Establishment of an Osteocyte-like Cell Line, MLO-Y4

Abstract

Although osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T-antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen-coated surfaces which are optimal for osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO-Y4 (for murine long bone osteocyte Y4). The properties of MLO-Y4 cells are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline-phosphatase. The cells produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on osteocyte function and for determining the means whereby osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.

Mechanism of FGF23 processing in fibrous dysplasia

“…(28) Although Western blotting demonstrated that MLO-Y4 cells produce FGF23, mouse FGF23 migrated faster than human FGF23 and failed to show any processing to cFGF23, suggesting that in mice FGF23 may be differently processed. In addition, in spite of the fact that FGF23 could be detected by Western blot, it could not be detected by the intact FGF23 assay, which is known to detect mouse FGF23 (data not shown).…”

Osteocyte Remodeling of the Perilacunar and Pericanalicular Matrix

“…Establishment of a cell line, MLO-Y4, captured the imagination of many investigators who now had access to a osteocyte-like cell (Kato et al, 1997) to examine osteocytic response to mechanical loading in the form of shear stress, osteocyte apoptosis, osteocyte signaling, and osteocyte communication through gap junctions and hemichannels (Bonewald, 2007). Breakthroughs were made in the identification of important markers for osteocyte differentiation which included E11/gp38，an early marker for embedding cells, Phex and dentin matrix protein 1, DMP1, important in metabolism, and Sost/sclerostin, a late marker for mature osteocytes (Bonewald, 2007).…”

Hypoxia mediates osteocyte ORP150 expression and cell death in vitro

“…The murine long bone osteocyte Y4 (MLO-Y4) cell line (supplied from University of Missouri, Kanas City, USA) was used and cultured as previously described (36,37). Briefly, the cells were cultured on collagen-coated (rat tail type I collagen; BD Biosciences, Franklin Lakes, NJ, USA) plastic ware and were grown at 37˚C, 5% CO 2 , 95% air with α-minimal essential medium (α-MEM; 1X; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 2.5% fetal bovine serum (FBS) (PAA; GE Healthcare Life Sciences, Pittsburgh, PA, USA), 2.5% bovine calf serum (BCS; GE Healthcare Life Sciences, Logan, UT, USA) and 100 U/ml penicillin/streptomycin at (Invitrogen; Thermo Fisher Scientific, Inc).…”