Kristen Lee
2/21/11
Measurement of Osteocyte-specific Markers
Perform reverse transcription and real-time PCR using Jacobs lab protocols
PRIMERS (1)
DMP1:
5’- GGCTGTCCTGTGCTCTCCCAG-3’ and 5’-GGTCACTATTTGCCTGTGCCTC-3’
Sclerostin*:
5’-CTTCAGGAATGATGCCACAGAGGT-3’ and 5’-ATCTTTGGCGTCATAGGGATGGTG-3’
MEPE:
5’-AGGACCCAAGGGCAAGAG-3’ and 5’-TGTCTTCATTCGGCATTGG-3’
Phex:
5’-GTGCATCTACCAACCAGATACG-3’ and 5’-TCTGTTCCCCAAAAGAAAGG-3’
E11/gp38
5’-CAGTGTTGTTCTGGGTTTTGG-3’ and 5’-TGGGGTCACAATATCATCTTCA-3’
ALP:
5’-CAGGGTACACCATGATCTCACC-3’ and 5’-CGCCCATACCATCTCCCAGG-3’
EF-1a:
5’-TGCTGCCATTGTTGATATGG-3’ and 5’-TCCACAGCTTTGATGACACC-3’
Notes:
E11/gp38- the earliest osteocyte-selective protein to be expressed as the osteoblast differentiates into an osteoid cell or osteocyte, first appearing on the forming dendritic processes of these cells (2)
DMP1- dentin matrix protein 1, although DMP1 is expressed in all tissues that undergo mineralization, its expression in osteocytes is much higher than in any other cell type (3)
* Sclerostin-a secreted glycoprotein, is produced at or after primary mineralization by osteocytes in newly forming osteons to restrict bone formation by osteoblasts.a sufficient but not excessive amount of cortical bone may be formed to fill in osteonal and other canals without compressing their contents. Likewise it is envisaged that trabeculae are maintained at appropriate thickness (4)
-not expressed by MLO-Y4 cells
-may not be secreted by primary osteocytes then if not sufficiently mineralizing?
MEPE- Matrix extracellular phosphoglycoprotein, associated with bone mineralization and is highly expressed by osteocytes (5)
Measurement of Osteoblast-specific Markers
ALP- Alkaline phosphatase, in bone, is produced specifically by osteoblasts allowing calcium to precipitate to build bone
-there should be low expression of ALP in MLO-Y4s and primary osteocytes
mRNA was normalized to EF-1a mRNA- eukaryotic elongation factor I, functions in the elongation step of protein synthesis, where it mediates the binding of a charged tRNA molecule into the P site of the ribosome
References:
(1) Tatsumi, et al. “Targeted Ablation of Osteocytes Induces Osteoporosis with Defective Mechanotransduction” Cell Metabolism, 2007; 5(6) 464-475.
For quantitative RT-PCR, total RNA (1 mg) was reverse transcribed using SuperScript III (Invitrogen), and samples were analyzed using a LightCycler system (Roche Diagnostics).
(2) Zhang, et al. “E11/gp38 Selective Expression in Osteocytes: Regulation by Mechanical Strain and Role in Dendrite Elongation” Molecular and Cellular Biology, 2006; 26(12) 4539-4552.
(3) Rios, et al. “DMP1 is essential for osteocyte formation and function” J Musculoskelet Neuronal Interact 2005; 5(4):325-327
(4) Poole, et al. “Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation” PNAS, 2005; 19, 1842-1844.
(5) Nampei, et al. “Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone” J Bone Miner Metab (2004) 22:176–184.