Elsevier

Experimental Cell Research

Volume 371, Issue 2, 15 October 2018, Pages 342-352
Experimental Cell Research

Pax2 is essential for proliferation and osteogenic differentiation of mouse mesenchymal stem cells via Runx2

https://doi.org/10.1016/j.yexcr.2018.08.026Get rights and content

Highlights

  • Knock-down of Pax2 inhibits the osteogenesis of mouse MSCs in vitro and in vivo.

  • Pax2 promotes the proliferation of mouse MSCs.

  • Pax2 enhances transcriptional activity of Runx2 and activates MAPK pathways.

  • Pax2 promotes the osteogenesis of mouse MSCs and may serve as a target for future clinical treatment.

Abstract

Mesenchymal stem cells (MSCs) have been widely studied in the field of regenerative medicine with the potential to solve osteoporosis. Paired box 2 (Pax2), as a transcription factor, is the master regulator of embryogenesis and oncogenesis. However, the function of Pax2 in osteogenesis is unknown. Here, we reported for the first time that the expression of Pax2 gradually increased during osteogenic differentiation of mouse MSCs, and osteoprogenitor cells. However, detected in osteoblastic cells of mouse tibia, the expression of Pax2 in the embryonic stage was higher than that in adulthood. In C3H/10/T1/2 cells and compact bone-derived mouse MSCs (mMSCs), Pax2 knock-down inhibited the proliferation of these cells, down-regulated the expression of osteogenic marker genes, as well as repressed the ALP activity and mineralization. In addition, Pax2 enhanced the transcriptional activity of Runx2, and activated the MAPK pathway genes (ERK, JNK and p38). Furthermore, knock-down of Pax2 repressed the mMSCs-mediated bone regeneration in an ectopic bone formation model. In conclusion, Pax2 promotes osteogenesis of mouse MSCs, suggesting that Pax2 has a role in the pathophysiology of bone related diseases, and has potential application in bone tissue regeneration.

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.

References (40)

  • E. Grimley et al.

    Are pax proteins potential therapeutic targets in kidney disease and cancer?

    Kidney Int.

    (2018)
  • O. Tabatabaei-Malazy et al.

    New horizons in treatment of osteoporosis

    DARU

    (2017)
  • K. Eimori et al.

    Disrupted bone metabolism in long-term bedridden patients

    PLoS One

    (2016)
  • X. Qi et al.

    Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells repair critical-sized bone defects through enhanced angiogenesis and osteogenesis in osteoporotic rats

    Int. J. Biol. Sci.

    (2016)
  • Z. Guo et al.

    In vitro characteristics and in vivo immunosuppressive activity of compact bone-derived murine mesenchymal progenitor cells

    Stem Cells

    (2006)
  • D. Blashki et al.

    Mesenchymal stem cells from cortical bone demonstrate increased clonal incidence, potency, and developmental capacity compared to their bone marrow-derived counterparts

    J. Tissue Eng.

    (2016)
  • G.A. Elghanam et al.

    Compact bone-derived multipotent mesenchymal stromal cells (mscs) for the treatment of sjogren's-like disease in nod mice

    Methods Mol. Biol.

    (2017)
  • Z. Shan et al.

    Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease

    J. Gen. Virol.

    (2017)
  • Q. Chen et al.

    Fate decision of mesenchymal stem cells: adipocytes or osteoblasts?

    Cell Death Differ.

    (2016)
  • G.R. Dressler et al.

    Pax2, a new murine paired-box-containing gene and its expression in the developing excretory system

    Development

    (1990)
  • Cited by (0)

    1

    These authors have contributed equally to this work.

    View full text