Cav 1.3 damages the osteogenic differentiation in osteoporotic rats by negatively regulating Spred 2‐mediated autophagy‐induced cell senescence

Abstract Cav 1.3 can affect the classical osteoclast differentiation pathway through calcium signalling pathway. Here, we performed cell transfection, real‐time fluorescence quantitative PCR (qPCR), flow cytometry, SA‐β‐Gal staining, Alizarin Red S staining, ALP activity test, immunofluorescence, Western blot and cell viability assay to analyse cell viability, cell cycle, osteogenesis differentiation and autophagy activities in vitro. Meanwhile, GST‐pull down and CHIP experiments were conducted to explore the influence of Cav 1.3 and Sprouty‐related EVH1 domain 2 (Spred 2) on bone marrow–derived mesenchymal stem cells (BMSCs). The results showed that OS lead to the decreased of bone mineral density and differentiation ability of BMSCs in rats. Cav 1.3 was up‐regulated in OS rats. Overexpression of Cav 1.3 inhibited the activity of BMSCs, the expression of alkaline phosphatase (ALP), runt‐related transcription factor 2 (RUNX2) and osteocalcin (OCN), as well as promoted the cell cycle arrest and senescence. Furthermore, the negative correlation between Cav 1.3 and Spred 2 was found through GST‐pull down and CHIP. Overexpression of Spred 2 increased the expressions of microtubule‐associated protein 1 light chain 3 (LC3) and Beclin 1 of BMSCs, which ultimately promoted the cell activity of BMSCs and ALP, RUNX2, OCN expression. In conclusion, Cav 1.3 negatively regulates Spred 2‐mediated autophagy and cell senescence, and damages the activity and osteogenic differentiation of BMSCs in OS rats.

fractures, but also lead to other diseases. 2 Currently, osteoporosis is mainly based on medical treatment of symptoms and complications.
However, it urgently needs to find a more effective treatment due to the high cost, poor treatment effect and adverse reactions. Gene therapy, as a treatment method emerging in recent years, can be targeted to gene defects and diseased tissues. 3 It is known that the differentiation ability of osteoclasts and osteoblasts is an important factor affecting the process of osteoporosis. 4,5 The amount of bone formed in old age is less than the amount of bone absorbed, which can lead to osteoporosis. However, its exact pathological mechanism remains unclear.
BMSCs are a kind of pluripotent cells derived from bone marrow tissues. They have self-renewal and multi-directional differentiation capabilities, and can be differentiated into various mesoderm cell types such as osteoblasts, chondrocytes and fat cells. 6,7 In the dynamics of bone, the osteogenesis process is initiated by the proliferation/mineralization of BMSCs after they are recruited to the site of bone remodelling. 8 Therefore, the osteogenic differentiation ability of BMSCs is crucial in bone remodelling. However, aging will not only reduce the number of BMSCs, but also reduce its osteogenic differentiation ability through overactivated autophagy. 9 Especially in senile osteoporosis, cell senescence may be another factor affecting osteoblast differentiation.
Autophagy is a conservative steady-state mechanism of lysosomal degradation of foreign substances in cells. When autophagy occurs, autophagosomes that form double-membrane or multi-membrane vesicles play a role in the cell. Studies have shown that Spred protein, as a negative regulator of growth factors, can induce caspase-dependent and autophagy-dependent cell death. 10 And autophagy-related proteins Atg5, Atg7, Atg4B and LC3 play an active role in osteoclast differentiation and bone resorption, 11,12 and ultimately affect the dynamic balance of bone. But its effect on osteoblast differentiation is not clear.
Previous studies have shown that Cav 1.3 can promote osteoclast differentiation from osteoclast precursor cells to osteoclasts and bone resorption. Cav 1.3 may affect the classical osteoclast differentiation pathway through calcium signalling pathway. 13 However, its differentiation effect on osteoblasts is not clear. In this study, we focused on investigating the interaction between Cav 1.3 and Spred 2, and their effects on autophagy, cell senescence and osteogenic differentiation of BMSCs derived from OS rats.

| Establishment of OS rat model
Thirty Sprague-Dawley (SD) rats of 3 months old, 6 rats in each group, were fed with 80 mg/(kg/d) for 15 days by retinoic acid (dissolved in vegetable oil) for 15 days. The bone density of the living femur was measured. Meanwhile, comparing the bodyweight of rats before and after gavage, the bone density was significantly reduced and the weight loss indicated that the model was established successfully. Normal rats served as a control group.

| BMSCs cell isolation, culture and osteogenic induction
3-week-old SD rats were selected in the OS group and the control group. The rats were injected with excessive chloral hydrate and cut the femurs of the hind limbs, and washed the limbs with phosphatebuffered saline (PBS) three times. The marrow cavity was flushed repeatedly by the culture medium to rinse out the cells. Subsequent the cells were centrifugated at 900 g for 5 minutes and inoculated into a Petri dish at 37°C, 5% CO 2 . The medium was changed every 3 days and passaged once every 7 days.

| Cell counting kit 8 (CCK8) test to detect the proliferation of cells
The 10 5 /ml cell suspension (100 μL/well) was inoculated in a 96-well plate, and the culture plate was placed in the incubator for pre-culture (37°C, 5% CO 2 ). 10 μL of CCK8 solution was added into the each well, and the plate incubated at 37 degrees for 1-4 hours. Finally, the absorbance at 450 nm was measured with a microplate reader.
The autophagy pathway was inhibited by 5 mmol/L/μL of 3-MA and detect cell activity were detected by a microplate reader.

| SA-β-Gal staining to detect cell senescence
The cells in the normal and OS groups in the 6-well plate were washed three times with PBS. One millilitre of staining fixative was added to the 6-well plate. The cells were fixed at room temperature for 20 minutes. After washing with PBS, the staining solution was added and incubated at 37 degrees overnight. The stained cells were observed with an optical microscope, and the positive rate was counted. Ten fields were selected under the microscope. The total number and positive number were counted (stained and developed in the cytoplasm). The number of cells in each field was controlled within 100-150.

| Alizarin Red S staining
The cells of the normal group and OS composed of bone differentiation were fixed for 30 minutes, washed with PBS 3 times and added with alizarin red dye for 5 minutes, washed with PBS, and finally observed under an optical microscope.

| ALP activity test
After the osteogenic differentiation culture on the 48-well plate, the medium was removed and washed 3 times with PBS.
In the normal group and the OS group, 0.05% Triton X (lysed cell membrane) was added to each well to perform freeze-thawfreeze-thaw. After centrifugation at 4 degrees/15 000 rpm, the supernatant was collected. After 100 μL substrate and 20 μL sample were added to 96-well plate and shaken for 1 minutes, and incubated at 37 degrees for 15 minutes, 80 μL top solution was added to stop the reaction. The microplate reader measures the absorbance at 450 nm.

| Immunofluorescence
The cells in the normal group and the OS group were fixed for 30 minutes; they were washed three times with PBS. 5% BSA was added to the fixed cells to block the cells for 30 minutes and then washed with PBS three times. Primary antibody (LC 3, 1:500, ab192890, Abcam) was added to the blocked cells and incubated at 4 degrees in the dark overnight. After the primary antibody is incubated, the cells were washed with PBS 3 times. Secondary antibody (1:1000, ab150113, Abcam) was added and incubated at room temperature for 30 minutes, and finally observed the cells under an optical microscope.

| Flow cytometry analysis
The cells in the normal group and OS group were digested with trypsin and centrifuged at 900 g for 5 min. The PBS was added to resuspend the cells. 0.5 mL of 50 μg/mL propidium iodide (PI) solution were added to 1*105 cells and stained in the dark for 30 minutes at room temperature. The cells in the centrifuge tube were filtered with a 300 μm nylon mesh into a new centrifuge tube to make a single cell suspension and inserted it into a flow cytometer.

| Total RNA extraction, reverse transcription and qPCR
The cells in the normal and OS groups were lysed with Trizol, chloroform was used to extract the total RNA, and the kit was used to reverse the RNA to cDNA. SLAN fluorescence quantitative PCR instrument was used to detect the expression of Cav1.3 and Spred 2 in BMSCs after osteogenic induction. The expression of osteogenic differentiation-related genes ALP, RUNX2 and OCN and the expression of autophagy-related genes LC3 II/I and Beclin1 were detected.

| Cell transfection
The overexpression plasmid and interference plasmid of Cav 1.

| Western Blot analysis
Cells in each group were collected and 200 μL of cell lysate was added to each well, and the cells were lysed on ice for 1 hour.
Subsequently, it was centrifuged at 11260 g for 15 minutes at 4°C. Developers are used for development and photography.

| CHIP
Cells in normal and OS groups were fixed with 4% paraformaldehyde for 30 minutes, collected and sonicated. CAV 1.3 protein antibody was added, followed by 60 μL of protein A agarose DNA and incubated at 4 degrees for 2 hours. After centrifugation, the supernatant was removed and washed. The complex was de-crosslinked, and the resulting complex was purified. Finally, the obtained complex was tested by PCR.

| Statistical analysis
The data were analysed using SPSS 19.0 software, and all data were expressed as mean ± standard deviation (SD). Prism 6.0 software was used for t test and one-way ANOVA or Student's t test for differences between groups and within groups. P < .05 was considered to have a significant statistical difference. And all experiments were carried out independently three times.

| The expression of Cav1.3 in BMSCs derived from OS rats increased while the expression of Spred2 decreased
In this study, the bone density and bodyweight of rats in the normal group and the OS group were first compared. As shown in Figure 1A, the bone density and bodyweight of the rats in the OS group were significantly lower than those in the normal group. Subsequently, BMSCs were extracted from the normal group and the OS group, and the surface markers CD34 + , CD44 + and CD90 + of BMSCs were identified by flow cytometry in the third generation. The positive expression of CD34 + , CD44 + and CD90 + was as high as 90%, indicating that the extracted and cultured BMSCs can be used for follow-up experiment ( Figure 1B).
We simultaneously induced osteogenic differentiation (ID) of two groups of BMSCs. Alizarin red S staining and ALP identification showed that the osteogenic differentiation ability of BMSCs in the OS group was significantly lower than that of the normal group ( Figure 1C). PCR results showed that the expression of osteogenic differentiation-related genes ALP, RUNX2 and OCN in OS group was also lower than that in normal group, indicating that the differentiation ability of BMSCs in OS group decreased. Western blotting results showed that the expression of Cav1.3 in BMSCs derived from OS rats increased while the expression of Spred2 decreased, and the difference was statistically significant (P < .05).

| The overexpression of Cav1.3 inhibited the osteogenic differentiation ability of BMSCs in OS group
In order to explore the effects of Cav1.

| Cav1.3 inhibits the autophagy ability of BMSCs and Spred2 promotes the autophagy ability of BMSCs
In order to explore the effect of Cav1.3 and Spred2 on the autophagy level of BMSCs, we overexpressed and inhibited the expression of

| Cav1.3 and Spred2 proteins bind to each other and Cav1.3 negatively regulates spred2
In this study, the Cav1.

| Spred2 promoted the activity, autophagy and osteogenic differentiation of BMSCs derived from OS rats by activating autophagy
In order to explore the effect of Spred2 on BMSCs in the OS group, Spred2 was successfully overexpressed and inhibited ( Figure 5A).
The results of CCK8 showed that the cell activity of OE-Spred2 was significantly higher than that of other groups, and using 3-MA sh-Spred2 group showed low calcium mineralization ability and decreased expression of genes related to osteogenic differentiation.

Western blotting and immunofluorescence staining analysis results
showed that LC 3I/LC 3II in the OE-Spred2 group were significantly increased. On the contrary, after Spred2 was inhibited, the expression levels of LC 3I/LC 3II were significantly lower than those in the OE-Spred2 (P < .05).

| Spred2 inhibited cell cycle arrest and cell senescence of BMSCs derived from OS rats by activating autophagy
In order to explore the effects of Spred2 on the cell cycle and senescence of BMSCs derived from OS rats, after overexpression and inhibition of Spred2, the results showed that the cell G1 phase and SA-β-Gal levels in OE-Spred2 group were significantly higher than those of other groups. The G1 phase of BMSCs decreased, and the S phase increased in OE-Spred2 + 3-MA ( Figure 7A,B). The S phase of the sh-Spred2 group was as high as 30%. SA-β-Gal staining was used to detect the cell senescence, and the result was consistent

| D ISCUSS I ON
OS is a decrease in bone mass caused by an imbalance between bone formation and bone resorption in the body. Bone formation is mainly regulated by osteoblasts. The proliferation of osteoblasts and the decline of bone formation ability greatly promote the occurrence of osteoporosis. 14 The osteoblast cell line is derived from differentiation, and many studies have found that the gene expression in osteoporosis patients is different from that in normal control groups.
Therefore, in this experiment, the whole bone marrow culture method was used to extract and purify rat BMSCs. The purpose was to study the effect of the relationship between Cav 1.3 and Spred 2 on the biological functions of OS rat BMSCs, and explore the specific mechanism of this effect.
At present, the identification of BMSCs is mainly based on the positive or negative expression of their specific surface markers, and the ability to target differentiation as a reference index. 15  There is increasing evidence that cell senescence is related to multiple pathways, including cell division, shortening of telomeres, protein aggregation, DNA damage and cellular stress response caused by increased reactive oxygen species. 18 These stress responses will further activate downstream p53/ p21 and other pathways, and produce a cascade reaction to trigger cell senescence. 19 Cell senescence will cause the body's function to decline, and with the body's aging, the cell subgroups of various lineages in the bone microenvironment will age, which is mainly manifested by the decline of bone density and the reduction of the differentiation ability of BMSCs. In this study, the up-regulated Cav1.3 was not only manifested as decreased osteogenic differentiation capacity of BMSCs, but also manifested as increased cellular senescence and the expression levels of P16, P21 and P53 proteins. This phenomenon was more obvious in the OS group.
Many studies have shown that Spred 2 is related to multiple pathways, and the interaction between Spred 2 and p85 affects the Ras/ ERK pathway. 20 The direct association of Spred 2 with DYRK1A modified the substrate/kinase interaction. 19   This research has broad prospects and provides molecular biology guidance for basic research and clinical application of osteoporosis. This study also has limitations and no further animal experiments. In our next work, we will do more research on the animal level.

| CON CLUS IONS
We systematically studied the influence of the relationship be-

ACK N OWLED G EM ENTS
We would like to acknowledge the everyone for their helpful contributions on this paper.

CO N FLI C T O F I NTE R E S T
All authors declare no conflict of interest.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
The ethic approval was obtained from the Ethic Committee of the First Affiliated Hospital of Xi'an Jiaotong University School of Medicine.

CO N S E NT TO PU B LI S H
All of the authors have Consented to publish this research.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are free access to available upon request.