key: cord-0754352-57qa23om
authors: Zhao, Zhen‐qun; Liu, Wan‐lin; Guo, Shi‐bing; Bai, Rui; Yan, Jing‐long
title: Mechanism of Methylprednisolone‐Induced Primary Cilia Formation Disorder and Autophagy in Osteoblasts
date: 2020-02-16
journal: Orthop Surg
DOI: 10.1111/os.12630
sha: 681a4dcd3ab332a84758adae27e1266e62e974aa
doc_id: 754352
cord_uid: 57qa23om

OBJECTIVE: To study the role of primary cilia formation disorder and osteoblasts autophagy in the pathogenesis of steroid‐induced avascular necrosis of the femoral head (SANFH). METHODS: Osteoblasts were isolated from rabbit bones and treated with 1 μM Methylprednisolone for 0, 12, 24, 48, and 72 h. The Beclin1, MAP1LC3, Atg‐5, Atg‐12, IFT20 and OFD1 mRNAs and proteins were detected by PCR and Western blotting, and their correlation was statistically analyzed. The lengths of osteoblast cilia were measured under a laser confocal microscope, and the autophagy flux was tracked by transfecting the osteoblasts with GFP‐RFP‐LC3 lentivirus. RESULTS: Methylprednisolone significantly upregulated Beclin1, MAP1LC3, Atg‐5, Atg‐12 and OFD1 mRNAs and proteins in a time‐dependent manner, and decreased that of IFT20 (P < 0.05). In addition, the autophagy flux in the osteoblasts also increased and the ciliary length decreased in a time‐dependent manner after Methylprednisolone treatment. The length of the cilia were 5.46 ± 0.11 um at 0 h, 4.08 ± 0.09 um at 12 h, 3.07 ± 0.07 um at 24 h, 2.31 ± 0.10 um at 48 h, and finally 1.15 ± 0.04 um at 72 h. Methylprednisolone treatment also affects primary cilium numbers in cultures, for 0 to 72 h. The autophagy regulatory genes, Beclin1, MAP1LC3, Atg‐5 and Atg‐12, were found to be negatively correlated with IFT20, with an average correlation coefficient of −0.81. A negative correlation was also found between OFD1 and IFT20, with an average correlation coefficient of −0.53. CONCLUSION: Methylprednisolone inhibits primary cilia formation and promotes autophagy, which could be the pathological basis of SANFH. The exact regulatory mechanism needs to be further studied in vivo.

O steonecrosis of the femoral head affects 20 mn people worldwide, with more than 200,000 cases diagnosed annually in China 1,2 . A multicenter review of 6395 patients with osteonecrosis of the femoral head in China found that 24.1% of the cases, especially those without trauma, were steroid induced 2 . In addition, steroid-induced avascular necrosis of the femoral head (SANFH) frequently occurs in young and middle-aged people, and is associated with a high risk of disability since it involves bilateral femoral heads. Interestingly, numerous cases of SANFH emerged after SARS in 2003 3, 4 , although the pathological basis remains unknown, thereby limiting prevention and treatment.

The loss of cartilage cells, osteocyte, and osteoblasts is hypothesized to drive SANFH 5, 6 , although the type of cell death is not completely clear. Autophagy is an auto-catabolic recycling process of eukaryotic cells that is frequently dysregulated in cancer, inflammatory diseases, autoimmune diseases, aging, etc [7] [8] [9] [10] . Our preliminary results show that SANFH is associated with autophagy, and the autophagyrelated Beclin 1 and microtubule-associated protein 1 light chain 3 (MAP1LC3) are upregulated in osteoblasts 11 . Primary cilia are sensitive cellular antennae that sense and transmit external signals 12 , and coordinate the responsive mechanisms 13 . Recent studies show that the formation of primary cilia depends on autophagy 14 . The intraflagellar transport 20 (IFT20) protein which regulates ciliary formation and length 15, 16 is upregulated during starvation or lysosomal inhibition, and its knockdown during autophagy inhibits cilia formation 17 . IFT20 also recruits non-ciliary proteins to the plasma membrane, which is the site of autophagosome formation. Furthermore, autophagy is significantly decreased when ciliary formation is disrupted, which is the basis of ciliated disease 18 . The oral facial digital syndrome-1 (OFD1) is a centrosome satellite protein that suppresses cilia formation, and its autophagic degradation promotes the biosynthesis of primary cilia 19, 20 .

It is not clear at present whether primary cilia growth is related to avascular necrosis of the femoral head, or whether osteoblast autophagy affects primary cilia formation during local ischemia and hypoxia of the femoral head. To this end, we investigated the relationship between osteoblast autophagy and primary cilia formation in the presence of steroids. The purpose of this study is to: (i) determine whether primary ciliary formation disorder is associated with SANFH; (ii) determine whether osteoblast autophagy is associated with SANFH; and (iii) examine the relationship between primary cilia formation disorder and autophagy in osteoblasts under the action of Methylprednisolone.

Osteoblast Twenty white rabbits from New Zealand aged 8 weeks and weighing 1.5 AE 0.2 kg (Jiangsu Yizheng Biotechnology Co. Ltd., Nanjing. China, Certificate No. SCXK [Su] 20160005) were raised in single cages exposed to natural light. The animal experiments conducted were in accordance with the 2009 "Ethical issues in animal experimentation," and approved by the Ethics Reviewing Council of Second Affiliated Hospital of Inner Mongolia Medical University (NO.YKD2016143, Inner Mongolia, China). The rabbits were anesthetized with intravenous injection of pentobarbital sodium (Sigma, Shanghai, China) into their ears. After swabbing the legs with alcohol (Nanjing Shengxing Biology, Nanjing, China), the animals were dissected and their femurs were removed. The bones were rinsed with sterile PBS (Nanjing Shengxing Biology, SN331, Nanjing, China), cut into small pieces, and washed once with DMEM (Hyclone, Shandong, China). The extraneous tissues were sequentially digested with 0.25% trypsin (Hyclone, Shandong, China) and 1 mg/mL collagenase I (Solarbio, C8140, Shanghai, China) for 20 min each at 37 C, and removed by centrifuging at 1000 rpm for 5 min. The marrow was flushed out of the cleaned bones with DMEM supplemented with 15% serum (Hyclone, Shandong, China), and incubated at 37 C with the medium replaced every 3 days. Once the cells were 50% confluent, the medium was replaced with DMEM containing 10% FBS and 1% penicillin + streptomycin (Hyclone, Shandong, China). The cells were harvested once they grew to 80%-90% confluency using 0.25% trypsin as per standard protocol.

Total RNA and miRNAs were isolated from the osteoblasts using TRIzol Reagent (Invitrogen, Shanghai, China) and miRNA Extraction Kit (Ribobio, Guangzhou, China) respectively, and the former was reverse transcribed to cDNA using PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). RT-PCR was performed using SYBR Premix Ex Taq and SYBR Green I PCR Kit (TaKaRa, Dalian, China) in the ABI Step One Plus Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Shanghai, China) with the following cycling conditions: initial denaturation for 5 min at 95 C and 40 cycles of denaturation at 95 C for 10 s, followed by annealing and extension at 60 C for 34 s. All steps were performed as per the manufacturers' instructions, and the experiment was repeated thrice. The primer sequences were as follows: Beclin1-forward 5 0 -GTACAGGATGGATGTGG AGAAAG-3 0 and reverse 5 0 -TGTCCACTGTTCCTCAGAA TTG-3 0 ; LC3-forward 5 0 -AGAACGATACAAGGGTGAGA AG-3 0 and reverse 5 0 -GGCGCCTCCTGATGATTT-3 0 ; Atg5-forward 5 0 -TGAAAGGGAAGCAGAACCATAC-3 0 and reverse 5 0 -TCAGTGGTGTGCCTTCATATTC-3 0 ; Atg12-forward 5 0 -CTGGCGAC ACCAAGAAGAAA-3 0 and reverse 5 0 -GGATGGTTCTTGTTCGCTCTAC-3 0 ; IFT20forward 5 0 -GACCATAGAGCTCAAGGAAGAG-3 0 and reverse 5 0 -GCAAGTTGGTCAACGAGTTC-3 0 ; OFD1forward 5 0 -TCAAAGACACGAGCGATGAC-3 0 and reverse 5 0 -CTGTCCTTCATCCTTGCTAAGT-3 0 ; GAPDH-forward 5 0 -GCGTGAACCACGAGAAGTAT-3 0 and reverse 5 0 -CCT CCACAATGCCGAAGT-3 0 .

Total protein was extracted by homogenizing each sample with 100 μL RIPA lysis solution (Aladdin Biochemistry, Shanghai, China) on ice for 30 min. The lysates were centrifuged at 12,000 rpm for 5 min at 4 C, and the protein content of the supernatants were measured using the BCA assay kit (Biyuntian, Shanghai, China). Equal amounts of protein per sample were separated by SDS-PAGE using the Mini-PROTEAN 3 system (Bio-Rad, Shanghai, China), and the protein bands were transferred to PVDF membranes using the Trans-Blot SD wet transfer system (Bio-Rad, Shanghai, China). After blocking the membranes with 5% skim milk powder in TBST buffer (Shanghai, China) at room temperature for 1 h, they were incubated overnight with the primary antibodies in TBST solution containing 5% BSA (Aladdin Biochemistry, Shanghai, China) at 4 C. The blots were washed thrice with TBST at room temperature, and then incubated with the secondary antibody at room temperature for 2 h. After washing thrice with TBST, the blots were developed using the Tanon chemiluminescence sensor system (Tanon, Shanghai, China). GAPDH was used as the loading control.

Osteoblasts were seeded in 3.5 cm glass bottom petri dishes at the density of 2 × 10 6 cells/dish, and allowed to adhere overnight. The complete medium was replaced with serumfree medium, and the cells were infected with GFP-RFP-LC3 lentivirus (Gima Pharmaceutical Co. Ltd., Shanghai, China) at the MOI of 1:50. After 6 h, the medium was removed and fresh complete medium was added. The cells were cultured for 48 h, and transduction efficiency was observed under a fluorescence microscope. The stably transduced cells were treated with 1 μM Methylprednisolone for 0, 12, 24, 48, or 72 h, and fixed with 4% paraformaldehyde at room temperature for 10 min. The green and red fluorescence respectively indicating autophagosome and autophagolysosome formation were observed by laser confocal microscope.

Glass slides were pre-coated with 0.1% agar either overnight at room temperature or at 37 C for 2 h, and sterilized under ultraviolet light for 30 min. The osteoblasts were seeded onto the slides and, after overnight culture, treated with 1 μM Methylprednisolone followed by serum starvation (0.5% FBS) for 24 h to induce ciliation. The cells were washed thrice with PBS and fixed with 4% paraformaldehyde for 15 min at 4 C. After washing again with PBS three times, the cells were permeabilized and blocked with 3% BSA/0.3% NP-40 for 30 min with constant shaking at room temperature. The cells were incubated overnight with the primary antibody against α-tubulin (1:1000) at 4 C, washed thrice with 0.1% PBS-Tween, followed by the secondary antibody (1:200) at room temperature for 1 h. After washing thrice with 0.1% PBST, the cells were counterstained with DAPI, sealed, and observed under a laser confocal microscope. The images were analyzed using the Zen software (Zeiss, Shanghai, China).

The methylprednisolone-treated cells were fixed overnight in 2.5% glutaraldehyde at 4 C, followed by 1% osmium tetroxide for 1 h at room temperature. The fixed cells were immobilized in 10% gelatin and fixed again with glutaraldehyde for 1 h at 4 C. After dehydrating across an ethanol gradient (30%, 50%, 70%, 90%, 95%, 100%, 100%), the samples were immersed in epoxy resin, cut into ultrathin sections with a Leica UC6 (Leica, EM UC6, Germany), and observed under a transmission electron microscope (TEM) (JEM1011, Japan).

The results were presented as mean AE standard deviation of three independent experiments. All statistical analyses were conducted using SPSS 20.0 software (IBM, Armonk, NY, USA). Beclin1, MAP1LC3, Atg-5, Atg-12 and OFD1 mRNAs and proteins, numbers and length of primary cilium were compared by one-way analysis of variance (ANOVA), followed by LSD test. The correlation between autophagy gene expression and cilia length was analyzed using linear correlation analysis. P values <0.05 were considered statistically significant.

Osteoblasts are arranged in a monolayer, and the cell bodies are either cubic or dwarf columnar in shape. There are many small processes on the cell surface, which form a gap junction with adjacent osteoblasts or osteocyte. The nucleus is large and round, the cell culture is scale-like, the cytoplasm is rich, and pseudopods are present (Fig. 1) .

Under an electron microscope, the autophagosome appears grayish white, and there is a double-layer or multi-layer membrane-like structure that has a tendency to wrap around the cytoplasm, and the vacuole-like structure containing cytoplasmic components is wrapped by a doublelayer or multi-layer membrane (with an average diameter of about 500 nm). The monolayer film structure surrounds broken and degraded components (Fig. 2) . After Methylprednisolone was added to osteoblasts, during an extension of time (0 to 72 h), the number of autophagosomes gradually increased in the osteoblasts, as seen under an electron microscopy, but this increase could not be statistically analyzed.

After the merge of red and green fluorescence, the visible yellow spots were autophagosomes. The red spots indicate autophagy lysosomes. If the phagosome and lysosome can fuse, then red fluorescence increases, compared with that of the yellow fluorescence. However, when there is an autophagy block downstream, phagosomes and lysosomes cannot fuse as normal, and only a yellow fluorescence is mainly visible. As shown in Fig. 3 , after treatment with Methylprednisolone for 12 to 72 h, compared with the control and the 0 h group, a gradually increasing number of RFP-GFP-LC3 double-label positive staining cells were found. For 0 to 72 h, the average positive staining cells were 3%, 8%, 10%, 21%, and 35%.

As shown in Fig. 4 , to prevent lentivirus from affecting autophagy flow results, autophagic flux was also analyzed by assessing p62 levels following treatment at the different time points. For 0 to 72 h, the average gray value of p62 was 1.56, 1.45, 1.26, 0.95, and 0.63, respectively. Compared with 0 h from 12 to 72 h, the P values were 0.081, 0.005, 0.0003, and 0.0001, respectively.

Primary Cilia Length When Methylprednisolone was added to the osteoblasts, the primary cilia of the osteoblasts gradually shortened along with an increase in time (Fig. 5) . The length of the cilia were 5.46 AE 0.11 um at 0 h, 4.08 AE 0.09 um at 12 h, 3.07 AE 0.07 um at 24 h, 2.31 AE 0.10 um at 48 h, and finally 1.15 AE 0.04 um at 72 h. The ANOVA analysis found significant differences among the four groups (P = 0.003), while the LSD test analysis also found significant difference within groups (P = 0.006). Compared with 0 h, the length of primary cilia from 12 to 72 h was 74.73%, 56.23%, and 42.31%, respectively.

Methylprednisolone treatment also affects primary cilium numbers in cultures (Fig. 6) . Each group counted 200 cells 

The results of the PCR show that in the key autophany genes, Beclin1, MAP1LC3, Atg-5 and Atg-12, and the primary cilia regulatory gene, OFD1, mRNA levels gradually increased at 0, 12, 24, 48, and 72 h after Methylprednisolone treatment, while in the primary cilia regulatory gene, IFT20, mRNA levels gradually decreased. Significant differences were found between levels of Beclin1, MAP1LC3, Atg-5, Atg-12, OFD1 and IFT20 at 12, 24, 48, and 72 h after Methylprednisolone treatment, compared with the GAPDH control group (P < 0.01) (Fig. 7-9 ).

The autophagy regulatory genes Beclin1, MAP1LC3, Atg-5 and Atg-12 were found to be positively correlated with OFD1, with an average correlation coefficient of 0.76. The autophagy regulatory genes, Beclin1, MAP1LC3, Atg-5 and Atg-12, were found to be negatively correlated with IFT20, with an average correlation coefficient of −0.81. A negative correlation was also found between OFD1 and IFT20, with an average correlation coefficient of −0.53. 

L ong-term use of glucocorticoids decreases bone mineral density, reduces the number of osteoblasts and osteoclasts, and prolongs osteoclast survival, eventually resulting in an imbalance between bone resorption and remodeling 21 . However, only 15% to 20% of the osteocytes in the femoral head were apoptotic in the mouse model of SANFH 22 , indicating other pathological mechanisms. Lane et al. found that glucocorticoids increase the gap between osteocytes, which results in the loss of minerals and disrupts bone metabolism independent of apoptosis 23 . Xia et al. first reported that dexamethasone increased autophagy in osteoblasts, which was verified in the primary osteoblasts isolated from prednisolone-treated mice 24 . In the current study, we detected increased Beclin 1 expression in the femoral head tissues of a rabbit model of SANFH, indicating that autophagy is also a pathological basis of SANFH. This is consistent with a previous study which showed that inhibition of autophagy by 3-MA retarded SANFH development in vivo 25 . Under physiological conditions with functioning osteoblasts, necrosis of the femoral head is repaired naturally. Therefore, glucocorticoids likely inhibit osteoblast function to aggravate SANFH. Luo et al. reported that Methylprednisolone increased autophagy in bone cells in a model of SANFH in a dosedependent manner, which also affects the interaction between osteoblasts and osteoclasts 6 . We found that Methylprednisolone upregulated the key autophagy genes in the osteoblasts, along with increasing autophagy flux.

IFT20 and OFD1 regulate primary cilia formation and length in various cell types [15] [16] [17] 19, 20 . Delaine-Smith et al.

found that the primary cilia of osteoblasts responded to fluid shear stress and interstitial flow-induced deposition of calcium in the bone 26 , which was confirmed by a later study 27 . We found that Methylprednisolone decreased the cilia length of osteoblasts by upregulating OFD1 and downregulating IFT20. This is consistent with the study of Tang et al., which showed that autophagy promoted cilia formation through selective degradation of OFD1. While LC3 specifically targeted OFD1 to the autophagy corpuscles for degradation, its centrosomal and basal parts were retained 28 . Pampliega et al. found that IFT20 was necessary for cilia formation and assembly, and was also degraded by autophagy to inhibit ciliation 29 . Taking both studies together, selective elimination of IFT20 during cell growth and proliferation hinders cilia formation while degradation of OFD1 during growth arrest promotes cilia formation. In this study, glucocorticoids gradually upregulated OFD1 and decreased IFT20 in the osteoblasts. We concluded, therefore, that glucocorticoids block the signal transduction of osteoblasts by selectively eliminating IFT20 during cell growth and proliferation.

Recent studies have shown a regulatory role of cilia in autophagy 30 . Wang et al. analyzed two renal cell lines with short and long cilia, and found that the former were activated by mTOR and resistant to autophagy 31 . In addition, Orhon et al. induced autophagy through the mechanical stress of fluid flow, which was regulated by the signal transduction pathway of primary cilia 32, 33 . Jang et al. found that inhibition of cilia-mediated autophagy partly hindered neuroectoderm differentiation, underscoring the functional role of cilia in autophagy regulation 34 . Taken together, autophagy and cilia formation regulate each other, but the exact regulatory mechanism needs to be further studied. We found that the autophagy genes were positively correlated with OFD1 and negatively correlated with IFT20.

Our study still has limitations. This study is an in vitro experiment and we cannot confirm whether the in vivo experiment has the same pathological process. This study is the effect of Methylprednisolone on osteoblasts and we cannot confirm whether other glucocorticoid have similar effects. Finally, this study can only confirm the relationship between osteoblast autophagy and primary cilia formation disorder in SANFH, but the exact regulatory mechanism is still unclear. To the best of our knowledge, this is the first study reporting that excessive autophagy and primary cilia disruption are potential mechanisms underlying osteoblast loss in SANFH. To summarize, glucocorticoid disturbs primary cilia formation in osteoblasts, which likely affects osteogenesis and destroys the dynamic balance between bone generation and resorption, eventually leading to SAFH.

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