F2r negatively regulates osteoclastogenesis through inhibiting the Akt and NFκB signaling pathways

Abstract

G-protein-coupled receptors (GPCRs) are pivotal drug targets for many diseases. Coagulation Factor II Thrombin Receptor (F2R) is an important member of GPCR family that is highly expressed in osteoclasts. However, the role of F2r in osteoclasts is still unclear. Here, to examine the functions of F2r on osteoclast formation, differentiation, activation, survival, and acidification, we employed loss-of-function and gain-of-function approaches to study F2r using F2r-targeted short hairpin RNA (sh-F2r) lentivirus and overexpression plasmid pLX304-F2r lentivirus respectively, in mouse bone marrow cells (MBMs) induced osteoclasts. We used three shRNAs targeting F2r which had the ability to efficiently and consistently knock down the expression of F2r at different levels. Notably, F2r knockdown trigged a significant increase in osteoclast activity, number, and size, as well as promoted bone resorption and F-actin ring formation with increased osteoclast marker gene expression. Moreover, F2r overexpression blocked osteoclast formation, maturation, and acidification, indicating that F2r negatively regulates osteoclast formation and function. Furthermore, we investigated the mechanism(s) underlying the role of F2r in osteoclasts. We detected RANKL-induced signaling pathways related protein changes F2r knockdown cells and found significantly increased pAkt levels in sh-F2r infected cells, as well as significantly enhanced phosphorylation of p65 and IKBα in early stages of RANKL stimulation. These data demonstrated that F2r responds to RANKL stimulation to attenuate osteoclastogenesis through inhibiting the both F2r-Akt and F2r-NFκB signaling pathways, which lead a reduction in the expression of osteoclast genes. Our study suggests that targeting F2r may be a novel therapeutic approach for bone diseases, such as osteoporosis.

GPR125 positively regulates osteoclastogenesis potentially through AKT-NF-κB and MAPK signaling pathways

“…The total RNA was extracted from primary monocytes or osteoclasts induced for 3 days or 5 days with TRIzol reagent (Invitrogen Corp., Carlsbad, CA) reagent according to the manufacturer's protocol to perform RNA-sequence analysis. These RNA samples were sequenced on an Illumina HiSeq 2000 system (Illumina, San Diego, CA) 30 to obtain 100 base-pair single-end reads per sample 31 . The modified results can be imported to Heatmapper web server to visualize the Heat map 32 .…”

“…The total RNA was reverse transcribed into cDNA using RevertAid RT Kit (K1691, Thermo Scientific) according to the manufacturer's instructions. We carried out qPCRs by using SYBR Green Reagents (4309155, Thermo Scientific) and Step-One Real-Time PCR System (Life Technologies, Applied Biosystems) 30 , 33 . The expression of glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) was used as an endogenous control for normalization.…”

Deletion of Coagulation Factor IX Compromises Bone Mass and Strength: Murine Model of Hemophilia B (Christmas Disease)

“…Absent levels or defective function of either FVIII or FIX impairs FX activation resulting in deficient thrombin generation. Both osteoblasts and osteoclasts express the thrombin receptor (F2R or PAR1) [10,[27][28][29], and in vitro studies indicate that thrombin induces differentiation of osteoblasts [29][30][31][32] and negatively regulates osteoclast formation and function [33,34]. In support of this mechanistic hypothesis, Aronovitch et al [35] demonstrated complete knockout of either FVIII or PAR1 in mice resulted in similar bone structural abnormalities.…”

Soft coral-derived Aspernolide A suppressed non-small cell lung cancer induced osteolytic bone invasion via the c-Fos/NFATC1 signaling pathway

“…Complex signaling pathways are involved in osteoclast formation and function. The binding of RANKL to RANK in osteoclast precursor cells results activation of osteoclast through signaling cascades including NFκBp65, MAPKs (ERK, JNK, and p38MAPK), and AKT pathways (31,32). Osteoprotegerin (OPG) prevents RANKL binding to RANK and inhibits bone resorption (33).…”