Regulation of Osteogenic Gene Transcription via Gap Junction-mediated Cell-cell Communication

BACKGROUND: Bone regenerative therapy with mesenchymal stem cells (MSCs) is now an area of intensive exploration. In this strategy, patient’s own cells were used to replace damaged bone tissue and, it reduces patients’ burden. However mechanisms of the transplanted MSCs differentiation into osteoblasts are not understood completely. Previous studies indicated that gap junction-mediated cell to cell communication between osteoblasts and neighbor cells such as osteocytes and osteoclasts underlies both bone formation and bone resorption. In this regard, gap junctional communication between cells is reportedly involved in the transmission of mechanical and chemical signals from one area of the bone to another. Therefore, gap junctional communication between MSCs and osteoblasts is likely to contribute to the regulation of MSC differentiation. However, few advances have been made toward elucidation of the communication networks linking MSCs with osteoblasts. Here, we performed an analytical study of cell to cell communication between osteoblasts and MSCs

MATERIALS AND METHODS: We investigated cell to cell communication between osteoblasts and MSCs using two types of cell line, MLO-A5 and C3H10T1/2. The MLO-A5 cell line was developed from transgenic mice in which the SV40 large T-antigen oncogene was expressed under the regulation of the osteocalcin promoter. MLO-A5 cells mineralize in culture and express high amounts of alkaline phosphatase (ALP) as an osteoblast marker. Thus, the MLO-A5 cell line is considered to be the mature osteoblasts. While the C3H10T1/2 cell was established from an early mouse embryo and is capable of differentiating into myocytes, adipocytes, chondrocytes, and osteoblasts. Therefore, C3H10T1/2 cells share quintessential characteristics with MSCs. In this study, we co-cultured MLO-A5 and C3H10T1/2 cells, and investigated the effect of the co-culture with MLO-A5 on the differentiation of C3H10T1/2 cells.

RESULTS: As a result of the co-culture, the mRNA expression levels of ALP and Bone sialoprotein (BSP) an osteoblast marker increased in co-cultured C3H10T1/2 cells. The elevated ALP and BSP expression levels occurred within just 6 h. The presence of gap junctions between MLO-A5 and 10-GFP cells was also demonstrated by dye transfer biocytin and patch clamp experiments. In addition, CBX, an inhibitor of gap junction, showed that gap junctions are required for the elevation in ALP and BSP mRNA levels in co-cultured C3H10T1/2 cells. Furthermore, histone acetylation levels were higher in the co-cultured C3H10T1/2 cells than in mono-cultured C3H10T1/2 cells.

CONCLUSIONS: The present study implies that osteoblasts and MSCs communicate via gap junctions. Furthermore, our results strongly suggest that chromatin remodeling followed by ALP and BSP transcription occur in MSCs as a result of gap junction-mediated intercellular communication. This indicates that osteoblasts induce osteogenic differentiation of MSCs. To the best of our knowledge, this is the first demonstration of gap junction-modulated transcription in stem cells. While these observations have enormous implications for understanding the mechanisms of bone remodeling and MSCs maintenance and differentiation.

REFERENCES

Mikami Y, Matsumoto T, Kano K, Toriumi T, Somei M, Honda MJ, Komiyama K. (2014) Current status of drug therapies for osteoporosis and the search for stem cells adapted for bone regenerative medicine. Anat Sci Int 89:1-10.

Marotti G and Palumbo C. (2001) The mechanism of transduction of mechanical strains into biological signals at the bone cellular level. Eur J Histochem 51:15-19.