Recombinant CRP inhibits the proliferation of OPCs by blocking the cell cycle in G1-S transition
To explore the role of CRP in osteogenesis, the proliferation of OPCs treated with various concentrations of recombinant CRP (5, 10, and 20 mg/mL) was determined, as described in the literature (14, 25, 63). Proliferation decreased significantly at recombinant CRP concentrations of 5, 10, and 20 mg/mL, inhibition that was dose-dependent (Fig. 1A). Apoptosis increased significantly at recombinant CRP concentrations of 10 and 20 mg/mL, increases that were dose-dependent (Supplemental Fig. 1). Staining of Ki67, a marker of proliferation, was dramatically lower in the CRP group than that of the control (Fig. 1B,C). To further explore the underlying mechanisms of CRP-mediated OPCs proliferation, cell cycling was investigated by immunofluorescence. Cyclins D1, D2, and E are G1 phase-associated proteins. Immunofluorescence indicated that incubation with recombinant CRP reduced the protein expression levels of Cyclins D1, D2, and E in the nuclear region and inhibited their nuclear translocation (Fig. 1D,E,F). Western blot analysis also indicated that CRP reduced the levels of G1 phase-associated proteins (Fig. 1G,H). Finally, flow cytometry indicated that the percentage of OPCs in the G2/M phase decreased markedly, while those in the G0/G1 phase increased accordingly in the recombinant CRP-treated group (Fig. 1I,J).
Recombinant CRP suppresses osteoblastic differentiation
Since inflammation leads to increased bone resorption and inhibition of bone formation (33), osteoblastic differentiation of OPCs treated with recombinant CRP was examined by culturing OPCs in OS medium both with and without recombinant CRP. After 14 days, OPCs treated with recombinant CRP had fewer mineralized nodules (Fig. 2A,B). Quantitative measurements demonstrated that calcium deposits were smaller than those of the control group (Fig. 2C). Western blot analysis revealed that levels of protein markers of osteoblastic differentiation were lower in recombinant CRP-treated OPCs than in the control, including ALP and OPN (Fig. 2D,E). Fluorescence staining was used to evaluate the expression levels of the osteoblastic differentiation marker, OCN. The expression of OCN on day 3 was significantly lower than that of the control group (Fig. 2F,G). Finally, the activity of the early osteoblastic differentiation marker ALP was evaluated. The data indicate that ALP activity was lower in OPCs treated with recombinant CRP after 7 days than in the control group (Fig. 2H).
Recombinant CRP abnormally activates the expression of primary cilia during the osteoblastic differentiation of OPCs
To determine the key biological processes involved in osteoblastic differentiation, the R package DESeq2 was used to identify a total of 778 differentially expressed genes (DEG) prior to performing GO and KEGG analysis. The results demonstrated that the DEGs were primarily relevant to three biological processes (BPs): negative regulation of apoptotic process, inflammatory response, signal transduction (Fig. 3A). The MCODE plugin in Cytoscape software was then used to identify the 20 + genes regulated in the key module, from which ten genes were identified as hub genes using the cytoHubba plugin in Cytoscape software. These may be the differentially expressed genes required for the process of osteoblastic differentiation and proliferation (Fig. 3B). Table 1 displays the description and function of the 10 Hub genes. Microtubule activity was shown to be essential for each of these biological processes and cellular components. Since microtubules are an important component of primary cilia, it has been reported that primary cilia also regulate cell cycle processes and can prevent abnormal cell growth by restriction of the cell cycle in studies of the effect of CRP on the primary cilia of OPCs (2, 12). In the present study, as expected, there were more primary cilia on recombinant CRP-treated OPCs than control OPCs, indicating that CRP abnormally activated primary cilia formation (Fig. 3C,D,E). Whether CRP influenced primary cilia disassembly or assembly was also investigated. The primary cilia on OPCs treated with 5 mg/mL recombinant CRP was longer (mean: 5.065 ± 1.45 µm) than those of control cells (mean: 2.195 ± 1.30 µm) (Fig. 3F,G), indicating that CRP positively influenced primary cilia assembly in OPCs. Similarly, Western blot analysis also confirmed that incubation in recombinant CRP was accompanied by higher expression of acetylated-α-tubulin and γ-tubulin (Fig. 3H,I).
Table 1
Descriptions and functions of the 10 Hub genes
Gene.symbol | Description | Function |
AURKA | Aurora kinase A | Cell cycle regulating kinases appear to be involved in the formation and/or stability of microtubules at the spindle pole during segregation of chromosomes. |
NUSAP1 | Nucleolar spindle associated protein 1 | NUSAP1 is a nucleolar-spindle-associated protein that plays a role in spindle microtubule organization. Proteins related to microtubules that promote the formation of mitotic spindles and microtubules. |
MAD2L1 | Mitotic arrest deficient 2-like protein 1 | Component of the spindle-assembly checkpoint that prevents the onset of anaphase until all chromosomes are correctly aligned at the metaphase plate. |
CDC45 | Cell division cycle 45 | The protein encoded by this gene was identified by its strong similarity with Saccharomyces cerevisiae Cdc45, an essential protein required for the initiation of DNA replication, important for early steps of DNA replication in eukaryotes. |
CDC20 | Cell division cycle 20 | Target of the spindle assembly checkpoint and positive regulator that promotes the complex after mitosis which guides the ubiquitination and degradation of certain proteins in the cell cycle and ensures the normal separation of chromosomes. |
TYMS | Thymidylate synthase | The encoded thymidylate-forming enzyme (TS) is the rate-limiting enzyme for pyrimidine nucleotide synthesis and an important factor for tumor growth. |
PRC1 | Protein regulator of cytokinesis 1 | Proteins involved in cytokinesis are the substrates of several cyclin-dependent kinases (CDK). It is necessary for polarizing parallel microtubules and concentrating factors responsible for contractile ring assembly. |
CCNA2 | Cyclin A2 | A protein that binds and activates cyclin-dependent kinase 2 and thus promotes transition through G1/S and G2/M. |
TRIP13 | Thyroid hormone receptor interactor 13 | Gene that encodes a protein that interacts with thyroid hormone receptors, also known as hormone-dependent transcription factors. The gene product specifically interacts with the ligand binding domain. This gene is one of several that may play a role in early-stage non-small cell lung cancer. |
TOP2A | DNA topoisomerase II alpha | This gene encodes a DNA topoisomerase, an enzyme that controls and alters the topologic states of DNA during transcription. This nuclear enzyme is involved in processes such as chromosome condensation, chromatid separation, and the relief of torsional stress that occurs during DNA transcription and replication. It catalyzes the transient separation and rejoining of the two strands of duplex DNA which allows the strands to pass through one another, thus altering the topology of DNA. |
Chloral hydrate removes longer primary cilia and rescues the reduction in osteoblastic differentiation of OPCs caused by treatment with recombinant CRP
To confirm the changes in primary cilia during the osteoblastic differentiation of OPCs, the cells were treated with chloral hydrate, damaging the junction of the primary cilia with the basal body. Compared with the control group, immunofluorescence staining confirmed that the chloral hydrate successfully removed primary cilia from the OPCs in the recombinant CRP-treated group (Fig. 4A,B,C,D). Whether chloral hydrate was able to promote osteogenesis was also investigated by evaluating the effect of chloral hydrate on the expression of genes involved in the osteogenesis of OPCs. Western blot analysis indicated that, compared with recombinant CRP treatment alone, treatment with recombinant CRP and chloral hydrate (10mM) for 3 days caused a marked increase in Collagen TypeIand OPN expression levels (Fig. 4E,F). Finally, ALP activity assays demonstrated that chloral hydrate significantly rescued the recombinant CRP-mediated decrease in ALP activity (Fig. 4G).
Recombinant CRP inhibits OPC osteogenesis via primary cilia/Hh signaling pathway
The Hh pathway has been shown to play a major role in regulating bone formation and osteoblastic differentiation. Recombinant CRP was found to increase protein expression levels of PTCH1 and SUFU in OPCs. However, treatment with SAG (10nM) resulted in the promotion of SUFU to be partially inhibited, but that of PTCH1 was unchanged (Fig. 5A). In contrast, recombinant CRP significantly decreased protein expression levels of Gli2 and SHH in OPCs, while treatment with SAG was partially rescued in Gli2 but the low expression of SHH was not altered (Fig. 5B). To clarify the relationship between primary cilia and the Hh pathway, OPCs were treated with chloral hydrate to remove primary cilia after which the difference in Hh pathway activity was observed with or without primary cilia. Chloral hydrate had the same effect as SAG on expression of the Hh signaling pathway (Fig. 5C,D). Gli2, SUFU, and primary cilia were co-stained using immunofluorescence experiments to further clarify their relationship. After co-staining acetylatedα-tubulin and Gli2, it was found that Gli2 was attached to the bottom of the primary cilia, and the location of its expression was highly coincident with the primary cilia (Supplemental Fig. 2A). By co-staining acetylatedα-tubulin and SUFU, the expression of SUFU was found to increase, but its site of expression was inconsistent with that of primary cilia (Supplemental Fig. 2B,C). The results described above indicated that the primary cilia may regulate the Hh signaling pathway via regulation ofGli2 expression. We also investigated whether SAG promoted osteogenesis. Fluorescence staining demonstrated that SAG markedly increased OCN expression levels which had been decreased as a result of recombinant CRP treatment (Fig. 5E,F). The influence of SAG on the expression of genes that participate in the osteogenesis of OPCs was investigated. Western blot analysis indicated that, compared with treatment using recombinant CRP alone, recombinant CRP combined with SAG (10 nM) for 3 days resulted in a marked increase in the expression of Collagen Type Iand OPN (Fig. 5G,H). Finally, ALP activity assays demonstrated that SAG significantly rescued a recombinant CRP-mediated decrease in ALP activity (Fig. 5I). In summary, the results indicated that recombinant CRP inhibited OPC proliferation by preventing G1-S transition of the cell cycle in vitro, inhibited the Hh pathway, and ultimately inhibited OPCs osteoblastic differentiation by abnormally activating the expression of the cilia.