Dentin matrix protein 1 gene cis-regulation: use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo.
Dentin matrix protein 1 (DMP1) is highly expressed in osteocytes and is mechanically responsive. To study osteocyte-specific and mechanically regulated DMP1 gene expression, the transcriptional activity of three cis-regulatory regions was first examined in an osteoblast differentiation model in vitro using a green fluorescent protein (GFP) reporter. Expression of the -9624 to +1996 bp (10 kb) and -7892 to +4439 bp (8 kb) DMP1 cis-regulatory regions dramatically increased in areas of mineralized matrix, in dendritic, osteocyte-like cells. Mineralizing cultures expressing the 8-kb construct show dramatic GFP increases in response to loading in cells with a dendritic morphology. Transgenic mice expressing the 8-kb DMP1-GFP and -2433 to +4439 bp (2.5 kb) DMP1-LacZ were generated. Osteocyte-specific expression was found with the 8 kb but not with the 2.5 kb in postnatal animals. However, the 2.5 kb could support expression in rapidly forming osteoblasts and pre-osteocytes in the embryo. Primary calvarial osteoblast cultures demonstrated that the 2.5 kb supports weak expression in a subset of osteoblasts and pre-osteocytes, but not in mature osteocytes. However, the 8 kb supports robust expression in primary bone marrow cultures. Therefore the region -7892 to -2433 bp, termed a 5.5-kb "Osteocyte Enhancer Module," appears to be required for osteocyte specificity. Ulnae of mice with the 8-kb DMP1-GFP were subjected to mechanical loading where GFP expression increased selectively and locally in osteocytes, distal to the mid-shaft and near the surface of the bone. Thus, the 8-kb region of the DMP1 gene is a target for mechanotransduction in osteocytes, and its cis-regulatory activity may be correlated to local strain in bone.
Yang
Wuchen
W
Department of Periodontics and Cellular and Structural Biology, University of Texas, San Antonio, Texas 78229, USA.
Lu
Yongbo
Y
Kalajzic
Ivo
I
Guo
Dayong
D
Harris
Marie A
MA
Gluhak-Heinrich
Jelica
J
Kotha
Shiva
S
Bonewald
Lynda F
LF
Feng
Jian Q
JQ
Rowe
David W
DW
Turner
Charles H
CH
Robling
Alexander G
AG
Harris
Stephen E
SE
D000818:Animals; D001696:Biomechanical Phenomena; D001854:Bone Marrow Cells; D001842:Bone and Bones / Q000502:physiology; D002454:Cell Differentiation; D002460:Cell Line; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D008822:Mice, Transgenic; D010006:Osteoblasts; D010011:Osteocytes / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid; D013314:Stress, Mechanical; D014162:Transfection; D014457:Ulna; D001616:beta-Galactosidase / Q000235:genetics
C513309:DMP1 protein, human; C513308:Dmp1 protein, mouse; D016326:Extracellular Matrix Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase