A Novel Fluorogenic Probe for Intravital Microscopic Imaging of Enzymatically Active Cathepsin K in Functional Osteoclasts

초록

Cathepsin K (CTSK) is a lysosomal cysteine protease that is essential to the osteolytic activity of functional osteoclasts. Several fluorescence-based probes for CTSK have been developed for preclinical studies on osteoclast inhibitors but the sensitivity and signal intensity are insufficient for real-time live imaging in mice. To overcome this limitation, we synthesized a novel fluorescence resonance energy transfer (FRET)-based fluorogenic CTSK probe and tested its efficacy in vitro using a confocal microscope and in vivo using an intravital microscope. A peptide substrate specific to CTSK was conjugated with a TAMRA fluorophore and a BHQ-2 quencher. Upon addition of a nano-molar range of recombinant CTSK, the TAMRA fluorophore was cleaved from the quencher, resulting in a strong fluorescence signal, which was fully suppressed by odanacatib, a CTSK inhibitor. Notably, our probe exhibited over 60-fold higher sensitivity than previously reported probes. For in vitro imaging, osteoclasts were formed by treating murine bone marrow monocytes with RANKL and M-CSF on collagen-coated plates or dentin discs, followed by fixation, permeabilization and staining with the probe, phalloidin and DAPI. Confocal microscopy demonstrated the subcellular localization of CTSK in osteoclasts (Figure Panel A). A strong fluorescence signal was localized in the ruffled border, indicating that the probe is sensitive and specific to functional CTSK in bone-resorbing osteoclasts. For intravital imaging, we used three different mouse models: an ovariectomy-induced osteoporosis model, a RANKL injection bone loss model, and a bone metastasis model using intra-tibial injection of breast tumor cells. The CTSK probe was administered by tail-vein injection (50?mol), and live real-time images were captured in the proximal tibia with a small skin incision under anesthesia. No significant toxicities were noted in multiple injection groups of mice. Two-photon intravital microscopy showed clear real-time images of osteoclasts on the endosteal surface or trabecular bone of live animals (Figure Panel B). The fluorescence signal was not detectable in negative control mice, and the signal was significantly reduced in mice treated with zoledronic acid or odanacatib. In conclusion, we developed a novel probe for sensitive and specific visualization of functional CTSK in real-time live mice that can be useful in bone biology research and in the development of therapeutics for CTSK or osteoclasts.