Identification of differentially expressed genes between osteoblasts and osteocytes.
Osteocytes represent the most abundant cellular component of mammalian bones with important functions in bone mass maintenance and remodeling. To elucidate the differential gene expression between osteoblasts and osteocytes we completed a comprehensive analysis of their gene profiles. Selective identification of these two mature populations was achieved by utilization of visual markers of bone lineage cells. We have utilized dual GFP reporter mice in which osteocytes are expressing GFP (topaz) directed by the DMP1 promoter, while osteoblasts are identified by expression of GFP (cyan) driven by 2.3 kb of the Col1a1 promoter. Histological analysis of 7-day-old neonatal calvaria confirmed the expression pattern of DMP1GFP in osteocytes and Col2.3 in osteoblasts and osteocytes. To isolate distinct populations of cells we utilized fluorescent activated cell sorting (FACS). Cell suspensions were subjected to RNA extraction, in vitro transcription and labeling of cDNA and gene expression was analyzed using the Illumina WG-6v1 BeadChip. Following normalization of raw data from four biological replicates, 3444 genes were called present in all three sorted cell populations: GFP negative, Col2.3cyan(+) (osteoblasts), and DMP1topaz(+) (preosteocytes and osteocytes). We present the genes that showed in excess of a 2-fold change for gene expression between DMP1topaz(+) and Col2.3cyan(+) cells. The selected genes were classified and grouped according to their associated gene ontology terms. Genes clustered to osteogenesis and skeletal development such as Bmp4, Bmp8a, Dmp1, Enpp1, Phex and Ank were highly expressed in DMP1topaz(+)cells. Most of the genes encoding extracellular matrix components and secreted proteins had lower expression in DMP1topaz(+) cells, while most of the genes encoding plasma membrane proteins were increased. Interestingly a large number of genes associated with muscle development and function and with neuronal phenotype were increased in DMP1topaz(+) cells, indicating some new aspects of osteocyte biology. Although a large number of genes differentially expressed in DMP1topaz(+) and Col2.3cyan(+) cells in our study have already been assigned to bone development and physiology, for most of them we still lack any substantial data. Therefore, isolation of osteocyte and osteoblast cell populations and their subsequent microarray analysis allowed us to identify a number or genes and pathways with potential roles in regulation of bone mass.
Paic
Frane
F
Department of Reconstructive Sciences, MC 3705, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06032, USA.
Igwe
John C
JC
Nori
Ravi
R
Kronenberg
Mark S
MS
Franceschetti
Tiziana
T
Harrington
Patrick
P
Kuo
Lynn
L
Shin
Dong-Guk
DG
Rowe
David W
DW
Harris
Stephen E
SE
Kalajzic
Ivo
I
D000818:Animals; D000831:Animals, Newborn; D002462:Cell Membrane / Q000378:metabolism; D002469:Cell Separation; D005109:Extracellular Matrix / Q000235:genetics; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009132:Muscles / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010011:Osteocytes / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012886:Skull / Q000166:cytology / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins