Elsevier

Bone

Volume 35, Issue 1, July 2004, Pages 74-82
Bone

Dentin matrix protein 1 expression during osteoblastic differentiation, generation of an osteocyte GFP-transgene

https://doi.org/10.1016/j.bone.2004.03.006Get rights and content

Abstract

Our previous studies have demonstrated that promoter-green fluorescent protein (GFP) transgenes can be used to identify and isolate populations of cells at the preosteoblastic stage (pOBCol3.6GFP) and at the mature osteoblastic stage (pOBCol2.3GFP) in living primary bone cell cultures. This strategy forms the basis for appreciating the cellular heterogeneity of lineage and relating gene function to cell differentiation. A weakness of this approach was the lack of a selective marker for late osteoblasts and mature osteocytes in the mineralized matrix.

In this study, we have examined the expression of DMP-1 mRNA in murine marrow stromal and calvarial osteoblast cultures, and in bone, and calvaria in vivo. Furthermore, we have generated transgenic mice utilizing a mouse DMP1 cis-regulatory system to drive GFP as a marker for living osteocytes. Transgene expression was directed to mineralized tissues and showed a high correlation with the expression of the endogenous gene. Osteocyte-restricted expression of GFP was observed in histological sections of femur and calvaria and in primary cell cultures. Generation of this transgenic model will facilitate studies of gene expression and biological functions in these terminally differentiated bone cells.

Introduction

Osteocytes, the cells embedded within the bone matrix, represent the largest cell population in bone and play important roles in bone biology and metabolism. They are originally derived from matrix-producing osteoblasts, which become embedded within the mineralizing matrix, take on a more dendritic phenotype, and decrease their synthetic output. One of the main morphological characteristics of osteocytes is the presence of numerous elongated cell processes or dendrites that lie in the channels or canaliculi throughout the bone. These processes connect to other osteocytic extensions, or to osteoblasts and lining cells on the endosteal and periosteal bone surface, thereby generating a complex communication network between different stages of cells within the osteoblast–osteocyte lineage. Due to their morphological characteristics and to their ability to sense mechanical stimulation, they have been called “nerve” cells of the bone [1], [2].

Studies of osteocyte biology have been hampered by their inaccessibility and by the lack of molecular and cell surface markers that could be used to isolate and characterize this cell population. A few cell lines, such as HOB-01-C1 and MLO-Y4, exhibit the osteocytic characteristics [3], [4]. However, rather than showing an absence of proliferation, which is one of main features of the osteocyte, these cell lines proliferate and express other markers that are not osteocyte-specific. In some cases, they do not synthesize significant levels of proteins that are known to be made by osteocyte in vivo. This indicates a potentially severe limitation of these particular cell lines.

Cell surface markers that have been useful in identifying immature progenitors (STRO-1, ALCAM, HOP-26) have not been effective in identifying matrix-embedded cells. Promoter-GFP reporter transgenic mice have been developed and used to identify matrix-embedded cells in cell culture and in histological sections of bone (pOBCol3.6GFP, pOBCol2.3GFP, and OC-GFP) [5], [6], [7], [8], [9], [10]. While these constructs identified subpopulations of osteoblast lineage cells, none of them showed expression restricted to osteocytes. Identifying an osteocyte-specific surface marker and generating a promoter transgene construct that would activate selectively in osteocytes would facilitate many experimental approaches for studying this cell type, both in in vivo and in in vitro systems.

Previous studies have described dentin matrix protein (DMP-1) as being restricted to mineralized tissues in rat and chicken and in the development of mouse bone [11], [12]. DMP1 gene is also responsive to mechanical stimulation in both the tooth movement model and in the rat ulnae loading models [13], [14]. The present study compares the expression of DMP-1 in adult murine long bone, mandible, and calvaria with DMP-1 expression during osteoblastic differentiation in primary bone cell cultures. Based on these results and others, we began to test various regions within the DMP1 promoter that regulates its osteocyte-specific expression. Several fragments ranging in size from 12 to 6.5 kb, which include all of intron 1, were stably transfected into osteoblastic cell line. One construct that encompassed a region from about −8 to +4 kb was preferentially expressed in mineralizing nodules in vitro and represented a good candidate for in vivo osteocyte-specific expression (submitted, [15]). To explore the possible use of this region of the DMP1 promoter to study the osteoblast–osteocyte lineage and target genes to the osteocyte, we have generated transgenic mice utilizing this −8 kb to +4 kb fragment of the mouse DMP1 promoter to drive GFP and demonstrated that this region of the DMP1 gene contains osteocyte-specific control elements that permit selective expression. Based on the expression pattern of this real time marker, we should be able to isolate osteocytes for further in vitro and in vivo studies.

Section snippets

RNA extraction and Northern blot analysis

Animals were sacrificed with CO2 asphyxiation and excised soft tissues were immediately frozen in liquid nitrogen in a 15-ml polypropylene tube (Falcon Cat 2059). The epiphyseal portions of the long bones (tibia and femur) were cleaned of attached muscle and the marrow flushed using a 25-gauge needle before freezing. Frozen samples, including soft tissues, were suspended in 4 ml of TRI Reagent (Molecular Research Center, Inc.) and homogenized as described below. Long bone samples were crushed

Analysis of DMP-1 mRNA expression

We analyzed the expression of DMP-1 by Northern blot analysis of various mineralized and soft tissues. Strong DMP-1 expression was observed in bones and calvaria, while a weak but detectable signal was found in brain. Besides the expression in brain, DMP-1 was restricted to tissues that expressed osteocalcin, while the other Col1a1-expressing tissues (tendon, skin, aorta, bladder, lung, ovary) did not have any detectable DMP-1 expression (Fig. 1).

To localize DMP-1 expression to a specific cell

Discussion

Osteoblast lineage heterogeneity has been well described and can be thought of in terms of vertical (stage of differentiation) and horizontal heterogeneity (different populations of cell within the same lineage stage). A number of approaches to overcome this obstacle while studying the osteoblast growth and differentiation have been employed. Partial success in identifying different populations of cells has been achieved using in situ hybridization and antibodies to matrix proteins, and more

Acknowledgements

Supported by NIH grant AR43457 and DK 63478 to D.W. Rowe. IK holds a fellowship from Children Brittle Bone Foundation and S.E. Harris is supported by NIH grants AR44728 and AR46798. This work is also supported by grant from Patterson Trust and NIH grant DE13363 to M.M.

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