Introduction
The family with sequence similarity 20C (FAM20C), the
bona fide “Golgi casein kinase”, answers for the phosphorylation of over 100 secreted phosphoproteins with a consensus motif Ser-x-Glu/pSer and is ubiquitously expressed in multiple tissues and bodily fluids [
1‐
4]. Moreover, FAM20C figures prominently in a wide range of cellular processes, including phosphate metabolism, lipid homeostasis, wound healing, biomineralization, cardiac function, cell adhesion, and migration [
1,
5‐
8]. These abundant and useful functions make FAM20C an important protein kinase. At the physiological and pathological level, FAM20C gene mutations or aberrant function of kinase lead to many diseases, such as Rains Syndrome, cancers, and other diseases. Notwithstanding, the exploration of the pivotal role of FAM20C in health and disease remains in an infant stage.
Aberrant FAM20C mutation causes lethal or non-lethal Raine syndrome in humans, which is clinically characterized by generalized osteosclerotic dysplasia and hypophosphatemia [
9,
10]. To better understand the pathogenesis of Raine syndrome in humans, from work in animal and cellular models established by ablating
Fam20c gene, diseases and phenotypes associated with Raine syndrome have been studied. Mice lacking
Fam20c present the phenotype similar to human non-lethal Raine syndrome, including growth retardation, rickets/osteomalacia, defects in the growth plate, defective osteocytes differentiation, reduction of serum phosphate, and elevation of serum fibroblast growth factor (FGF23) [
11]. This has been partially attributed to elevated FGF23, which regulates serum phosphate levels, and its activity is regulated by FAM20C-mediated phosphorylation at Ser180 and O-glycosylation [
6,
12]. Furthermore,
Fam20c−/− mice develop abnormal dental morphological changes and differentiation defects, which can be caused by the suppressing of BMP signaling pathways [
13‐
15]. Thus, considering
Fam20c-deficient mice develop hard tissue dysplasia and FAM20C is highly expressed in bone and tooth tissues [
2,
16,
17], studies have sought to identify the cellular changes in FAM20C ablation cells. Intriguingly, in
Fam20c-deficient dental mesenchymal cells and osteoblasts, changes in gene expression of key factors related to osteogenesis and mineralization are consistent with
Fam20c−/− mice, and impaired cellular mineralization could not be rescued by the addition of normal bone-derived extracellular matrix proteins [
18,
19]. These findings suggest that FAM20C regulates osteoblast behaviors in a cell-autonomous manner. However, little is known regarding the mechanisms by which FAM20C influences cell autonomous behaviors.
A comprehensive understanding of human diseases requires exploration of their complexity and heterogeneity of the pathogenesis at the epigenetic, genomic, and proteomic levels [
20,
21]. In the study of Raine syndrome regard, only 70 cases have been reported so far due to its extremely rare and low incidence rate [
22], making it difficult to apply high-throughput sequencing technology in the research field of Raine syndrome. To investigate the mechanisms underlying the FAM20C-associated phenotype in Raine syndrome, some studies have been conducted to study the biological functions of FAM20C by using high-throughput sequencing technology. Zhang et al. [
23] analyzed the proteins that interact with FAM20C in the endoplasmic reticulum (ER) and Golgi apparatus through immunoprecipitation and mass spectrometry, revealing that FAM20C could phosphorylate ER oxidoreductin 1α (Ero1α) to maintain the ER redox homeostasis. Besides, our previous research conducted a transcriptional analysis of
Fam20c-deficient osteoblasts, indicating the cells undergo mesenchymal to epithelial transformation [
24]. Besides a single sequencing method, the combination of multi-omics analysis provides a more comprehensive picture linking “molecular information” to “phenotype” through functional and signaling networks [
25‐
27]. Little is known, however, about multi-omics analysis of FAM20C remains to be determined, especially chromatin sequencing.
Here, we use established
Fam20c-deficient cells in our previously published work [
28] to conduct a comprehensive omics analysis. To probe the relationship between chromatin, transcriptional and protein changes response to
Fam20c ablation in osteoblasts, we performed integrated omics analyses using an assay for transposase-accessible chromatin using sequencing (ATAC-seq), RNA sequencing (RNA-seq), proteomics, and phosphoproteomics.
Fam20c ablation specifically regulates a subset of genes comprising Wnt signaling pathway molecules and TCF4 was highly enriched in the chromatin opened of
Fam20c deficient osteoblasts, as revealed by ATAC-seq. We identified Calpastatin/Calpain proteolysis system as a novel target axis for FAM20C to regulate cell migration and F-actin cytoskeleton. Subsequently, we demonstrated that Calpain agonist treatment rescued
Fam20c-deficient cells migration in vitro, and negatively manage the Wnt signaling pathway. Further, our results suggested that the homeostatic imbalance of
Fam20c knockout osteoblasts may involve changes in multiple signaling pathways in the conduction system.
Materials and methods
Cell lines and cell culture
Immortalized mouse
Fam20cf/f osteoblast cells (referred as “OB
Fam20cf/f”) were purchased from Applied Biological Materials Inc. (Lot# T0038).
Fam20c-deficient osteoblasts (referred as “OB
Fam20cKO”) were established with infection of Cre recombinase lentivirus as previously published work described [
28]. OB
Fam20cf/f and OB
Fam20cKO were cultured at 37 °C with 5% CO
2. All cells were cultured in α-Minimum Essential Medium (α-MEM) (Biosharp, BL306A) containing 10% fetal bovine serum (Gibco, #10091148) and 1% penicillin–streptomycin (Gibco, #15140122). Additionally, cells were passaged with 0.25% trypsin (Gibco, #12604013).
ATAC-seq
Cell samples for ATAC-seq were prepared as described in
Current protocols in molecular biology by Buenrostro et al. [
29]. In brief, 50,000 cells were obtained from each sample, resuspended in cold PBS, and centrifugated at 500 g for 5 min at 4 °C. Cell pellets were washed once with 50 μl cold PBS on ice, followed by a 5 min centrifugation at 4 °C. Pelleted nuclei were taken for transposition reaction with Tn5 enzyme, and the DNA fragments after transposition were recovered with MinElute PCR Purification Kit (Qiagen). Accessible DNA was amplified by PCR with 1 X NEBNext High-Fidelity Pcr Master Mix (New England Biolabs, MA). Libraries were sequenced on a Novaseq 6000 (Illumina).
ATAC raw reads data from each sample was filtered using Trimmomatic software (Version 0.36) [
30], after data filtering, the overall quality of the resulting clean reads was assessed. The clean reads were then aligned to reference genome
mm10_gencode using the BWA program (Version 0.7.13-r1126). Three repeated samples for each group were used for callpeak by MACS2 (Version 2.1.2) [
31] with the parameters qvalue < 0.05. Reads distributions across genes were presented using deeptools (Version 3.4.3)[
32], and genes were represented as lines sorted in descending order based on the signal strength in the heatmap. Motif analysis was performed using the HOMER's findMotifsGenome.pl tool (v4.11). Differential accessible peak analysis was performed by using bedtools software [
33] and DESeq2 (Version 1.16.0)[
34], log2 fold change ≥ 1 or ≤ -1, and p-value < 0.05 were considered as the cutoff values. The raw data and processed data were uploaded into the Gene Expression Omnibus (GEO) database (
https://www.ncbi.nlm.nih.gov/geo/) with an accession number GSE233445.
Gene Ontology (GO) and Kyoto gene and genome encyclopedia (KEGG) signal pathway analysis of differential peaks nearest genes were performed using ClusterProfiler with a False Discovery Rate (FDR)-adjusted p-value cutoff of 0.05. Thus the significant GO categories and pathways were identified.
Integration Analysis of ATAC-seq and RNA-seq
RNA-seq analysis was as previously reported [
28]. The associated genes in open chromatin regions with enhanced and attenuated ATAC-seq signal overlapped with the up and down differentially expression genes (DEGs) in the transcriptomes, respectively. Further, GO and KEGG pathways analyses were performed as described above.
Proteomic and phosphoproteomic analysis
Cells were washed three times with cold PBS and lysed in lysis buffer containing 50 μl 50 mM Tris–HCl (pH 8.5), 57 μl ddH2O, 10 μl 400 mM 2-chloroacetamide (CAA) (Sigma, 22,790), 2 μl tris (2-carboxyethyl) phosphine (Sigma, 646547) and 1 μl phosphatase inhibitor cocktail. Samples were then boiled in a 95 °C water bath for 10 min in darkness. Subsequently, cooled at room temperature and diluted five-fold. The protein concentration was measured by using BCA protein assay, and the rest part of protein samples was digested with Lys-C (Wako) at 37 °C for 3 h. Protein samples underwent trypsin digestion (1:50) at 37 °C overnight. Afterward, samples were acidified by 1% trifluoroacetic acid (TFA) and ethyl acetate. The mixture of lysates was centrifuged at 15000g for 3 min, the bottom aqueous phase was collected followed by vacuum-dried centrifugation and desalting. 95% and 5% portions of each sample were used for phosphoproteomics and proteomics experiments, respectively.
Peptide samples were analyzed on the nanoElute coupled online with a timsTOF Pro mass spectrometer (Bruker). Peptides were re-dissolved in 0.1% formic acid (FA) and loaded onto a 25 cm in-house column n (360 μm OD × 75 μm inner diameter) packed with C18 resin (particle size 2.2 μm, pore size 100 Å, Michrom Bioresources). All peptides were separated onto the analytical column with a 120 min gradient (buffer A: 0.1% FA in ultrapure water; buffer B: 0.1% FA in acetonitrile) at a constant flow rate of 300 nL/min (90 min, 0 to 37% buffer B using a linear AB gradient of 2 to 22% of buffer B; 10 min, 22 to 37% of buffer B, 10 min, 37 to 80% of buffer B; 10 min, 80% of buffer B). Mass spectrometry was set under a data-dependent acquisition mode. Mass range was 100–1700 m/z at a resolution of 40,000.
MS raw files produced by LC–MS/MS were searched against the Uniprot human proteome database using PEAKS Studio X + software (Bioinformatics Solutions Inc.). Mass tolerances were 15 ppm for initial precursor mass and 6 ppm for final tolerance. Carbamidomethyl of cysteine (+ 57.0214 Da) was considered a fixed modification. Variable modifications were acetylation (+ 42.011 Da) at the N terminus of proteins, oxidation (+ 15.9949 Da) on methionine residues, and phosphorylation (+ 79.996 Da) on serine, threonine, or tyrosine residues. Up to 2 missed cleavages were allowed. The cutoff of FDR was 1% for all proteins, peptides, and phosphosites. Differential expression analyses were determined based on p-value < 0.05 and |log2(Fold change)|> 1.
Cell migration assays.
To measure the relative migration ability of cells, scratch-wound healing assays were performed. Cells were plated in a 6-well plate at a density of 50*104/well. After cells reached confluency, manually scraped the monolayer cell with a 10 μl pipette tip to create the wounds. The cells were then washed with phosphate saline buffer (PBS), replenished with serum-free α-MEM, photographed with the inverted phase-contrast microscope, and recorded the exact location of each wound. Afterward, the cells were placed back in the cell-culture incubator, and the recorded scratch regions were photographed 24 h after wounding. For statistical analysis, using Image J to outline the pictures, measure the area of the scratch, analysis and plot were performed using GraphPad Prism 8.
Phalloidin-fluorescent staining
Cells were seeded in a 24-well plate at approximately 100% confluence and subsequently scraped with the pipette tip to create wounds. For scratch-wound healing experiments, cells were treated as described above. At 24 h after wounding, cells were fixed by 4% formaldehyde solution for 10 min, and washed three times with PBS, each time for 5 min. Cells were then permeabilized with 0.1% Triton X-100 for 5 min, and washed three times with PBS. To reduce nonspecific background staining, cells were blocked in 1% bovine serum albumin (BSA) for 30 min at room temperature. The rhodamine phalloidin staining solution (MESGEN, MF8204) was diluted in 1% BSA and incubated cells for 20 min at room temperature. The cells were rinsed by PBS three times, each time for 5 min. 200 μl per well of Hoechst 33,258 (Beyotime, C1018) was used to label the nuclear of cells. Cells were observed on fluorescence microscope (Nikon), and photographed at least three different representative regions at 4× magnification or 10× magnification.
Quantitative real-time PCR analysis
RNA was extracted from cells by using RNAiso Plus reagent (Takara, 9108) following the manufacturer’s protocols. RNA quality and concentration were determined by using a Nanovue spectrophotometer (GE Healthcare Life Sciences, Marlborough, MA, USA). cDNA was prepared by RNA reverse transcripted with PrimeScript RT reagent kit (Takara, RR047A). Subsequently, SYBR TB Green Premix Ex Taq™ kit (Takara, RR820A) was used for one-step real-time RT-PCR analysis on the MxPro-Mx3000P real-time PCR System. The experiment was repeated three times.
Normalize the expression value of the target gene in a given sample to the corresponding GAPDH expression. The relative expression value of the targeted genes was calculated by the 2 − △ △Ct method. The primers were: RhoA-F, 5’- GAAACTGGTGATTGTTGGTGATG-3’, RhoA-R, 5’- ACCGTGGGCACATAGACCT-3’, Rac1-F, 5’- ACGGAGCTGTTGGTAAAACCT-3’, Rac1-R, 5’- AGACGGTGGGGATGTACTCTC-3’, Cdc42-F, 5’- CCCATCGGAATATGTACCAACTG-3’, Cdc42-R, 5’- CCAAGAGTGTATGGCTCTCCAC-3’, Tcf4-F, 5’-GATGGGACTCCCTATGACCAC-3’, Tcf4-R, 5’- GAAAGGGTTCCTGGATTGCCC-3’, β-catenin-F, 5’- ATGGAGCCGGACAGAAAAGC-3’, β-catenin-R, 5’- TGGGAGGTGTCAACATCTTCTT-3’, Calpain1-F, 5’- ATGACAGAGGAGTTAATCACCCC-3’, Calpain1-R, 5’- GCCCGAAGCGTTTCATAATCC-3’, Calpain2-F, 5’- GGTCGCATGAGAGAGCCATC-3’, Calpain2-R, 5’- ATGCCCCGAGTTTTGCTGG-3’, Calpastatin-F, 5’- GGAAGGACAAACCAGAGAAGC-3’, Calpastatin -R, 5’- AGGGGCAGCTATCCAAATCTT-3’, GSK-3β-F, 5’- TTGGACAAAGGTCTTCCGGC-3’, GSK-3β-R, 5’- AAGAGTGCAGGTGTGTCTCG-3’, GAPDH-F, 5’- AACTCCCACTCTTCCACCTTC-3’, GAPDH-R, 5’- CCTGTTGCTGTAGCCGTATTC-3’.
Western blot and antibodies
Cells were collected and lysed in radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, P0013B) containing phenylmethanesulfonyl fluoride (PMSF) (Beyotime, ST506) and phosphatase inhibitor cocktail (Sigma, P5726). Total proteins were extracted in the supernatant. To prepare the nuclear and cytoplasmic extracts, the nuclear and cytoplasmic protein extraction kit (Beyotime, P0028) was applied to lyse the cells following the manufacturer’s instructions. After vigorous vortexing, the lysates were centrifuged at 12000g for 5 min at 4 °C. The supernatant was collected as the cytoplasmic protein. Add the nuclear protein extraction reagent to the pellet, vortexing and ice bathing alternately for 30 min, the lysates were centrifuged at 12000g for 10 min at 4 °C, and the supernatant was collected as the nuclear fraction. All extracted proteins were quantified using the Enhanced BCA Protein Assay Kit (Beyotime, P0010). The protein concentration between different groups was adjusted to be consistent before boiling.
For western blot assay, 40 µg of protein lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, P0012AC) and transferred to polyvinylidene difluoride (PVDF) membranes (Biosharp, BS-PVDF-45/BS-PVDF-22). After blocking in 5% non-fat milk (Biosharp, BS102), PVDF membranes were then incubated overnight at 4 °C with primary antibodies and probed for Calpastatin (Cell Signaling Technology, #4146), Calpain 1 (Proteintech, 10538-1-AP), Calpain 2 (Proteintech, 11472-1-AP), β-catenin (Proteintech, 51067-2-AP), Tcf4 (Proteintech, 22337-1-AP), GSK3-β (Proteintech, 22104-1-AP), Lamin B (Wanleibio, WL01775), Rho A (Wanleibio, WL02853), Cdc42 (Proteintech, 10155-1-AP), β-Tubulin (Abmart, M20005), and GAPDH (Proteintech, 10494-1-AP). Peroxidase-conjugated IgG (Biosharp, BL003A) was used as the secondary antibody, and blots were developed using the enhanced chemiluminescence kit (Meilunbio, MA0186) detection.
Immunofluorescence analysis
Cells were seeded in a 24-well plate at approximately 60% confluence. Cells were fixed by 4% paraformaldehyde (PFA) for 30 min at room temperature, and washed three times with PBS, each time for 5 min. Cells were then blocked in immunol staining blocking buffer (Beyotime, P0102) for 1 h at room temperature. The primary antibodies were diluted in primary antibody dilution buffer for Immunol staining (Beyotime, P0262) and incubated the cells for overnight at 4 °C. PBS rinsed the cells five times, each time for 5 min. Secondary antibodies (Cell Signaling Technology, #8890S) were diluted in antibody dilution buffer (1:400) and incubated for 2 h at room temperature. Hoechst 33258 was used to tag the nuclear of cells. Cells were observed on fluorescence microscope, and photographed at least three different representative regions at 4× magnification or 10× magnification.
Casein zymogram
To determine calpain activity, casein zymography analysis was performed. The main principle is that the enzymes in the lysates are separated by the polyacrylamide gel containing casein. Casein molecules in zymography gels act as substrates for calpain in solutions containing calcium ions, and their degradation reflects enzymatic activity [
35,
36]. In resolving gel, 0.21 mg casein was copolymerized with 4.9 ml ddH
2O, 2.5 ml 1.5 mol/L Tris–HCl (pH 8.8), 2.5 ml polyacrylamide solution (4:0.16), 40 μl ammonium persulfate solution (APS), and 28 μl N,N,N’,N’-Tetramethylethylenediamine (TEMED). For stacking gel, it contained 6.5 ml ddH
2O, 2.5 ml 1.32 mol/L Tris–HCl (pH 6.8), 1 ml polyacrylamide solution, 50 μl APS, and 10 μl TEMED. The casein gel was pre-run at 160 V for 30 min at 4 °C with electrode running buffer. The running buffer (10x) contained 25 mM Tris–HCl (pH 8.3), 192 mM glycine, 1 mM ethylene glycol-bis (β-aminoethyl ether)-N’,N’,N’,N’-tetraacetic acid (EGTA), 0.05% (v/v) 2-mercaptoethanol (2-MCE), and 1 mM ethylenediaminetetraacetic acid (EDTA). Subsequently, lysates were loaded with sample buffer (1:3, v/v), which included 150 mM Tris–HCl (pH 6.8), 0.04% (w/v) bromophenol blue, 20% (v/v) glycerol, and 0.75% (v/v) 2-MCE. The gel with samples loaded was run for electrophoresis at 125 V for 3 h at 4 °C. At the end of the electrophoresis, the gel was incubated in proteolysis buffer for 24 h at 20 °C. The proteolysis buffer (10x) was comprised of 20 mM Tris–HCl (pH 7.4), 10 mM dithiothreitol (DTT), and 4 mM CaCl
2. The gel was stained with Coomassie Blue Fast Staining Solution (Beyotime, P0017) for 60 min, and the thermal cycler imaging system was applied to photograph the bands of the gel.
Mouse models
All mouse models are on a C57BL6/J background and breading in a SPF grade facility. The generation of conditional knockout mice was as follows: (1) Fam20cflox/flox mice (Texas A&M University College of Dentistry, America) mated with Osx-Cre mice (Biocytogen, China) from the progeny of which the Fam20cflox/+; Osx-Cre mice were selected. (2) By mating these mice with Fam20cflox/flox mice, the Osx-Cre; Fam20cflox/flox mice were obtained from the progeny, whose gene Fam20c was deleted in the pre-osteoblast. In this research, Osx-Cre; Fam20cflox/flox (conditional knockout, cKO) mice were used as the experimental group, and their littermates with Fam20cflox/flox genotype were put into the control group. The genotype identification was performed on the tails of postnatal mice by polymerase chain reaction, the genotyping of each mouse was repeated three times.
Immunohistochemical staining
The bone tissue samples of 4-week-old mice were collected and fixed for 48 h, then placed in the decalcified fluid (15% EDTA). After the end of decalcification, the mouse tissues were placed in the automatic tissue dehydrator. The wax-soaked tissues were placed into the embedding machine. Conventional sections of tissue wax blocks were cut with a thickness of 3 µm, and the position and depth of the tissue sections in the cKO group and the control group were basically the same as far as possible. Immunohistochemical staining was performed on 3 µm thickness paraffin sections, the sections were sequentially deparaffinized, washed with pure water, and soaked in 3% H2O2 to block endogenous peroxidases. Antigen retrieval was performed using citrate buffer under high pressure, and then the slides were incubated in 10% normal goat serum and 5% bovine serum albumin (Solarbio, China) for 60 min, and incubated with the antibody for 2 h at room temperature. The sections were washed in PBST (PBS plus 0.1% Tween) and incubated with the secondary antibody at room temperature for 60 min. Then the slides were washed in PBST, and immunopositive reactions were visualized using a 3,3’-diaminobenzidine tetrahydrochloride solution. The nuclei were counterstained with hematoxylin. The slides were washed in distilled water and dehydrated in graded alcohol, cleared in xylene. Images were taken with a biological microscope (Nikon, Japan).
Statistical analysis
Each experiment was repeated at least three times. Statistical analysis was performed with GraphPad Prism8.0 software. Student’s t test was used for univariate comparison between two groups. P < 0.05 was considered to be different and statistically significant and presented as * P < 0.05, ** P < 0.01, or *** P < 0.001.
Discussion
Increasing evidence has pointed to essential roles for FAM20C phosphorylated substrates within S-x-E/pS motifs in regulating many physiological processes [
1,
7,
51]. Despite this intrigue, the knowledge about FAM20C has remained in the infant stage resulting from its complex regulatory mechanisms. The widespread multiple FAM20C substrates, ranging from the secreted proteins to intracellular proteins, of which phosphorylated levels are altered [
52,
53]. Accordingly, the complex regulatory network by FAM20C, including epigenetics, gene expression, and protein interactions, is attracting broad attention. Comprehensive multi-omics analysis could broaden our knowledge of the molecular events relevant to biological processes [
54,
55]. Here, we hypothesized that the joint profiling analysis of chromatin accessibility, gene expression, proteomics, and phosphorylated proteomics data between OB
Fam20cf/f and OB
Fam20cKO, may capture some new regulatory mechanism of FAM20C.
ATAC-seq allows the identification of genomic regions associated with gene-regulatory activity, thus providing a method to infer transcription factor (TF) activity [
37]. Our data demonstrated that lack of
Fam20c generally intensified 12% of the accessible regions in osteoblasts, leading to a global activation of downstream TFs (Fig.
1B). Motif analysis illuminated that TCF4 was highly enriched in the chromatin opened of
Fam20c deficient osteoblasts (Fig.
1E). TCF4, a member belonging to the bHLH TF family, was regarded to play a vital role in many developmental processes [
56,
57]. Furthermore, when Wnt ligands bind to the Frizzled-Lrp5/6 receptor complex, β-catenin degradation is blocked and translocated to the nucleus, allowing TCF to bind and to activate Wnt target genes [
58,
59]. Consistently, we also found a significant enrichment of the KEGG pathway in the Wnt signaling pathway through joint profiling ATAC-seq and RNA-seq (Fig.
2D). Previous studies have shown that tissue homeostasis and cell maintenance could be impacted by Wnt signaling pathway [
60,
61]. In this research, PCR, Western blot, and Immunofluorescence both elucidated that
Fam20c knockout significantly activated the transcriptional activity of
β-catenin and
Tcf4 signaling, as well as elevated the protein expression levels of them, especially, the increased expression levels in the nucleus. While GSK-3β displayed a contrary trend, that is, the total expression level and the nuclear expression level decreased (Fig.
7A–F).
Moreover, Qin’s research found that TCF1 and LEF1 were down-regulated in the vertebrae of
Sox2-Cre;
Fam20cfoxl/flox mice [
62]. Further, TCF4 is combined with
β-catenin upstream of BMP. In our research, knockout of
Fam20c in osteoblasts in vivo, changes were only found in the hypertrophic cartilage layer, indicating that there were differences in vivo and in vitro, which might be caused by the paracrine and juxtacrine functions of cells. In addition, cartilage thickening has been observed in several condition knockout animal models, whether this phenomenon was related to the Wnt signaling pathway remains to be further investigated. Furthermore, studies have shown that Wnt signal can inhibit the expression of activated Col2a1, and the overexpression of
β-catenin in chondrocytes can cause serious congenital chondrosis, indicating that its overexpression affects the bone development process, supporting the results of our study [
63]. Therefore, we believe that after
Osx-Cre knockout of
Fam20c, the expression of downstream GSK-3β in the cytoplasm is inhibited, thus increasing the entry of
β-catenin into the nucleus, and the overexpression of
β-catenin in the nucleus overactivated TCF4. Finally, the proliferation of chondrocytes is induced through the Wnt signaling pathway, thus affecting the process of bone development.
In the current study, extensive Mass Spectrometry (MS)-based proteomics and phosphoproteomics analysis permitted in-depth screening of the changes in protein expression, post-translational phosphorylated modification, and biological processes alteration in
Fam20c deficient osteoblasts. These included but were not limited to: (I) weakened changes in the cell growth, where the predominant changes were the regulation of mitotic cell cycle, cell division, and regulation of cell cycle G2 M phase transition; (II) a decrease in alteration in regulation of proteolysis involved in protein catabolic process; (III) alteration in cell migration (Fig.
3C, D). The deficiency of cell growth in OB
Fam20cKO was proved in previous studies [
18,
24]. Of note, in this research, we verified the major driver protein was Calpastatin, which was less expressed and decreased phosphorylated levels in OB
Fam20cKO than in OB
Fam20cf/f (Fig.
4). We also performed experiments to knock out FAM20C in human 293 T cells and obtained a similar trend (Additional file
6: Figure S6). Calpastatin belongs to the calpain system; mediates cell motility, cell cycle, signal pathways transduction, apoptosis, and regulates gene expression via proteolytic degradation [
43,
64,
65]. The other two members of the calpain system, Calpain 1 and Calpain 2, activity was mediated by Calpastatin phosphorylation state [
42,
66,
67]. Specifically, the phosphorylated Calpastatin presented relatively static, located near the cell nucleus, and presented an aggregation state, that is, the activity of Calpastatin was inhibited, thus enhancing the activity of Calpain. On the contrary, the dephosphorylation of Calpastatin made its distribution in cells relatively favorable, and the activity of Calpastatin increased, which then weakens the activity of Calpain. Consistently, we observed
Fam20c-deficient osteoblasts displayed a diminished expression of Calpain 1 and Calpain 2, and attenuation of Calpain activity (Fig.
4D). OB
Fam20cKO cells under the influence of impaired Calpastatin/Calpain proteolysis system exhibited weak mobility and guide differential remodeling of cytoskeletons at the leading edge versus the OB
Fam20cf/f, with the inhibition of RhoA, Rac1, and Cdc42 (Fig.
5). The Rho family of small guanosine triphosphatases (GTPases), particularly RhoA, Rac1, and Cdc42, play essential roles in regulating the cell cycle and actin cytoskeleton, affecting cell adhesion and migration [
68‐
70]. For cell migration, specifically, RhoA participated in the assembly of stress fibers, Rac1 was responsible for the formation of lamellar pseudopods, and Cdc42 regulated the shape of filamentous pseudopods [
71,
72]. Moreover, we observed the weakened mobility was rescued by CaCl
2 in OB
Fam20cKO, accompanied by a concentration-dependent elevation of Calpain 1 and Calpain 2 expression and activity but no significant changes in Calpastatin phosphorylation level (Fig.
6), further indicating that cells migration barriers were associated with Calpastatin/Calpain proteolysis system. Calpain could regulate a variety of signaling pathways through proteolysis of target proteins, including cell cycle (cyclin D1 and cyclin E), cell survival (nuclear factor- κ B), and apoptosis (Bcl2 family and caspases) [
73]. It was also possible that Calpain mediated cell migration through multiple mechanisms, as noted in previous studies, including the cleavage of integrin-related complex, adhesive plaque, Focal adhesion kinase (FAK), Paxillin, Talin, and other components, resulting in reduced cell adhesion and increased mobility [
74,
75]. Similar to our in vitro results, we observed the expression of Calpastatin, Calpain 1, and Calpain 2 in the growth plate region in animal models at the time when articular cartilage changes were most visible (i.e. 4 weeks after birth), and the results were consistent with cytological trends. In the knockout group, the expression of Calpastatin in the calcified area of growth plate cartilage, the pre-osteoblasts on the surface of bone trabeculae and hypertrophic cartilage decreased significantly. Calpain 1 and Calpain 2 were expressed in different positions in the cartilage, among which Calpain 1 was mainly expressed in the surface and middle layers of articular cartilage, and Calpain 2 was mainly expressed in the deep layer. The expressions of both decreased significantly in the knockout group. Real-time quantitative PCR detection showed that the expression levels of
Calpain 1 and
Calpain 2 genes in bone tissue decreased. These results further prove that the Calpastatin/Calpain proteolysis system is inhibited overall after the conditional knockout of
Fam20c, and the dysfunction of this system can cause abnormalities in its downstream signaling pathway, inhibit the functions of pre-osteoblasts and hypertrophic chondrocytes, and ultimately affect the process of cartilage osteogenesis and cancellous bone formation.
Regarding Calpastatin/Calpain proteolysis system’s influence on Wnt signaling pathway, we took note of some previous studies showing more than 100 substrates were downstream of Calpain, including β-catenin and GSK-3β of Wnt signaling pathway [
76]. Briefly, Calpain activity could regulate β-catenin expression level, intracellular localization, and function [
77].
The down-regulated of Calpain 2 expression, resulted in elevated β-catenin expression and accumulate in the nucleus. Conversely, Calpain activation could promote β-catenin degradation to achieve negative regulation of Wnt signaling pathway [
45]. Lade et al. [
50] reported that Calpain 1 induced N-terminal truncation of β-catenin in the mouse liver development process, this might play a vital role in regulating the differentiation of hepatoblasts. In addition, Goni-Oliver and Ma et al. [
78,
79] reported that Calpain truncated C- and N-terminal self-suppressing domains of GSK-3β to activate GSK-3β. In this regard, we added CaCl
2 to OB
Fam20cKO with weak calpain activity to activate calpain activity, and then the cell migration ability was enhanced, β-catenin and Tcf4 expression levels decreased, a decreased location of Tcf4 and β-catenin in OB
Fam20cKO nuclei (Fig.
7K–N). Another key to note in our study is that previous studies have indicated that activation of Wnt signaling pathway could promote cell proliferation and migration [
80,
81]. However, activating Wnt signaling pathway had less pronounced benefits, we do not detect accelerated cell migration in OB
Fam20cKO. Given that we found
Fam20c depletion to cause a weakened Calpastatin/Calpain proteolysis system, which could promote the hydrolysis of migration-related proteins to accelerate cell migration and negatively manage the Wnt signaling pathway. Thus, in the absence of
Fam20c, this phenomenon could be explained by the possible explanation that Calpastatin/Calpain proteolysis system acts predominantly in regulating cell migration.
Additional analysis of the Wnt ligands, as shown here, only
Wnt7a was observed significant changes in canonical Wnt ligands (Fig.
7G). The overlapped genes obtained from ATAC-seq and RNA-seq were projected into the KEGG pathway and visualized, which also showed the enrichment of
Wnt7a (Additional file
4: Figure S4). Several studies have highlighted the importance of Wnt7a in regulating cell migration and invasion [
82,
83], however, its role was cell-specific. Xie et al. reported that Wnt7a promoted oral squamous cell carcinoma cell migration induced by epidermal growth factor via the activation of the β-catenin/Matrix metalloproteinase-9 (MMP-9) signal pathway [
83]. Nevertheless, Lan et al. proposed the overexpression of Wnt7a inhibited the growth and migration of hepatocellular carcinoma in β-catenin-independent manner [
84]. Taken together, our findings supported
Fam20c depletion led to Wnt ligands down-regulated in osteoblasts, however, β-catenin increasingly translocated to the nucleus, triggering Wnt signaling pathway ligands-independent manner.
Furthermore, a series of elegant papers have reported that FAM20C could phosphorylate bone morphogenetic protein 4 (BMP4), while BMP2 and BMP7 might be possible substrates of FAM20C due to possessing S-x-E/pS motifs [
7,
85]. Intriguingly, we have previously observed that in
Fam20c salivary gland conditional knockout mice, the transcriptional and protein levels of BMP2 and BMP7 were up-regulated, while protein expression of BMP4 was reduced but did not change its transcript level [
86]. The distribution pattern of BMP4 expression in salivary glands was almost not observed in the extracellular matrix, but concentrated in the cytoplasm, indicating that knockout of
Fam20c caused abnormal secretion of BMP4. Also, Liu et al. showed mRNA levels of BMP2 and BMP7 were similarly increased in OB
Fam20cKO, however, their downstream remains in a repressed state, implying that phosphorylation may affect the activity of BMP ligands [
18]. In addition, as illustrated by our analyses,
Fam20c depletion induced Calpastatin dephosphorylation, decreased Calpain activity, elevated β-catenin expression levels, and translocated into nucleus to combine with Tcf4, leading to the activation of Wnt signaling pathway in a ligands-independent manner. Since Wnt signaling pathway regulated the transcription of BMP [
87,
88], thus, our findings provided a possibility to explain the enhanced transcription of BMP in
Fam20c-deficient animal and cell models. On the other hand, increased transcription of BMP, but not activation of BMP signaling pathway, indicating that conduction inhibition occurred in BMP signaling pathway.
Finally, several prior studies have shown the loss of bone maturation phenotype in
Fam20c cKO mice and loss-of-function of the in vivo gained osteoblasts [
11,
15,
89]. Also, the absence of
Fam20c distorted cell biological processes. Osteogenic differentiation blockade occurs in OB
Fam20cKO, which could not be rescued by the addition of extracellular matrix proteins extracted from normal bone tissue, suggesting that FAM20C might regulate cell behaviors in a cell-autonomous manner [
18]. Another characteristic of OB
Fam20cKO, as above elucidated, was their paucity of BMP signaling pathway activation even with the elevated BMP ligands transcription, indicating that there was conduction abnormality in BMP signal pathway. Our integrated analysis revealed alterations of Calpastatin/Calpain proteolysis system and Wnt signaling pathway among the most dramatic differences between OB
Fam20cf/f and OB
Fam20cKO. The aberrant Calpastatin/Calpain proteolysis system could alter the cleavage of specific proteins, regulating pathological and normal physiological processes that are thought to play critical roles in governing homeostasis [
90,
91]. Also, the coordination of cell fates and tissue homeostasis is controlled by Wnt signaling pathway that regulates cell-to-cell communication [
92,
93]. Then again, we found the signaling pathways related to cell signal transduction were significantly influenced in OB
Fam20cKO, including mitogen-activated protein kinases (MAPK) signaling pathway, Ras signaling pathway, mammalian target of rapamycin (mTOR) signaling pathway, cAMP signaling pathway, etc. [
24] (Additional file
5: Figure S5). These data provide experimental support for a paradigm in which OB
Fam20cKO appears the phenomenon of cellular homeostasis imbalance. Homeostasis is the basis of maintaining all kinds of important functions of the body, which involves tissue, cell, signal pathway, molecule, and others, breaking cell homeostasis will cause various pathological processes [
94,
95]. These results comprise that the homeostatic imbalance of FAM20C knockout osteoblasts may involve changes in multiple signaling pathways in the conduction system.
Taken together, our current work provides an integrated and comprehensive analysis of FAM20C using multiple omics, including ATAC-seq, RNA-seq, proteomics, and phosphoproteomics. We revealed that Calpastatin/Calpain proteolysis system and Wnt signaling pathway alterations were possibly the key factors between OB Fam20cf/f and OB Fam20cKO. Targeting Calpastatin phosphorylation may provide a promising research direction to study the role of FAM20C in development. However, this research still existed restrictions. Initially, the protein–protein interaction between FAM20C and Calpastatin needs to be examined in follow-up studies. In the future experiment, we will add FAM20C recombinant protein to OB Fam20cKO to observe the change of phosphorylation of Calpastatin. Applying the radiolabeled technique to test the phosphorylation site of Calpastatin, constructing a mutant vector of the Calpastatin site to transfect OB Fam20cKO to activate the phosphorylation of Calpastatin, and observing the expression and activity of Calpastatin/Calpain proteolytic system and its effect on osteoblast function. Second, we performed phosphorylated proteomics, and detected the difference in Calpastatin phosphorylation and the changes in the phosphorylated site after Fam20c gene knockout. Nevertheless, the exact phosphorylated site of Calpastatin has not been verified. Third, in the present study, we found the Calpastatin/Calpain proteolysis system could negatively regulate the Wnt signaling pathway. Further studies are needed to conduct experiments associated with Calpain activators/ inhibitors as well as activators/antagonists of the Wnt pathway to further define this regulatory relationship.