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Reduced SIRPα phosphorylation and concommitant SHP-2–PI3K–Akt2 signaling decrease osteoblast differentiation
Umeå University, Faculty of Medicine, Department of Odontology.
Umeå University, Faculty of Medicine, Department of Odontology.
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(English)Article in journal (Other academic) Submitted
Abstract [en]

Normal differentiation of bone forming osteoblasts is a prerequisite for maintenance of skeletal health and is dependent on an intricate cellular signaling including the essential transcription factor Runx2. The cell surface glycoprotein CD47 and its receptor signal regulatory protein alpha (SIRPα) are suggested to regulate bone cell differentiation. In the present study, we investigated osteoblastic differentiation of bone marrow stromal cells from SIRPα mutant mice lacking the cytoplasmic signaling domain of SIRPα. Moreover, we compared downstream signaling events of SIRPα in wild-type and CD47-deficient mouse bone marrow stromal cells. SIRPα-mutant stromal cells showed significantly less expression of Runx2, Sp7 (osterix), Bglap (osteocalcin), and Akp1 (alkaline phosphatase) mRNA compared to stromal cells from wild-type mice. An impaired osteoblastogenesis in SIRPα-mutant cell cultures was demonstrated by lower alkaline phosphatase activity and less mineral formation compared to wild-type cultures. Western blot analyses showed that CD47 expression was required for Src homology-2 domain containing protein tyrosine phosphatase- 2 (SHP-2) to associate with SIRPa. As a result, SHP-2 and Akt2 in stromal cells from CD47 deficient mice were less phosphorylated, as compared to that in wild-type stromal cells. In conclusion, we here show that CD47-dependent SIRPα signaling through SHP-2–PI3K–Akt2 strongly influences osteogenic differentiation of bone marrow stromal cells.

National Category
Medical and Health Sciences
Research subject
cellforskning
Identifiers
URN: urn:nbn:se:umu:diva-85948OAI: oai:DiVA.org:umu-85948DiVA: diva2:696292
Available from: 2014-02-13 Created: 2014-02-13 Last updated: 2014-02-21Bibliographically approved
In thesis
1. CD47–SIRPα: an interaction of importance for bone cell differentiation
Open this publication in new window or tab >>CD47–SIRPα: an interaction of importance for bone cell differentiation
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[en]
CD47–SIRPα : en interaktion av betydelse för skelettcellers differentiering
Abstract [en]

Bone tissue is continuously remodeled by bone-forming osteoblasts and bone-resorbing osteoclasts, in processes tightly regulated by hormones, cytokines and growth factors. CD47, a ubiquitously expressed protein, and one of its ligands, signal-regulatory protein alpha (SIRPα), are two cell-surface proteins belonging to the immunoglobulin (Ig)-superfamily. The interaction between CD47 and SIRPα is important for, amongst other processes, the fusion of macrophages into giant cells, which are closely related to osteoclasts.

The aim of the present study was to gain knowledge about the role of CD47–SIRPα interaction and resultant downstream signaling pathways in bone cell differentiation, formation and function.

The addition of antibodies against CD47 or SIRPα inhibited the formation of multinucleated osteoclasts from bone marrow monocytes (BMMs) in culture. Moreover, a significant decrease in the number of osteoclasts was detected in CD47-/- BMM cultures compared to CD47+/+ cultures. In line with these in vitro results, we found fewer osteoclasts in vivo in the trabecular bone of CD47-/- mice, as compared to CD47+/+ bone. Interestingly, an extended analysis of the trabecular bone of CD47-/- mice revealed that the bone volume, mineralizing surface, mineral apposition rate, bone formation rate and osteoblast number were also significantly reduced compared with CD47+/+ mice, indicating the importance of CD47 in osteoblast differentiation. In vitro studies of bone marrow stromal (BMS) cells from CD47-/- mice or SIRPα-mutant mice (mice lacking the signaling domain of SIRPa) showed a blunted expression of osteoblast-associated genes. Moreover, these altered genotypes were associated with reduced activity of the bone mineralization-associated enzyme alkaline phosphatase as well as a reduced ability to form mineral. To reveal the molecular mechanisms by which CD47 activation of SIRPα is important for BMS cell differentiation, we studied signaling downstream of SIRPα in the absence of CD47. In BMS cells lacking CD47, a considerable reduction in the levels of tyrosine phosphorylated SIRPα was detected, and the subsequent recruitment of the Src-homology-2 (SH2) domain-containing protein tyrosine phosphatase (SHP-2)–phosphoinositide 3-kinase (PI3K)–Akt2 signaling module was nearly abolished.

In conclusion, the interaction between CD47 and SIRPα results in the activation of the SHP-2–PI3K–Akt2 pathway, which is necessary for normal osteoblast differentiation. In CD47-/- mice and SIRPα-mutant mice, this interaction is perturbed, which prevents normal osteoblast differentiation and subsequent mineral formation. In addition, the altered BMS cell phenotype results in an impaired ability to stimulate osteoclast differentiation. 

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2014. 50 p.
Series
Umeå University odontological dissertations, ISSN 0345-7532 ; 132
Keyword
CD47, SIRPalpha, osteoblast, osteoclast, SHP-2, Akt2
National Category
Basic Medicine
Research subject
cellforskning
Identifiers
urn:nbn:se:umu:diva-85912 (URN)978-91-7601-000-6 (ISBN)
Public defence
2014-03-14, Sal B, 9 tr Tandläkarhögskolan, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
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Available from: 2014-02-21 Created: 2014-02-13 Last updated: 2014-02-21Bibliographically approved

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Koskinen, CeciliaSulniute, RimaOldenborg, Per-ArneLundberg, Pernilla
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