Role of osteoclast-derived exosomal miR-214 in regulating osteoblastic bone formation

A tight coupling between bone resorption and bone formation is essential for the homeostasis of the skeletal system. When this coupling is lost, pathologies raging from systemic diseases (e.g. osteoporosis) to local bone destruction (e.g. osteonecrosis) ensue. The maintenance of this equilibrium requires the coordinated work by the bone-resorbing osteoclast and bone-forming osteoblast, highlighting the cross-talk between these two types of cells. Apart from the well- documented regulatory mechanism of osteoblast-dependent bone resorption, emerging evidences over the last decade unveil the unique role of osteoclast in directing osteoblastic bone formation. A direct osteoclast-to-osteoblast signaling has been reported in the following two paradigms in cell contact-dependent manner. One is the bi-directional signal (e.g. the ephrins/ephs system) between osteoclast and osteoblast. The other is the transmembrane protein (e.g. Sema4D) found in osteoclast that participates in regulating osteoblast differentiation and bone formation. Besides the cell-contact dependent manner, the growth factors (e.g. TGF-β and IGF-1) released from bone matrix during bone resorption could stimulate osteoblast differentiation, representing an indirect paradigm for osteoclast-dependent bone formation in cytokine-dependent manner. However, it remains unclear whether there are other efficient ‘paracrine’ approaches for osteoclast-osteoblast communication. The recently published evidences have demonstrated that circulating microRNAs (miRNAs) are stable in body fluids (e.g. serum) and transported within extracellular vesicles (e.g. exosome), indicating their potential role as extracellular genetic molecules for cell-cell communication. Thus far, studies addressing the role of osteoclast-derived miRNAs in regulating osteoblastic bone formation have not been reported.

In the previous study, the applicants showed that increased microRNA-214 (miR-214) in osteoblast could suppress bone formation (Wang X, et al., Nat Med 2013). Recently, the applicants’ preliminary data from human samples documented that increased miR-214 in serum exosomes was accompanied by decreased expression of bone formation marker gene (BGLAP mRNA) in bone specimens during aging. Furthermore, the serum exosomal miR-214 level was found to be positively correlated with the miR-214 level in osteoclasts and negatively correlated with bone formation rate in aged mice during aging. In addition, our in vitro data showed that high abundance of both intra-cellular and supernatant exosomal miR-214 were presented in the cultured osteoclasts rather than osteoblasts. Thus, we speculate that the exosomal miR-214 in bone microenvironment could be contributed by osteoclast rather than osteoblast. We also found that miR-214 was indispensable for maintaining the activity of osteoclast in vitro. Further, we observed that the miR-214 could be transferred from osteoclast to osteoblast in exosome-encapsulated form, and the osteoclast-derived miR-214 could inhibit the osteoblast activity in vitro (Li D, et al., ASBMR Annual Meeting, 2014). Moreover, we have generated the osteoclast-specific miR-214 knockin (Ctsk/miR-214) mice and developed an osteoclast-targeting delivery system (Liu J et al., Biomaterials 2015, Zhang G et al., Nat Med 2012). We also have successfully established the protocols to harvest exosomes from the above Ctsk/miR-214 mice-derived osteoclast (Li D, et al., ASBMR Annual Meeting, 2015).Taken all together, we raised our hypothesis that osteoclast-derived exosomal miR-214 could inhibit osteoblastic bone formation.

To test the above hypothesis, we have the following four specific aims: (1) To examine bone formation and bone mass/structure by dynamic bone histomorphometry and micro-CT in adult Ctsk/miR-214 mice. (2) To detect bone formation, bone mass/structure and serum exosomal miR-214 by dynamic bone histomorphometry, micro-CT and real-time PCR in adult Ctsk/miR- 214 mice after treatment with antagomir-214 encapsulated in the osteoclast-targeting delivery system. (3) To investigate bone formation and bone mass/structure by dynamic bone histomorphometry and micro-CT in adult mice after administration with osteoclast-derived exosomes from Ctsk/miR-214 mice. (4) To determine bone formation, bone mass/structure and serum exosomal miR-214 by dynamic bone histomorphometry, micro-CT and real-time PCR in aged mice after treatment with antagomir-214 encapsulated in the osteoclast-targeting delivery system during aging.

This proposal would provide a new insight into miRNA-mediated molecular communication from osteoclast to osteoblast. It might also indicate a novel therapeutic target within osteoclast to promote osteoblast activity in skeletal disorders with impaired bone formation.