Pax2 is essential for proliferation and osteogenic differentiation of mouse mesenchymal stem cells via Runx2
Introduction
Bone metabolism balance is primarily regulated by osteoblasts (bone-forming cells) and osteoclasts (bone-reabsorbing cells). When the balance between bone resorption and deposition leads to excessive resorption, bone loss occurs, which is a precursor to osteoporosis [1]. As the most prevalent metabolic bone disorder, millions of people, such as postmenopausal women, elderly, and long-term bed-ridden patients suffer from osteoporosis, however, treatment for osteoporosis is still a challenge worldwide [1], [2].
Mesenchymal stem cells (MSCs), the origin of osteoblasts, have been widely investigated in the field of regenerative medicine [3]. Compact bone-derived mouse mesenchymal stem cells (mMSCs) were reported to have similar phenotypic properties and superior proliferative, as well as differentiation capabilities compared to bone marrow-derived MSCs [4], [5]. MSCs from compact bone have been given more attention recently and applied to explore its potential efficacy in various cellular therapies, such as in nonalcoholic steatohepatitis, Sjogren's-like Disease, and prion diseases [6], [7], [8]. The commitment and differentiation of MSCs toward osteogenic cell fate depend on a variety of transcription factors and signaling pathways, such as runt-related transcription factor 2 (Runx2), mitogen-activated protein kinases (MAPK), bone morphogenic protein (BMP), wingless type (Wnt), transforming growth factor-beta (TGF-β) and so on [9].
Paired box 2 (Pax2) is a member of subgroup II (Pax2/5/8) of the paired box-containing gene family, and acts as a transcription factor regulating developmental processes [10]. The Pax family is involved in bone development given that skeletal abnormalities have been seen in some Pax protein mutant mice, including Pax1, Pax5, Pax7, and Pax9 [11]. Among them, Pax5 has been reported to positively regulate osteoblastogenesis through direct induction of osterix (Osx) and bone gamma carboxyglutamate protein (OC) [11]. Pax2, characterized by the similar structure (PD-OP-PTHD structure) with Pax5, is well known to play a crucial role in regulating the development of urogenital, sensory, central nervous system and tumorigenesis [12], [13], [14], [15], [16], [17]. However, the role of Pax2 in osteogenesis is little known. According to a recent case report, a new skeletal deformity was found in a patient suffering from Papillorenal syndrome (PRS) with a heterogeneous Pax2 mutation. In this case, the patient showed a decline of bone mineral density of her left middle finger [18]. All these findings above suggest that Pax2 is a likely participant in skeletal development and plays a potential role in MSCs osteogenesis.
Here, we aimed to investigate whether Pax2 is involved in mechanisms underlying the regulation of osteogenesis in MSCs using the mouse pluripotent stem cell line C3H/10T1/2 and mMSCs in vitro and a heterotopic bone regeneration model based on mMSCs in vivo. Our results showed that Pax2 promoted the proliferation and osteogenic differentiation of mouse MSCs via up-regulation of Runx2 and MAPK signaling pathways, suggesting Pax2 has a role in the pathophysiology of bone related diseases, and has potential application in bone tissue regeneration.
Section snippets
Ethics statement
All protocols and experiments with animals performed meet the guideline of the Animal Care Committee of Nanjing Medical University and were approved by the Ethics Committee of Nanjing Medical University (permit number; IACUC-1801009).
Cell culture and osteogenic differentiation
Primary mouse MSCs were isolated from mouse compact bone according to the protocol described previously [19], with modifications. Briefly, 4–6-week C57BL/6 mice were sacrificed by cervical dislocation, tibiae and femurs were dissected. To deplete hematopoietic
Expression of Pax2 is increased during osteogenic differentiation
The C3H/10T1/2 cells, mMSCs, osteoprogenitor MC3T3-E1 cells and mouse calvarial osteoprogenitor cells were used to investigate the expression of Pax2 during osteogenic differentiation. We analyzed the Pax2 protein levels with an osteogenic differentiation inducer cocktail for up to 14 days. Western blotting analysis showed that Pax2 protein levels were gradually increased during the culture periods prior to 14 days in C3H/10T1/2 cells, mMSCs, as well as MC3T3-E1 cells and mouse calvarial cells,
Discussion
Understanding of the mechanisms that regulate MSCs osteogenic differentiation is quite important for the development of therapeutic strategies that promote bone formation and regeneration to treat bone-related diseases. In this study, we demonstrate that Pax2 is essential for osteogenesis as it promotes the proliferation and osteogenic differentiation of C3H10/T1/2 cells and mMSCs. Pax2 may promote osteogenesis by enhancing the activation of Runx2, in which the MAPK pathways are involved.
Pax2
Acknowledgments
This research was funded by the National Natural Science Foundation of China (81771029), Project Funding by the Priority Academic Program Development of Jiangsu Higher Education Institution (2014-037).
Author contributions
ML, FH, YZ and LY performed the experiments and analyzed the data. ML, SG, CM and JM designed the study. ML, SG, CM and JM wrote the manuscript. All authors reviewed and revised the manuscript.
Conflicts of interest
The authors declare no conflict of interest.
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These authors have contributed equally to this work.