IL-34, the second ligand for CSF-1R, was identified on a functional screening of a library of proteins secreted by the embryonic kidney cell line transfected with recombinant cDNAs . Although, structurally, it is markedly different from any other proteins, IL-34 binds to the CSF-1 receptor strongly and is similar to CSF-1 in the ability to enhance monocyte viability and osteoclast generation [12, 13, 32, 34]. In addition, IL-34 has been regarded as a promising clinical biomarker and therapeutic target for IA [14, 20, 21]. Studies using concentrations of IL-34 above 2.5 ng/ml have reported effects on the differentiation, proliferation, and survival of osteoclasts [13, 22, 23, 35]. However, the effect of IL-34 on the bone metabolism, particularly when low doses are used, has rarely been described. Similarly, IL-34 has not been linked to the effects of osteogenic differentiation in hBMSCs. Finally, there is no description to date of a role for IL-34 in the molecular mechanisms of the osteogenesis. Here, to our knowledge, we first revealed the underlying mechanisms of low-dose IL-34 on the regulation of bone homeostasis. IL-34 promoted the bone formation of hBMSCs partly via the PI3K/AKT and ERK signaling pathways in vitro. Meanwhile, a rat tibial bone defect model with a local injection of IL-34 produced a better recovery in vivo. However, low-dose IL-34 did not contribute to the differentiation of mBMMs in vitro, and a rat OVX model gave results consistent with this conclusion. These observations demonstrate that low-dose IL-34 enhances the osteogenic differentiation of hBMSCs, at least by partial activation of the PI3K/AKT and ERK signaling pathways, but it has no effect on osteoclastogenesis.
Serum IL-34 levels in healthy people were found to be 152.0 (92.0–234.0) pg/ml, 56.74 ± 2.30 pg/ml or 49.1 ± 78.5 pg/ml [13, 15–17, 22, 23, 35]. Thus, in the present study, we regarded the concentration of IL-34 in a range from 0.0001 to 0.01 ng/ml as a low dose. Chen et al. revealed that IL-34 together with RANKL can induce the formation of murine osteoclasts from not only splenocytes but also bone marrow cells in a dose-dependent manner (2.5 ng/ml, 25 ng/ml, and 100 ng/ml) and these cells have bone resorption activity . According to Nakamichi et al. IL-34 appears to play a pivotal role in the generation and storage of osteoclast precursors in the spleen and osteoclastogenesis in CSF-1op/op mice . A study conducted by Cheng et al. first demonstrated that IL-34 was conducive to the survival of osteoclast progenitors and further promoted RANKL-induced osteoclast formation by the JAK2/STAT3 pathway in vitro . Furthermore, it has been reported that TNF-α up-regulates osteoclastogenic cytokine IL-34 production through the activation of NF-κB and JNK signaling in the synovial cells of rheumatoid arthritis (RA) patients . In the present study, we revealed that a low-dose IL-34 obviously heightened the expression of osteogenic-specific genes and proteins in hBMSCs, which filled a blank by showing that IL-34 plays an important role in osteogenesis. However, no significant differences were observed during osteoclastogenesis with the low-dose IL-34. This may be associated with the concentration of IL-34, which was too low to work for osteoclast formation both in vivo and in vitro. These results demonstrated that in low dose, IL-34 contribute to osteoblastogenesis rather than osteoclastogenesis.
Given that IL-34 has been demonstrated to play dominant roles in synovial inflammation and bone erosion, it is possibly leaded to RA and osteoarthritis (OA) pathology . Plenty of studies have concentrated on the underlying correlation between the concentrations of IL-34 in the circulation or joint fluid and clinical parameters of RA patients. A case-control study containing with 100 RA patients and 59 healthy controls not only measured serum IL-34 levels in RA patients and healthy controls but also observed that serum IL-34 levels were significantly greater in RA patients than in healthy controls (603.5 [123.3–1,673.0] vs. 152.0 [92.0–234.0] pg/ml) . These conclusions agreed with the results conducted by Wang et al., who pointed out that serum IL-34 levels in RA patients were markedly higher than in healthy controls (269.72 ± 14.71 pg/ml vs. 56.74 ± 2.30 pg/ml) . The concentrations of IL-34 levels in serum were found to be correlated with several clinical variables [15, 16]. Moon et al. indicated that the serum IL-34 levels in RA patients were much higher than in OA patients and healthy controls. The mean serum IL-34 levels were 49.1 ± 78.5 pg/ml, 36.6 ± 38.0 pg/ml, and 188.0 ± 550.3 pg/ml in healthy controls, OA, and RA patients, respectively . All of these previous findings supported the concept that a high-dose serum IL-34 level is a risk factor for both RA and OA. Thus, IL-34 has the classical actions, including a possibility to generate bone erosion, and may play a key role in the formation of RA and OA. However, the role of low-dose IL-34 in bone metabolism was still unclear. In our study, we focused on the relationship between low-dose IL-34 and bone metabolism, revealing that IL-34 from 0.0001 to 0.01 ng/ml contributed to osteoblastogenesis. The effects of IL-34 on hBMSCs during osteogenesis were evaluated by qRT-PCR and Western blotting analysis, revealing that IL-34 increased osteo-specific genes and proteins at lower concentrations, especially 0.001 ng/ml. ALP staining and ARS are early and late markers, respectively, of osteoblastic differentiation [31, 37, 38]. We found that IL-34 intensified ALP activity and deepened mineralization at lower concentrations, especially at 0.001 ng/ml. Those results suggested that low-dose IL-34 promoted the osteogenesis of hBMSCs in vitro. Meanwhile, we also observed that low-dose IL-34 has no effect on osteoclastogenesis both in vivo and in vitro (Fig. 7).
The specific tyrosine residues increasedly dimerized and autophosphorylated intracellularly by the association of IL-34 with the extracellular domain of CSF-1R, leading to the accomplishment of kinds of kinases and adaptor proteins. Such players can be found in signaling pathways, including ERK and AKT . These pathways enhance the pleiotropy of IL-34-mediated CSF-1R when cells differentiated, attached, migrated and proliferated. Furthermore, they stimulate cellular cytoskeletal organization and survival, subsequently modulate the specific genes expression . As shown in Fig. 2, our research described an obvious increase in the expressions of P-AKT and P-ERK during the hBMSCs-driven differentiation with endogenous IL-34.
Several studies have emphasized the key role of the PI3K/AKT signaling pathway for all of the periods of osteogenic differentiation, maturation, and bone formation [38–41]. Not only chondrocyte differentiation would be impaired, but also longitudinal bone growth would be inhibited by blocking the PI3K/AKT signaling pathway [3, 42]. With the activation of the PI3K/AKT signaling pathway, IL-34 switched the phenotype of Kupffer cells from M1 to M2 in vitro . Chen et al. mentioned that IL-34, which is expressed and secreted by embryonic stem cells, may be responsible for ESC-promoted macrophage survival by activating the ERK1/2 and PI3K/AKT pathways . In this study, we found that IL-34 enhanced bone formation by activating the PI3K/AKT signaling pathway. An inhibitor specific for the PI3K/AKT pathway significantly inhibited P-AKT. Western blotting analyses, ALP staining, ARS, and IF analyses further confirmed the regulatory role of the IL-34-PI3K/AKT axis in the osteogenic differentiation of BMSCs.
The ERK pathway, one of MAPK signaling pathways, is an important signal transducer in the regulation of the osteoblastogenesis of MSCs and bone metabolism . It has been reported that major secreted ligands that regulate osteoblast activity seem to serve partly via the ERK pathway . The expression levels of RUNX2 and Osterix are firmly associated with ERK phosphorylation . Matsushita et al. found a critical role for ERK in osteoblast mineralization because mice with Erk1 and Erk2 deletions display dramatically reduced bone mineralization . Further, IL-34 modulates rheumatoid synovial fibroblasts proliferation and migration via the ERK/AKT signaling pathway . According to the experimental results, such as western blotting, ALP staining, ARS, and IF analyses, we found ERK signaling pathway is firmly correlated to osteogenesis in hBMSCs. In order to further verify our results, an inhibitor (U0126) specific for ERK signaling pathway was applied. Treatment with U0126 blocked ERK1/2 phosphorylation and significantly decreased RUNX2, COL1A1, P-ERK, ALP activity, and mineralized nodule formation when compared with the control group. Based on the results above, we demonstrated that ERK signaling pathway is quiet important in IL34-induced osteogenesis in hBMSCs.
The relationship between cytokines and bone metabolism has been demonstrated by several studies [13, 21, 23, 35]. Nevertheless, this is the first study, to the best of our knowledge, to demonstrate the effect of low-dose IL-34 on the dynamic balance of bone metabolism, as shown in Fig. 6H. Unfortunately, we did not investigate the impact of IL-34 on signaling molecules, such as IL-1, IL-6, and TNF-a, that shape the inflammatory microenvironment. Moreover, the mechanisms of crosstalk between PI3K/AKT and ERK/MAPK signaling are not fully clarified and require further investigation in future studies. Finally, a rat OVX model used in vivo and mBMMs used for in vitro experiments may reveal the effect of low-dose IL-34 on osteoclastogenesis. Even though the percentage identity of human IL-34 with the rat and mouse IL-34 are 72% and 71%, respectively , there are still biological structural difference among different races. Thus, further studies are needed.