Membrane Regulation of Bone Morphogenetic Protein 2 Signaling in Pre-osteoblastic Cells

Bone morphogenetic protein 2 (BMP2) is a potent growth factor crucial for the differentiation of bone forming cells (osteoblasts) and it directs bone mineral density (BMD). BMP2 signals through two receptors named, BMP receptor type Ia (BMPRIa) and BMP receptor type II (BMPRII). When BMP2 binds with these receptors at the cell surface multiple signaling pathways are activated including the Smad pathway which is crucial for osteoblast differentiation and bone development. These receptors are localized to distinct areas of the cell surface called clathrin coated pits (CCPs) and caveolae. Currently the model for BMP2 signaling suggests the BMP2 receptors that are localized to caveolae initiate signaling pathways that are independent of the Smad pathway, while BMP2 receptors that localization to CCPs activate the Smad signaling pathway. Yet there are published data conflicting with this model.

In order to define the role CCPs and caveolae have in BMP2 signaling the Family of Image Correlation Spectroscopy (FICS) was used to identify the regions at the cell surface where BMPRIa was localized to. Caveolae were identified to be centers where BMP2 interacts with its receptors and regulates the Smad pathway. Additionally the movement of BMPRIa at the cell surface in response to BMP2 was characterized. It suggested that it was involved in osteoblast differentiation and also affected BMD. Further utilizing the collected receptor localization results by FICS and Smad signaling data from reporter gene assays, a mathematical model was derived which suggested caveolae contributed 78% of the Smad-dependent signal. In addition, the proteins Adaptor protein 2 (AP2) and Casein Kinase II (CK2), were identified to interact with BMPRII or BMPRIa. The results suggested that these two proteins regulate the movement of the BMP receptors at the cell surface. Further, the interaction between CK2 and BMPRIa led to the development of blocking peptides which induced the Smad pathway in the absence of BMP2. Injection of the peptide into a mouse model led to increased bone growth. These results indicated BMP2 signaling through BMPRIa and BMPRII was highly regulated at the cell surface by both CCPs and caveolae. The movement of these receptors between both caveolae and CCPs are needed for BMP2 signaling to drive osteoblast differentiation, mineralization, and BMD.