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CD47–SIRPα: an interaction of importance for bone cell differentiation
Umeå University, Faculty of Medicine, Department of Odontology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
CD47–SIRPα : en interaktion av betydelse för skelettcellers differentiering (English)
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 [en]
CD47, SIRPalpha, osteoblast, osteoclast, SHP-2, Akt2
National Category
Basic Medicine
Research subject
cellforskning
Identifiers
URN: urn:nbn:se:umu:diva-85912ISBN: 978-91-7601-000-6 (print)OAI: oai:DiVA.org:umu-85912DiVA: diva2:696297
Public defence
2014-03-14, Sal B, 9 tr Tandläkarhögskolan, Norrlands universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2014-02-21 Created: 2014-02-13 Last updated: 2014-02-21Bibliographically approved
List of papers
1. Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha-interaction
Open this publication in new window or tab >>Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha-interaction
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2007 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 352, no 2, 444-448 p.Article in journal (Refereed) Published
Abstract [en]

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPalpha) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPalpha-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPalpha strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)+ osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47-/- mice were strongly reduced, and bones of CD47-/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPalpha interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47-/- mice.

Keyword
Animals, Antigens; CD47/*metabolism, Cell Differentiation, Cells; Cultured, Male, Mesenchymal Stem Cells/*cytology/*metabolism, Mice, Mice; Inbred C57BL, Osteoclasts/*cytology/*metabolism, Osteogenesis/*physiology, Protein Binding, Receptors; Immunologic/*metabolism
National Category
Cell and Molecular Biology Dentistry
Identifiers
urn:nbn:se:umu:diva-12288 (URN)10.1016/j.bbrc.2006.11.057 (DOI)17126807 (PubMedID)
Available from: 2007-11-27 Created: 2007-11-27 Last updated: 2014-02-21Bibliographically approved
2. Lack of CD47 impairs bone cell differentiation and results in an osteopenic phenotype in vivo due to impaired signal regulatory protein α (SIRPα) signaling
Open this publication in new window or tab >>Lack of CD47 impairs bone cell differentiation and results in an osteopenic phenotype in vivo due to impaired signal regulatory protein α (SIRPα) signaling
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2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 41, 29333-29344 p.Article in journal (Refereed) Published
Abstract [en]

Here, we investigated whether the cell surface glycoprotein CD47 was required for normal formation of osteoblasts and osteoclasts and to maintain normal bone formation activity in vitro and in vivo. In parathyroid hormone or 1α,25(OH)2-vitamin D3 (D3)-stimulated bone marrow cultures (BMC) from CD47(-/-) mice, we found a strongly reduced formation of multinuclear tartrate-resistant acid phosphatase (TRAP)(+) osteoclasts, associated with reduced expression of osteoclastogenic genes (nfatc1, Oscar, Trap/Acp, ctr, catK, and dc-stamp). The production of M-CSF and RANKL (receptor activator of nuclear factor κβ ligand) was reduced in CD47(-/-) BMC, as compared with CD47(+/+) BMC. The stromal cell phenotype in CD47(-/-) BMC involved a blunted expression of the osteoblast-associated genes osterix, Alp/Akp1, and α-1-collagen, and reduced mineral deposition, as compared with that in CD47(+/+) BMC. CD47 is a ligand for SIRPα (signal regulatory protein α), which showed strongly reduced tyrosine phosphorylation in CD47(-/-) bone marrow stromal cells. In addition, stromal cells lacking the signaling SIRPα cytoplasmic domain also had a defect in osteogenic differentiation, and both CD47(-/-) and non-signaling SIRPα mutant stromal cells showed a markedly reduced ability to support osteoclastogenesis in wild-type bone marrow macrophages, demonstrating that CD47-induced SIRPα signaling is critical for stromal cell support of osteoclast formation. In vivo, femoral bones of 18- or 28-week-old CD47(-/-) mice showed significantly reduced osteoclast and osteoblast numbers and exhibited an osteopenic bone phenotype. In conclusion, lack of CD47 strongly impairs SIRPα-dependent osteoblast differentiation, deteriorate bone formation, and cause reduced formation of osteoclasts.

Keyword
Bone, CD47, Cell Differentiation, Osteoblasts, Osteoclast, SIRPalpha, Stromal Cell
National Category
Dentistry
Identifiers
urn:nbn:se:umu:diva-82921 (URN)10.1074/jbc.M113.494591 (DOI)000330615300013 ()23990469 (PubMedID)
Available from: 2013-11-13 Created: 2013-11-13 Last updated: 2017-12-06Bibliographically approved
3. Reduced SIRPα phosphorylation and concommitant SHP-2–PI3K–Akt2 signaling decrease osteoblast differentiation
Open this publication in new window or tab >>Reduced SIRPα phosphorylation and concommitant SHP-2–PI3K–Akt2 signaling decrease osteoblast differentiation
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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:nbn:se:umu:diva-85948 (URN)
Available from: 2014-02-13 Created: 2014-02-13 Last updated: 2014-02-21Bibliographically approved

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Koskinen, Cecilia

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