MACF1 alleviates aging‐related osteoporosis via HES1

Abstract Ageing‐related osteoporosis is becoming an emerging threat to human health along with the ageing of human population. The decreased rate of osteogenic differentiation and bone formation is the major cause of ageing‐related osteoporosis. Microtubule actin cross‐linking factor 1 (MACF1) is an important cytoskeletal factor that promotes osteogenic differentiation and bone formation. However, the relationship between MACF1 expression and ageing‐related osteoporosis remains unclear. This study has investigated the expression pattern of MACF1 in bone tissues of ageing‐related osteoporosis patients and ageing mice. The study has further elucidated the mechanism of MACF1 promoting bone formation by inhibiting HES1 expression and activity. Moreover, the therapeutic effect of MACF1 on ageing‐related osteoporosis and post‐menopausal osteoporosis was evaluated through in situ injection of the MACF1 overexpression plasmid. The study supplemented the molecular mechanisms between ageing and bone formation, and provided novel targets and potential therapeutic strategy for ageing‐related osteoporosis.


| INTRODUC TI ON
Ageing-related osteoporosis is a high incidence disease, which was identified as deterioration in bone mass and bone strength, and it would increase the risk of fracture. 1 The incidence of ageing-related osteoporosis is enhancing along with the ageing of human population and had become a major threat to the health of senile human.
The causes of ageing-related osteoporosis are variable and complex, and researchers have found that osteogenic differentiation is inhibited via multiple signalling pathways during ageing processes both in male population and in post-menopausal female population, which resulted in declined bone formation and osteoporosis gradually. 2,3 Among all the ageing-related signalling pathways, Wnt/β-catenin pathway and Notch signalling pathway have been reported to highly correlate with ageing-related osteoporosis. [4][5][6] Wnt/β-catenin pathway is one of the most essential pathways regulating osteogenesis, it activates the expression of multiple osteogenic factors by its transcription factor TCF7/LEF1. 3,4 Notch signalling pathway also played an important role on osteoblast differentiation. Hairy/enhancer of split 1 (HES1) is the transcription factor, which functions as the downstream of the Notch signalling pathway and inhibits bone formation, 6 and Hilton's study proved that HES1 physically interacted with osteogenic factor RUNX2 and diminished its activity, thus inhibited osteoblast differentiation. 7 Microtubule actin cross-linking factor 1 (MACF1) also named ACF7 (actin cross-linking factor 7), is a member of the plakin family. 8 It is a 600 kDa cytoskeletal protein that cross-links with actin and microtubules, and MACF1 regulates multiple physiological and pathological processes. [8][9][10][11][12][13] In cellular level, MACF1 regulates intracellular material transportation and thus plays an important role in signalling pathways. Chen has found MACF1 participates in Wnt/β-catenin pathway by facilitating β-catenin to dissociate from the β-catenin/AXIN/APC complex and thus transport to cell nucleus. 14 Burgo's study has found that MACF1 manipulates vesicles transporting from the trans-Golgi network to cell periphery. 15 Previous studies have proved that MACF1 promotes osteogenic differentiation and bone formation. [16][17][18][19] In Hu's study, MACF1 promoted osteoblast differentiation by stabilizing β-catenin. 16 In Zhang's study, MACF1 overexpression plasmid was transfected to osteoblast in vitro and in vivo, thus enhanced osteoblast differentiation and bone formation. 17 These studies implied that MACF1 plays an essential role on regulating osteogenesis; however, reports on the effects of MACF1 on ageing-related osteoporosis are still limited.
In this study, we explored the expression levels of MACF1 in femur tissues of ageing patients and ageing mice. And we have found that MACF1 expression was associated with the reduction in osteogenesis in ageing-related osteoporosis. The mechanism of MACF1 promoting bone formation was also investigated. The results implied that HES1 expression and activity might involve in aging related osteoporosis. Enhancing the expression of MACF1 by plasmids would efficiently rescue bone formation in ageing-related osteoporosis and post-menopausal osteoporosis mice.  and Institution Review Board of the Northwestern Polytechnical University (12 September 2016). All participants were provided with informed written consent before their participation in this study.

| Cell culture, human sample and mice model
Ageing and ovariectomized (OVX) mice were adopted to construct the osteoporosis model. All mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University (Xi'an, China). For ageing male mice model, eighty 6-month-old C57BL/6 mice were maintained under standard animal housing conditions (12-h light, 12-h dark cycles and free access to food and water). 61 mice were kept until 21 months old and selected as ageing group, whereas 19 mice were used as normal group. 38 mice (19 from ageing male group and 19 from normal group) were killed, and femurs were collected and processed for bone marrow mesenchymal stem cell isolation (n = 5/group), RT-PCR (n = 5/group), Western blot (n = 3/group), immunohistochemical staining (n = 3/group) and RNAseq (n = 3/group). The other 42 mice were processed for MACF1 overexpression plasmid (PEGFP-C1A-ACF7) in vivo transfection.
For ageing female mice model, six 6-month-old C57BL/6 mice were maintained under standard animal housing conditions. Three mice were kept until 21 months old and used as ageing group, and other 3 mice were used as normal group. All mice were killed, and femurs were collected for RNA-seq (n = 3/group

| Isolation of bone marrow mesenchymal stem cells (BMSCs)
Mice bone marrow mesenchymal stem cells were isolated for investigating the effect of ageing on osteogenic differentiation. 6-and 21-month-old mouse were killed, and mice femurs were immediately harvested. Attached soft tissues on femoral bones were carefully removed. Bone marrow was washed and collected by flushing several times with phosphate-buffered saline (PBS) using a 25G syringe needle. The collected PBS with bone marrow was centrifuged (1200 g, 8 minutes) and dissociated by culture medium (α-MEM, Gibco supplemented with 10% foetal bovine serum, 1% L-glutamine, 1% penicillin and streptomycin) using a 29G syringe needle. The suspension was cultured in a 60-mm plate for 3 hours (37°C, 5% CO 2 ) followed by careful wash with culture medium. Cells were cultured for another 36 hours with culture medium changed every 12 hours. The cells were transferred into a new plate as the 1st passage cells. Third passage cells were used for characterization and experiments.

| RNA-seq analysis
RNA-seq was used to assess expression levels of ageing-related mRNAs. Total RNA was extracted from 6 to 21 months of both male and female mouse femurs, respectively, using TRIzol Reagent

| Real-time PCR
RT-PCR was used to assess expression levels of MACF1 and osteogenic markers. Total RNA was extracted from femur tissues or cultural cells using TRIzol reagent. Femur tissues were harvested, washed by RNAlater solution, grind with liquid nitrogen and then digested by TRIzol reagent. 1μg of total RNA was used for cDNA synthesis using One Step PrimeScript RT Reagent Kit (Takara, RR037A, Dalian, China). Quantitative PCR amplification was performed with the Thermal Cycler C-1000 Touch System (Bio-Rad CFX Manager, Hercules, CA) and SYBR Premix Ex Taq II Kit (Takara, RR820A).
Gapdh was used as internal control gene. The quantitative PCR conditions included initial denaturation step at 95°C for 30 seconds, followed by 42 cycles at 95°C for 10s, 60°C for 30s and 72°C for 5s. Data were calculated using the comparative Ct method (2 −ΔΔCt ) and expressed as fold change compared with corresponding control. Primers (sequences;

| Western blot
For the detection of protein levels, Western blot analysis was performed as previously described. 21  China) and visualized using chemiluminescence detection system (Thermo Fisher Scientific, NCI5080). Protein bands were exposed to X-ray film (Kodak, 6535876). GAPDH was adopted as internal control.

| Immunohistochemical staining
To investigate the effect of ageing on osteogenic gene and transcription factor expression levels, and to determine the rescue effect of MACF1 on osteoporosis, immunohistochemical staining analysis was performed as previously described. 22 Human and mouse femur samples, along with mouse calvarias, were dissected and fixed in 4% paraformaldehyde, and decalcified in

| Micro-CT analysis
To investigate the rescue effect of MACF1 on ageing-related osteoporosis, mice distal femoral was scanned by the micro-CT system

| Therapeutic effect of MACF1 ageing and OVX mice
To investigate rescue effect of MACF1 to ageing-related osteoporosis, forty-two 21-month-old mice were randomly di- In the baseline and OVX group, mice were given no treatment.
All mice received the same standard diet during the experimental period. All mice of the baseline group and 3 mice from other groups were killed 15 days after OVX treatment. All other mice were killed 38 days after OVX, and calvarias were collected. For baseline group, mice were killed 10 days after OVX treatment.

F I G U R E 1
The decreased MACF1 level is associated with bone formation reduction in ageing-related osteoporosis patient femur tissues. A-C, Expression of MACF1, OCN and OSTERIX in femur tissues of ageing-related osteoporosis patients with age of 60-79 and 80-95, respectively, as detected by RT-PCR (up) and Western blot (below) (mean ± SD, ***P < .001). D, Expression of MACF1, OCN and OSTERIX in femur tissues of ageing-related osteoporosis patients with age of 60-79 and 80-95, as detected by immunohistochemical staining. Scale bar: 50 μm. E, Quantification of relative integrated optical density (IOD) values of MACF1, OCN and OSTERIX immunostaining using Image-Pro Plus 6.0 software (mean ± SD, **P < .01, ***P < .001). F-G, Correlation analysis between Macf1 levels and Ocn or Oxterix mRNA levels in femur tissues from ageing-related osteoporosis patients, respectively, as detected by RT-PCR

| Statistical analysis
All experiments were independently repeated at least three times with each done in triplicate. The statistical analyses of the data were performed with GraphPad Prism version 6.0 software (GraphPad Software Inc), and a Student t-test was used. The data are presented as mean ± standard deviation (SD). P values <.05 were considered statistically significant for all comparisons.

| Reduced MACF1 level is associated with the reduction in osteogenesis in elderly people
The

| MACF1 promotes bone formation by inhibiting the activities of HES1 in ageing-related osteoporosis
The mechanism of how MACF1 regulated bone formation was further explored. We established RNA-seq for femur tissues collected from both male and female ageing mice. We found the expression levels of Hes1 were significantly increased in 21-month ageing mice compared with 6-month control ( Figure 4A). The results were further confirmed by RT-PCR, Western blot and immunohistochemical staining in ageing human and mice femur tissues ( Figure 4B-C, Figure S1). Moreover, in femurs of different ages of C57BL/6 mice, expression level of Hes1 was positively correlated with P16 ( Figure 4D) convincing that HES1 expression was increased with the ageing process.
We then investigated the regulation effect of MACF1 on HES1.

| Rescue effects of MACF1 on ageing-related osteoporosis
For further evaluating the effects and mechanism of MACF1 on ageing-related osteoporosis, 21-month ageing C57BL/6 mice received a periosteal injection into medullary cavity of femur with MACF1 overexpression plasmid and transfection reagent [25][26][27] ( Figure 5A and β-C-telopeptides of type 1 collagen (β-CTX) level showed no significant differences in MACF1-treated ageing mice and revealed the treatment of MACF1 showed no effect on femoral bone resorption ( Figure S6A-D). Serum level of type 1 amino-terminal pro-peptide F I G U R E 2 The decreased MACF1 level is associated with bone formation reduction in ageing mice femur tissues. A-C, Expression of MACF1, OCN and OSTERIX in femur tissues of 6-and 21-mo male C57BL/6 mice, respectively, as detected by RT-PCR (up) and Western blot (below) (mean ± SD, ***P < .001). D, Expression of MACF1, OCN and OSTERIX in distal femur tissues of 6-and 21-mo male C57BL/6 mice, as detected by immunohistochemical staining. Scale bar: 50 μm. E, Quantification of relative integrated optical density (IOD) values of MACF1, OCN and OSTERIX immunostaining using Image-Pro Plus 6.0 software (mean ± SD, ***P < .001). F, Representative images showing distal femur mineral apposition rate of 6-and 21-mo male C57BL/6 mice, as detected by double calcein labelling (mean ± SD, ***P < .001). Scale bar: 10 μm. G-I, Correlation analysis between Macf1 levels and P16, Ocn or Oxterix mRNA levels in femur tissues from C57BL/6 mice, respectively, as detected by RT-PCR Expression levels of HES1 in MACF1-treated ageing mice were detected by immunohistochemical staining, and HES1 level in the trabecular bone osteogenic cells was reduced by MACF1 ( Figure 6A-B). In MACF1-treated mice BMSCs, HES1 mRNA and protein levels were both significantly decreased, as compared with normal ageing mice and control plasmid-treated mice ( Figure 6C). Moreover, the activity of HES1 in mice BMSCs also presented a 52.8% (P < .001) decrease after MACF1 treatment ( Figure 6D). The results implied that MACF1 may relieve ageing-related osteoporosis through inhibition of HES1.

| Rescue effects of MACF1 on post-menopausal osteoporosis
We showed that average calvarial thickness was decreased by ovariectomy and increased by MACF1 treatment (Figure 7A-B). Meanwhile, the mineral apposition rate in the OVX mice was decreased by 41.8% (P < .001) and increased by 139.8% (P < .001) after MACF1 overexpression plasmid treatment ( Figure 7C-D). Immunohistochemistry staining results showed the protein levels of OCN were significantly down-regulated after OVX surgery, while was up-regulated by MACF1 ( Figure 7E-F). These data suggested that enhancing the expression level of MACF1 could promote bone formation and therefore rescue the minus consequence of menopause. The differentiation of osteogenic cells can be impacted by many factors. MACF1 is a cytoskeletal protein that regulates actin and microtubule dynamics. [8][9][10][11][12] It also plays a role in regulating intracellular transport. 14,15 Our previous studies have shown that MACF1 positively regulated osteoblast differentiation and bone formation.

| D ISCUSS I ON
Qian has proved that MACF1 highly expressed in pre-osteoblast cell line MC3T3-E1. 28  was associated with the reduction in osteogenic differentiation and bone formation as well (Figures 1-3). Through all these results, we have firstly proved that MACF1 act as an important regulator for ageing-related osteoporosis.
Reports have shown that during the process of ageing, the expression of both Wnt/β-catenin and NOTCH signalling pathways was F I G U R E 4 MACF1 promoted bone formation by inhibiting the activities of HES1 in ageing-related osteoporosis. A, Selected area of RNA-seq heat map for femur tissues of 6-and 21-mo male (up) and female (below) C57BL/6 mice. B, Expression of HES1 in femur tissues of ageing-related osteoporosis patients (left) and ageing C57BL/6 mice (right), as detected by RT-PCR (up) and Western blot (below) (mean ± SD, ***P < .001). C, Expression of HES1 in femur tissues of ageing-related osteoporosis patients (up) and ageing C57BL/6 mice (below), as detected by immunohistochemical staining. ageing-related osteoporosis. [5][6][7] In this study, we also proved that HES1 expression was increased in femur tissues of ageing human and mice. We have also found MACF1 negatively regulated HES1 expression and activities (Figure 4), which indicated a potential mecha- B, Quantification of relative integrated optical density (IOD) values of HES1 immunostaining using Image-Pro Plus 6.0 software (mean ± SD, **P < .01). C, Expression of HES1 in BMSCs of ageing C57BL/6 mice distal femur after MACF1 overexpression plasmid treatment, as detected by RT-PCR (up) and Western blot (below) (mean ± SD, ***P < .001). D, Activities of HES1 in BMSCs of ageing C57BL/6 mice distal femur after MACF1 overexpression plasmid treatment, as detected by luciferase reporter assay (mean ± SD, ***P < .001) of mice calvaria. 17 Yet, the symptoms in ageing-related osteoporosis mostly affect weight-bearing bones, especially femur, lumbar vertebra and tibia. 29,30 In Li, Yuan and Zhao's studies, miRNAs or siRNAs targeting osteogenic genes were administrated into the bone marrow of mice femur by periosteal injection and enhanced bone formation. [25][26][27] In this study, MACF1 overexpression plasmid was injected into medullary cavity of mice femur by the same technique, and the treatment had successfully inhibited HES1 level and mice after OVX treatment and MACF1 overexpression plasmid transfection (mean ± SD, ***P < .001). C, Representative images showing calvarial mineral apposition rate of C57BL/6 mice after OVX treatment and MACF1 overexpression plasmid transfection, as detected by double calcein labelling. Scale bar: 10 μm. D, Calvarial mineral apposition rates of C57BL/6 mice after OVX treatment and MACF1 overexpression plasmid transfection (mean ± SD, **P < .01). E, Expression of OCN in calvarial tissues of C57BL/6 mice after OVX treatment and MACF1 overexpression plasmid transfection, as detected by immunohistochemical staining. Scale bar: 50 μm. F, Quantification of relative integrated optical density (IOD) values of OCN immunostaining using Image-Pro Plus 6.0 software (mean ± SD, ***P < .001) enhanced ageing mice bone formation ( Figures 5-6), which firstly proved plasmids could be adopted in periosteal injection rescue, and also suggested that overexpression of MACF1 would be a potential therapeutic strategy for ageing-related osteoporosis.
However, MACF1 was also proved enhancing the activities of osteoclast. 31 The overexpression of MACF1 might also aggravate bone resorption of ageing mice. We have measured the bone resorption levels of MACF1-treated ageing mice and found no significant differences. To clarify this conflict, we analysed the transfection efficiency of the large MACF1 overexpression plasmid (21kb) on osteoclast. Result showed the transfection efficiency was extremely low in both monocytic RAW264.7 cell line and extracted primary osteoclast, which may explain the reason why MACF1 overexpression plasmid treatment showed no effect on ageing mice bone resorption.
Moreover, the effect of MACF1 on post-menopausal osteoporosis was also proved. MACF1 overexpression plasmid was injected subcutaneously over the calvarial surface. 22 We found that MACF1 Through this study, we have discovered the relationship and mechanism between MACF1 and ageing-related osteoporosis.
MACF1 expression was reduced during the ageing process. The reduction in bone formation in ageing-related osteoporosis was caused by decreased MACF1 level leading to disabled suppression of HES1 expression. Through these findings, we have proved that enhancing MACF1 expression would rescue ageing and post-menopausalrelated osteoporosis. This study has discovered a new molecular mechanism for ageing-related osteoporosis and also provided new insights for therapeutic strategy of osteoporosis.