Cathepsin K Localizes to Equine Bone In Vivo and Inhibits Bone Marrow Stem and Progenitor Cells Differentiation In Vitro

Abstract

Selective inhibition of Cathepsin K (CatK) has a promising therapeutic potential for diseases associated with bone loss and osseous inflammation, such as osteoarthritis, periodontitis, and osteoporosis. In horses, stress-related bone injuries are common and accompanied by bone pain and inflammation resulting in excessive bone resorption and periostitis. VEL-0230 is a highly selective inhibitor of CatK that significantly decreased bone resorption and increased bone formation biomarkers. The goal of this study was to demonstrate the presence of CatK in equine bone and a simultaneous influence on the bone marrow cellular components including function and differentiation. Our objectives were: 1) to investigate the tissue localization of CatK protein in equine bone using immunohistochemistry, and 2) to determine the effect of CatK inhibition on osteoclastogenic, chondrogenic and osteogenic differentiation potential of equine stem and progenitor cells in vitro using histochemical staining and differentiation-related gene expression analyses. Bone biopsies, harvested from the tuber coxae and proximal phalanx of six healthy horses, were processed for immunostaining against CatK. Sternal bone marrow aspirates were cultured in 0, 1, 10, or 100 μM of VEL-0230 and subsequent staining scoring and gene expression analyses performed. All cells morphologically characterized as osteoclasts and moderate number of active bone lining osteoblasts stained positive for CatK. Histochemical staining and gene expression analyses revealed a significant increase in the osteoclastogenic, chondrogenic and osteogenic differentiation potential of equine bone marrow cells, which was VEL-0230-concentration dependent for the latter two. These results suggested that CatK inhibition may have anabolic effects on bone and cartilage regeneration that may be explained as a feedback response to CatK depletion. In conclusion, the use of CatK inhibition to reduce inflammation and associated bone resorption in equine osseous disorders may offer advantages to other therapeutics that would require further study.

In Vitro Effects of Weissella cibaria CMU and CMS1 on Receptor Activator of NF-κB Ligand (RANKL)-Induced Osteoclast Differentiation

“…Treating RAW 264.7 cells with RANKL initiates osteoclast differentiation signaling through RANK, thereby inducing the expression of key differentiation-related genes, including c-Fos, NFATc1, OSCAR, DC-STAMP, cathepsin K, and TRAP [ 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. Furthermore, c-Fos and NFATc1 work synergistically to induce the expression of several key osteoclastogenesis-related genes [ 7 , 8 , 9 , 10 ]. Specifically, NFATc1 is known as a key transcriptional regulator of osteoclasts and is an essential factor for osteoclast differentiation [ 7 , 8 , 9 , 10 ].…”

“…Furthermore, c-Fos and NFATc1 work synergistically to induce the expression of several key osteoclastogenesis-related genes [ 7 , 8 , 9 , 10 ]. Specifically, NFATc1 is known as a key transcriptional regulator of osteoclasts and is an essential factor for osteoclast differentiation [ 7 , 8 , 9 , 10 ]. OSCAR is an osteoclast-specific immune receptor that serves as a costimulatory signal essential for RANKL-mediated NFATc1 activation [ 11 ].…”

“…RANKL binds to RANK present in osteoclast precursors, activating the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways and sequentially activating nuclear factor of activated T cells c1 (NFATc1), which is essential for osteoclast differentiation [ 8 , 9 ]. Furthermore, it is known to promote osteoclast differentiation and bone resorption by inducing the expression of several genes [ 9 , 10 , 11 , 12 , 13 ]. Therefore, inflammatory periodontal disease associated with osteoclasts can be alleviated by suppressing osteoclast differentiation [ 14 ].…”

Cathepsin K+ Non-Osteoclast Cells in the Skeletal System: Function, Models, Identity, and Therapeutic Implications

“…Random sequencing of expressed sequence tags from cDNA libraries and Ctsk immunostaining indicated that Ctsk is predominantly stained in mature osteoclasts. Moreover, Ctsk is granularly located at a single pole of the osteoclasts ( Drake et al, 1996 ; Hussein et al, 2017 ; Debnath et al, 2018 ). Using the same methodology, Ctsk is moderately expressed in osteoblasts and in some osteocytes underlying the matrix of metaphyseal trabecular femoral heads.…”

“…Using the same methodology, Ctsk is moderately expressed in osteoblasts and in some osteocytes underlying the matrix of metaphyseal trabecular femoral heads. However, the Ctsk expression of the protein was reduced during bone matrix formation ( Mandelin et al, 2006 ; Hussein et al, 2017 ; Lotinun et al, 2019 ). Immunohistochemical analysis conducted in the degradation area, weight-bearing areas of knee cartilage, and menisci in osteoarthritis patients showed that chondrocytes and fibroblast-like synoviocytes expressed Ctsk.…”