Ulinastatin Inhibits Osteoclastogenesis and Suppresses Ovariectomy-Induced Bone Loss by Downregulating uPAR

Abstract

Recent studies indicate that uPAR acts a crucial part in cell migration and the modulation of bone homeostasis. As a natural serine protease inhibitor, ulinastatin owns the capacity to reduce proinflammatory factors, downregulate the activation of NF-κB and mitogen-activated protein kinases (MAPKs) signaling pathways. Osteoclastogenesis has been demonstrated to be related with low-grade inflammation which involves cell migration, thus we speculate that ulinastatin may have a certain kind of impact on uPAR so as to be a potential inhibiting agent of osteoclastogenesis. In this research, we investigated the role which ulinastatin plays in RANKL-induced osteoclastogenesis both in vivo and in vitro. Ulinastatin inhibited osteoclast formation and bone resorption in a dose-dependent manner in primary bone marrow-derived macrophages (BMMs), and knockdown of uPAR could completely repress the formation of osteoclasts. At the molecular level, ulinastatin suppressed RANKL-induced activation of cathepsin K, TRAP, nuclear factor-κB (NF-κB) and MAPKs, and decreased the expression of uPAR. At the meantime, ulinastatin also decreased the expression of osteoclast marker genes, including cathepsin K, TRAP, RANK, and NFATc1. Besides, ulinastatin prevented bone loss in ovariectomized C57 mice by inhibiting the formation of osteoclasts. To sum up, this research confirmed that ulinastatin has the ability to inhibit osteoclastogenesis and prevent bone loss, and uPAR plays a crucial role in that process. Therefore, ulinastatin could be chosen as an effective alternative therapeutics for osteoclast-related diseases.

uPAR antibody (huATN-658) and Zometa reduce breast cancer growth and skeletal lesions

“…36 A recent study demonstrated that uPAR expression increases during osteoclast formation, and knockdown of the gene encoding for uPAR completely inhibits the formation of osteoclasts. 37 Therefore, in this study, we evaluated the potential additive benefit of . The total number of osteoclasts was counted, and the bar graph on the right panel represents the mean ± SEM of osteoclasts counted from three animals per group.…”

“…5a to demonstrate that decreased osteolysis following these treatments has a net increase in bone volume. Since uPAR is also involved in osteoclast formation, 37 the addition of huATN-658 in the combination cohort further inhibits osteoclastogenesis. Moreover, the RNA-Seq results showed that the huATN-658 + Zometa combination treatment significantly increases the expression of COL1A2 and SPOCK3 genes encoding for type 1 collagen and osteonectin proteins.…”

PKM2 regulates angiogenesis of VR‐EPCs through modulating glycolysis, mitochondrial fission, and fusion

“…Cell Counting Kit‐8 (CCK‐8) assay was carried out as described previously (J. Guo et al, 2017; Huang et al, 2018). VR‐EPCs were seeded in 96‐well plates (1 × 10 4 cells/well), 24 hr later, the adherent cells were cultured with M199 medium containing different concentrations of C3k (0.1, 0.3, 0.5, and 1 μM) or DASA‐58 (1, 10, 30, and 50 μM) for 4 days.…”

“…Quantitative real‐time reverse transcription‐polymerase chain reaction (qRT‐PCR) was performed as described before (J. Guo et al, 2017; Huang et al, 2018). VR‐EPCs were plated into 6‐well plates (1 × 10 5 cells/well) and then cultured with M199 medium containing DASA‐58 (30 μM) or C3k (0.3 μM) or only DMSO for 2 days.…”

“…Western blot analysis was performed as described before (J. Guo et al, 2017; Huang et al, 2018). VR‐EPCs were seeded into 6‐well plates (1 × 10 5 cells/well) and then cultured with M199 medium containing DASA‐58 (30 μM), C3k (0.3 μM), VEGF (10 ng/ml), or only DMSO for 2 days, then lysed with radioimmunoprecipitation assay lysis buffer (Boster) with the addition of broad‐spectrum phosphatase inhibitors and phenylmethylsulfonyl fluoride (Boster).…”