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.

Osteoblast cell lines capable of undergoing bone formation in vitro would provide useful models for understanding gene expression during bone cell differentiation. To that end, transgenic mice were produced using a 2.9-kilobase bone morphogenetic protein 2 (BMP-2) promoter fragment, driving simian virus 40 T antigen as the transgene. The expression of simian virus 40 T antigen driven by the BMP-2 promoter immortalizes the cells. From the calvaria of the transgenic mouse, several osteoblastic cell lines were isolated and cloned. One clonal osteoblast cell line, called 2T3, has been characterized and shown to produce mineralized bone nodules. Recombinant human BMP-2 (rhBMP-2) accelerates the formation of these mineralized bone nodules. 2T3 cells express alkaline phosphatase, collagen type I, osteocalcin, and endogenous BMP-2 messenger RNA (mRNA) in a similar chronological order as normal freshly isolated fetal rat calvarial cells during early nodule formation and subsequent mineralization. The 2T3 cells also exhibit extensive growth and multilayering during differentiation, as demonstrated by growth curves and transmission electron microscopy. As with freshly isolated fetal rat calvarial cells, 1,25-dihydroxyvitamin D3 inhibited alkaline phosphatase activity and alkaline phosphatase mRNA expression, but stimulated osteocalcin mRNA expression, but stimulated osteocalcin mRNA expression. rhBMP-2 also accelerated the expression of alkaline phosphatase activity and mRNA, osteocalcin mRNA, and BMP-2 mRNA in 2T3 cells along with the formation of larger and more mineralized bone nodules. The 2T3 cell exhibits autoregulation at the mRNA and transcriptional levels. The 2T3 osteoblast cell line offers a system for examining autoregulation of the BMP-2 gene and downstream gene expression during osteoblast differentiation. 2T3 cells are reclonable and maintain their differentiation capabilities.

Cellular and molecular characterization of osteoclasts (OCL) has been extremely difficult since OCL are rare cells, and are difficult to isolate in large numbers. We used the tartrate-resistant acid phosphatase promoter to target the bcl -X L and/or Simian Virus 40 large T antigen (Tag) genes to cells in the OCL lineage in transgenic mice as a means of immortalizing OCL precursors. Immunocytochemical studies confirmed that we had targeted Bcl-X L and/or Tag to OCL, and transformed and mitotic OCL were readily apparent in bones from both Tag and bcl -X L /Tag mice. OCL formation in primary bone marrow cultures from bcl -X L , Tag, or bcl -X L /Tag mice was twofold greater compared with that of nontransgenic littermates. Bone marrow cells from bcl -X L /Tag mice, but not from singly transgenic bcl -X L or Tag mice, have survived in continuous culture for more than a year. These cells form high numbers of bone-resorbing OCL when cultured using standard conditions for inducing OCL formation, with ‫ف‬ 50% of the mononuclear cells incorporated into OCL. The OCL that form express calcitonin receptors and contract in response to calcitonin. Studies examining the proliferative capacity and the resistance of OCL precursors from these transgenic mice to apoptosis demonstrated that the increased numbers of OCL precursors in marrow from bcl -X L /Tag mice was due to their increased survival rather than an increased proliferative capacity compared with Tag, bcl -X L , or normal mice. Histomorphometric studies of bones from bcl -X L /Tag mice also confirmed that there were increased numbers of OCL precursors (TRAP ϩ mononuclear cells) present in vivo. These data demonstrate that by targeting both bcl -X L and Tag to cells in the OCL lineage, we have immortalized OCL precursors that form bone-resorbing OCL with an efficiency that is 300-500 times greater than that of normal marrow. ( J. Clin. Invest. 1998. 102:88-97.)

Osteoclasts are terminally differentiated cells that express tartrate-resistant acid phosphatase (TRAP) at a higher level than other normal cells. Therefore, in an attempt to develop immortalized osteoclasts, we produced two lines of transgenic mice in which expression of the simian virus 40 T antigen oncogene was targeted to osteoclasts using the TRAP gene promoter. Osteoclasts were increased in number in bones from both lines. More than 50% of them appeared morphologically transformed, 2-5% were mitotic, but, unexpectedly, 5% were apoptotic. Osteoclast tumors were observed occasionally in one line of mice (line 4), and sheets of TRAP-positive cells (tumorlets) developed in most mice in both lines. Although cells isolated from these tumorlets formed multinucleated TRAP-positive cells that resorbed bone in vitro, to date we have been unable to develop an immortalized osteoclast cell line from them. Osteoclasts from one line (line 5) had reduced ruffled border formation and a higher level of T-antigen expression than osteoclasts in the other line (line 4), and these features were associated with the presence of osteopetrosis. However, osteoclasts from these osteopetrotic mice and from line 4 mice resorbed bone normally when the mice were treated with interleukin-1. These findings indicate that T antigen can be targeted to osteoclasts in transgenic mice and causes osteoclast transformation, tumors, mitosis, and apoptosis. When T antigen is expressed at high levels, functional impairment of osteoclasts can be detected. Furthermore, these results suggest that T antigen is insufficient on its own to immortalize cells in the osteoclast lineage.

Because it is one of the few autoimmune disorders in which the target autoantigen has been definitively identified, myasthenia gravis (MG) provides a unique opportunity for testing basic concepts of immune tolerance. In most MG patients, Abs against the acetylcholine receptors (AChR) at the neuromuscular junction can be readily identified and have been directly shown to cause muscle weakness. T cells have also been implicated and appear to play a role in regulating the pathogenic B cells. A murine MG model, generated by immunizing mice with heterologous AChR from the electric fish Torpedo californica, has been used extensively. In these animals, Abs cross-react with murine AChR; however, the T cells do not. Thus, to study tolerance to AChR, a transgenic mouse model was generated in which the immunodominant Torpedo AChR (T-AChR) α subunit is expressed in appropriate tissues. Upon immunization, these mice showed greatly reduced T cell responses to T-AChR and the immunodominant α-chain peptide. Limiting dilution assays suggest the likely mechanism of tolerance is deletion or anergy. Despite this tolerance, immunization with intact T-AChR induced anti-AChR Abs, including Abs against the α subunit, and the incidence of MG-like symptoms was similar to that of wild-type animals. Furthermore, evidence suggests that this B cell response to the α-chain receives help from T cells directed against the other AChR polypeptides (β, γ, or δ). This model offers a novel opportunity to elucidate mechanisms of tolerance regulation to muscle AChR and to clarify the role of T cells in MG.