliferation and differentiation of bone cells, coincident with both aberrant expression of transcription factors and cytoskeletal organization, are typical uPAR-dependent molecular phenotypes, and we have now shown their function in osteoblasts and osteoclasts function in vivo.
Furlan
Federico
F
H San Raffaele Scientific Institute and Università Vita-Salute San Raffaele, Department of Molecular Biology and Functional Genomics, Milan, Italy.
Galbiati
Clara
C
Jorgensen
Niklas R
NR
Jensen
Jens-Erik B
JE
Mrak
Emanuela
E
Rubinacci
Alessandro
A
Talotta
Francesco
F
Verde
Pasquale
P
Blasi
Francesco
F
D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000166:cytology / Q000000981:diagnostic imaging / Q000502:physiology*; D017931:DNA Primers; D006706:Homeostasis / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D009929:Organ Size; D010006:Osteoblasts / Q000166:cytology*; D010010:Osteoclasts / Q000166:cytology*; D011956:Receptors, Cell Surface / Q000235:genetics / Q000502:physiology*; D055293:Receptors, Urokinase Plasminogen Activator; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014057:Tomography, X-Ray Computed
D017931:DNA Primers; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator