Abstract
Background: Osteoporosis is a degenerative skeletal disease with a limited number of treatment options. CK2.3, a novel peptide, may be a potential therapeutic. It induces osteogenesis and bone formation in vitro and in vivo by acting downstream of BMPRIA through releasing CK2 from the receptor. However, the detailed signaling pathways, the time frame of signaling, and genes activated remain largely unknown. Methods: Using a newly developed fluorescent CK2.3 analog, specific inhibitors for the BMP signaling pathways, Western blot, and RT-qPCR, we determined the mechanism of CK2.3 in C2C12 cells. We then confirmed the results in primary BMSCs. Results: Using these methods, we showed that CK2.3 stimulation activated OSX, ALP, and OCN. CK2.3 stimulation induced time dependent release of CK2β from BMPRIA and concurrently CK2.3 colocalized with CK2α. Furthermore, CK2.3 induced BMP signaling depends on ERK1/2 and Smad1/5/8 signaling pathways. Conclusion: CK2.3 is a novel peptide that drives osteogenesis, and we detailed the molecular sequence of events that are triggered from the stimulation of CK2.3 until the induction of mineralization. This knowledge can be applied in the development of future therapeutics for osteoporosis.
Highlights
Osteoporosis is a skeletal disorder and is the leading cause of fractures in the USA. 10 million Americans are diagnosed with osteoporosis and another 44 million Americans are at an increased risk of a fracture due to their low bone density
Teriparatide, a recombinant human- parathyroid hormone, composed of 1–34 amino acid fragments of the parathyroid hormone (PTH) [13,14], is an anabolic treatment that addresses the issue of increasing bone mineral density (BMD) in osteoporotic patients, but excess concentration of serum PTH
We identified an interaction between protein kinase 2 (CK2) and bone morphogenetic protein receptor type IA (BMPRIA) and determined the site on BMPRIA between the amino acids 213–217 (SLKD) to be a potential CK2 phosphorylation site
Summary
Osteoporosis is a skeletal disorder and is the leading cause of fractures in the USA. 10 million Americans are diagnosed with osteoporosis and another 44 million Americans are at an increased risk of a fracture due to their low bone density. To determine the signaling pathway utilized by CK2.3 to induce osteoblast differentiation, C2C12 cells were either left unstimulated or stimulated with 100 nM of CK2.3 for 6 h, 12 h, and 18 h over days 1–5. C2C12 cells were either left unstimulated or stimulated with 500 nM of U0126-EtOH, 5 μM of MKK-2206 2HCl, and 10 μM of SB202190 to inhibit ERK1/2, Akt1/2/3, and p38 MAPK signaling pathways, respectively. Our Western blot data (Figure 4) revealed that stimulation of C2C12 cells with 100 nM of CK2.3 activated Smad1/5/8, ERK1/2, and Akt1/2/3 signaling pathways; this can be attributed to the fact that most signaling pathways share common mediators and there is extensive cross-talk amongst one another [55]. PPrrooppoosseedd mmeecchhaanniissmm ooff CCKK22..33 mmeeddiiaatteedd oosstteeooggeenneessiiss.. ((AA)) IInn uunnssttiimmuullaatteedd cceellllss,, pprrootteeiinn kkiinnaassee CCKK22 iiss hhyyppootthheessiizzeedd ttoo bbee iinntteerraaccttiinngg wwiitthh tthhee rreecceeppttoorr BBMMPPRRIIAA.. ((BB)) SSttiimmuullaattiioonn ooff cceellllss wwiitthh CCKK22..33 rreessuullttss iinn iittss iinntteerrnnaalliizzaattiioonn ssttaarrttiinngg aatt 66 hh vviiaa ccaavveeoollaaee--mmeeddiiaatteedd eennddooccyyttoossiiss.. ((CC)) CCKK22..33 tthheenn iinntteerraaccttss wwiitthh eennddooggeennoouuss CCKK22,, ccaauussiinngg iittss rreelleeaassee ffrroomm rreecceeppttoorr BBMMPPRRIIAA.. ((DD)) TThheerreebbyy aaccttiivvaattiinngg BBMMPPRRIIAA ddoowwnnssttrreeaamm ssiiggnnaalliinngg ppaatthhwwaayyss aanndd ggeenneess
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