Roles of SDF-1/CXCR4 axis in cartilage endplate stem cells mediated promotion of nucleus pulposus cells proliferation

Highlights

• NPCs proliferation was promoted by co-cultured CESCs and CESCs-CM in vitro.

• SDF-1 and CXCR4 were expressed in a complementary pattern in CESCs and NPCs.

• CESCs promoted NPCs proliferation partially via SDF-1/CXCR4 axis.

• SDF-1/CXCR4 axis mainly activated ERK1/2 pathway rather than Akt pathway in NPCs.

• CESCs upregulated TIMP1 expression in NPCs in a SDF-1/CXCR4 axis independent manner.

Abstract

Stem cells transplantation has shown considerable promise in intervertebral disc repair and low-back pain release. Cartilage endplate stem cells (CESCs) also showed potential for nucleus pulposus (NP) regeneration in a rabbit disc degeneration model, the precise mechanism was unclear. Here we investigated the effects of CESCs on NP cells (NPCs) proliferation and the mechanism in vitro. CESCs and NPCs were isolated from surgical specimens of degenerative human lumbar disc. NPCs were co-cultured with CESCs at a 1:1 ratio or cultured in CESCs conditioned medium (CESCs-CM). NPCs proliferation was evaluated by Ki-67 staining, CCK-8 assay and cell cycle analysis. Gene expressions were detected by qRT-PCR and activation of Akt and ERK1/2 was detected by western blot. CXCR4 antagonist AMD3100 was used to block SDF-1/CXCR4 axis. ERK1/2 and Akt inhibitors were used to block Akt and ERK1/2 activation. Results showed that NPCs proliferation was promoted by direct-contact co-culturing with CESCs as well as culturing in CESCs-CM. SDF-1 expression level in CESCs was significantly higher than that in NPCs, while CXCR4 was the opposite. Promotion of NPCs proliferation mediated by CESCs-CM could be partially attenuated by AMD3100. CESCs-CM activated both Akt and ERK1/2 in NPCs, while rhSDF-1 scarcely activated Akt but obviously activated ERK1/2. Akt and ERK1/2 inhibitors could partially inhibited CESCs-CM mediated promotion of NPCs proliferation and showed cumulative effect, while ERK1/2 inhibitor and AMD3100 could significantly abrogate SDF-1 mediated promotion of NPCs proliferation. Our results suggested that CESCs might promote NPCs proliferation in a paracrine pathway, which was partially mediated by SDF-1/CXCR4 axis via ERK1/2 signaling transduction pathway.

Introduction

Intervertebral disc degeneration (IVDD), usually characterized by decreased number of functional nucleus pulposus cells (NPCs) and imbalance of synthesis and catabolism, is the initial factor of degenerative disc disease [1,2]. In the past decade, cell therapy has been extensively studied to prevent, slow down or reverse the degenerative changes of IVDD besides the available conservative and surgical treatments [3]. In clinical trials, autologous or allogeneic mesenchymal stem cells (MSCs) transplantation has shown great potential in IVD repair and low-back pain release [4,5].

Previously, we found progenitor cells in human degenerative cartilage endplate (CEP) and named them as cartilage endplate stem cells (CESCs), which shared mesenchymal stem cell markers with bone marrow-MSCs (BM-MSCs), and could be induced into osteoblasts, adipocytes and chondrocytes [6]. They showed better ability of chondrogenesis and osteogenesis compared to BM-MSCs [6]. We also demonstrated that CESCs performed powerful ability for NP regeneration in a rabbit disc degeneration model [7]. However, the precise mechanism remains unclear. MSCs were reported to promote proliferation and synthesis of proteoglycan of NPCs in vitro studies, and protect NPCs from apoptosis and improve the balance of extracellular matrix (ECM) metabolism in degenerative disc [[8], [9], [10]]. The mechanisms linking MSCs and their effects on NPCs and IVDD are not fully understood. Thus, we intended to investigate the effects of CESCs on NPCs proliferation and the mechanisms in vitro.

Broad repertoire of bioactive and regulatory growth factors produced by MSCs play important roles in MSCs regenerative and regulatory properties [11]. MSCs spontaneously express high levels of stromal cell-derived factor-1 (SDF-1) [12], of which signals transducted through the G-protein-coupled receptor CXCR4 and functions in regulating bone marrow microenvironment and hematopoietic stem cells homing, as well as survival and proliferation of MSCs [13,14]. Binding of SDF-1 to CXCR4 activates multiple signaling pathways to regulate cell proliferation, differentiation, survival and apoptosis [15]. Both SDF-1 and CXCR4 were up-regulated during IVDD, which may suggested a feedback to meet the demand of sufficient functional cells in the degenerative NP [16]. Therefore, we hypothesized that CESCs might regulate NPCs proliferation via SDF-1/CXCR4 axis and its relevant downstream signaling pathways.

To test our hypothesis, we established primary cultures of CESCs and NPCs from surgical specimens of degenerative human lumbar disc, and then, NPCs were direct-contact co-cultured with CESCs or cultured using CESCs conditioned medium (CESCs-CM). We found that CESCs showed potential to promote NPCs proliferation and paracrine pathways may play a role in this process. We then detected SDF-1 and CXCR4 expression levels in CESCs and NPCs and investigated the role of SDF-1/CXCR4 axis and downstream signaling pathways in CESCs mediated promotion of NPCs proliferation. We concluded that CESCs mediated NPCs proliferation was partially modulated via SDF-1/CXCR4 axis, which activated intracellular ERK1/2 signaling pathway in NPCs.