Foxf1 Knockdown Promotes BMSCs Osteogenesis Partly Through Activation of the Wnt/β-Catenin Signaling Pathway and Prevents Ovariectomized-Induced Bone Loss

Abstract

Background: Forkhead box protein f1 (Foxf1) is associated with cell differentiation, and may act as a key player in bone homeostasis. However, the effect of Foxf1 on osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) and ovariectomized-induced bone loss, as well as its clinical implication, remains largely unknown. Methods: By qRT-PCR and western bloting analysis, we first test Foxf1 expression level in bone tissues, BMSCs, and BMMs, derived from ovariectomized (OVX) mice, then further investigate its expression during osteogenic differentiation and osteoclast differentiation. Using loss-offunction approach (small interfere RNA [siRNA]-mediated knockdown) in vitro, we examined whether Foxf1 regulated osteoblast differentiation of BMSCs via Wnt/β-catenin signaling pathway. Furthermore, we assessed the anabolic action of Foxf1 knockdown (siFoxf1) in OVX mice in vivo. Clinically, we investigated the expression of Foxf1 in bone tissues derived from postmenopausal osteoporosis (PMOP) patients and the connection between Foxf1 expression and bone mineral density (BMD). Finally, we examined the role of Foxf1 knockdown on the osteoblastic differentiation of human BMSCs. Findings: Foxf1 expression was significantly increased in bone extracts and BMSCs derived from OVX mice and gradually decreased during osteoblast differentiation BMSCs, whereas no significant difference in Foxf1 expression was observed in OVX mice-derived BMMs and during osteoclast differentiation. In vitro, Foxf1 knockdown markedly increased osteoblast specific gene expression, alkaline phosphatase (ALP) activity, and mineral potential. Moreover, Wnt/β-catenin signaling pathway was activated with siFoxf1 treatment. The increased osteogenic differentiation induced by siFoxf1 treatment was partly rescued by an Wnt signaling inhibitor (DKK1). In the OVX mice model, Foxf1 siRNA delivery in vivo significantly improved the bone loss through enhancing bone formation. Clinically, Foxf1 expression was increased in bone tissues derived from PMOP patients and was continuously increased along with the decreasing BMD. Also, Foxf1 knockdown significantly promoted the osteogenesis of human BMSCs. Interpretation: These findings indicated that Foxf1 is a novel molecular that its knockdwn promotes BMSCs osteogenesis and prevents OVX-induced bone loss. Foxf1 could be an osteogenic biomarker and a potential therapeutic target for PMOP. Funding Statement: This work was supported in part by the following grants: National Natural Science Foundation of China [81503591, 81774338, 81674000]; Natural Science Foundation of Guangdong Province [2016A030313645]; Science and Technology Projects of Guangdong Province [2016A020226006]; Special Research Project for the Construction of the National TCM Clinical Research Base of the State Administration of Traditional Chinese Medicine [JDZX2015078]; Excellent Young Scholars Project of China Association of Traditional Chinese Medicine [CACM-2017-QNRC1-01]; Excellent Doctor Project of the First School of Clinic Medicine of Guangzhou University of Chinese Medicine [YB201702]; Scientific Research Project of Guangdong Traditional Chinese Medicine Bureau [20180330134046]; Excellent Young Scholars Project of the First Affiliated Hospital of Guangzhou University of Chinese Medicine [2015QN03, 2017TD08, 2017QN08]. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine. All protocols performed on patients were approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou University of Chinese Medicine.