Synergistic effects of Nell-1 and BMP-2 on the osteogenic differentiation of myoblasts.
Osteogenesis is synergistically enhanced by the combined effect of complimentary factors. This study showed that Nell-1 and BMP-2 synergistically enhanced osteogenic differentiation of myoblasts and phosphorylated the JNK MAPK pathway. The findings are important because of the osteochondral specificity of Nell-1 signaling and the potential therapeutic effects of coordinated BMP-2 and Nell-1 delivery.


BACKGROUND
BMPs play an important role in the migration and proliferation of mesenchymal cells and have a unique ability to alter the differentiation of mesenchymal cells toward chondrogenic and osteogenic lineages. Signaling upstream of Cbfa1/Runx2, BMPs effects are not limited to cells of the osteoblast lineage. Thus, additional osteoblast-specific factors that could synergize with BMP-2 would be advantageous for bone regeneration procedures. NELL-1 (NEL-like molecule-1; NEL [a protein strongly expressed in neural tissue encoding epidermal growth factor like domain]) is a novel growth factor believed to preferentially target cells committed to the osteochondral lineage.


METHODS
C2C12 myoblasts were transduced with AdLacZ, AdNell-1, AdBMP-2, or AdNell-1+AdBMP-2 overexpression viruses. Effects were studied by cell morphology, alkaline phosphatase activity, osteopontin production, and MAPK signaling. Additionally, in a nude mouse model, viruses were injected into leg muscles, and new bone formation was examined after 2 and 8 wk.


RESULTS
C2C12 myoblasts co-transduced with AdNell-1+AdBMP-2 showed a synergistic effect on osteogenic differentiation as detected by alkaline phosphatase activity and osteopontin production. Nell-1 stimulation on AdNell-1 + AdBMP-2 preconditioned C2C12 cells revealed significant activation of the non-BMP-2 associated c-Jun N-terminal kinase (JNK) MAPK signaling pathway, but not the p38 or extracellular signal-regulated kinase (ERK1/2) MAPK pathways. Importantly Nell-1 alone did not induce osteogenic differentiation of myoblasts. In a nude mouse model, injection of AdNell-1 alone stimulated no bone formation within muscle; however, injection of AdNell-1+AdBMP-2 stimulated a synergistic increase in bone formation compared with AdBMP-2 alone.


CONCLUSIONS
These findings are important because of the confirmed osteochondral specificity of Nell-1 signaling and the potential therapeutic effects of enhanced BMP-2 action with coordinated Nell-1 delivery.
Cowan
Catherine M
CM
Department of Bioengineering, University of California, Los Angeles, California, USA.
Jiang
Xinquan
X
Hsu
Tiffany
T
Soo
Chia
C
Zhang
Beiji
B
Wang
Joyce Z
JZ
Kuroda
Shun'ichi
S
Wu
Benjamin
B
Zhang
Zhiyuan
Z
Zhang
Xinli
X
Ting
Kang
K
D000256:Adenoviridae / Q000235:genetics; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D055396:Bone Morphogenetic Protein 2; D052005:Bone Morphogenetic Protein Receptors, Type I / Q000378:metabolism; D052006:Bone Morphogenetic Protein Receptors, Type II / Q000378:metabolism; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006609:High Mobility Group Proteins / Q000032:analysis / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008297:Male; D051379:Mice; D008819:Mice, Nude; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010012:Osteogenesis / Q000502:physiology; D053495:Osteopontin / Q000378:metabolism; D051381:Rats; D055758:SOX9 Transcription Factor; D015398:Signal Transduction / Q000502:physiology; D014157:Transcription Factors / Q000032:analysis / Q000378:metabolism; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
C525719:Bmp2 protein, mouse; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D006609:High Mobility Group Proteins; C484480:Nell1 protein, rat; D009419:Nerve Tissue Proteins; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053495:Osteopontin; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C498215:Bmpr1b protein, mouse; C498219:Bmpr2 protein, mouse; D052005:Bone Morphogenetic Protein Receptors, Type I; D052006:Bone Morphogenetic Protein Receptors, Type II; D000469:Alkaline Phosphatase