Once bone is formed, bone matrix constantly undergoes local synthesis and resorption. Turnover of bone matrix is important for maintaining the proper amount, structure, and physical properties of bone.
Bone matrix is synthesized by osteoblasts, and is degraded by the hematopoietically-derived osteoclasts. Osteoclasts are critical cells since they help regulate bone structure and are often targeted by diseases that affect bone (osteoporosis, metastatic cancer). Consequently, osteoclasts also provide a very convenient entry point from which to treat bone pathologies. Cell signaling processes that include tyrosine phosphorylation events play critical roles in regulating production and function of osteoclasts.
We have shown that the non-receptor isoform of protein tyrosine phosphatase epsilon (cyt-PTPe) supports the adhesion and activity of osteoclasts. Lack of cyt-PTPe causes structural defects in podosomes, the subcellular structures by which osteoclasts adhere to bone. At the molecular level, osteoclast contact with bone matrix activates signaling by the integrin receptor molecules, the mechano-sensory receptors of the cell. Activation of integrin signaling in osteoclasts induces phosphorylation of cyt-PTPe at its C-terminal Y638. This phosphorylation event allows the adaptor molecule Grb2 to bind cyt-PTPe and to recruit Src to its vicinity. cyt-PTPe then dephosphorylates the Src tyrosine kinase at its inhibitorry Y527, thus activating Src. Proper Src kinase activity is essential for osteoclasts to adhere to and degrade bone properly. Lack of cyt-PTPe in PTPe-deficient mice reduces Src activity in osteoclasts and hence harms the function of these cells, both in culture and in vivo.
Additional studies have revealed that, surprisingly, the closely-related PTP Alpha (RPTPa) does not play a unique role in osteoclasts. This is because the isoform of PTPe that is expressed in osteoclasts is the non-receptor, predominantly cytosolic form of PTPe, while PTP Alpha is expressed in these cells as a receptor-type PTP that is present at the cell membrane. Removing the PTP Alpha catalytic domains from their association with the membrane allows them to function in osteoclasts.
Wealso study a second phosphoatase, the receptor-type PTPROt. PTPROt regulates Src in a more complex manner.the phosphatse undergoes phosphorylation at its C-terminal Y399, which enables it to activate Src by targeting Y527 of Src. When it is not phosphorylted at Y399, PTPROt down-regulates Src activity. Phosphorylation of PTPROt at Y399 is thus a molecular switch that toggles its activity as an activator vs. oinhibitor of Src, in vivo.
Accordingly, mice that completely lack PTPROt do not exhibit bone or osteocalst phenotypes since both functions of PTPROt towards Src are disrupted, causing no net change in Src or osteoclast activities. On the other hand, mice homozygous for Y399F PTPRTOt, in which the Y399 phosphorylation site has been destroyed, exhibit reduced Src activity, reduced osteoclast activity, and increased bone mass. This is caused by selectively inhibiting only the Src-activating role of PTPROt, thereby inhibiitng Src. These mice were produced locally by us using CRISPR methodology.
This study was publiched in Science Signaling, 2019.