SPARC is a decoy counterpart for c‑Fos and is associated with osteoblastic differentiation of bone marrow stromal cells by inhibiting adipogenesis
- Authors:
- Published online on: January 10, 2023 https://doi.org/10.3892/mmr.2023.12937
- Article Number: 50
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Copyright: © Hatori et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Secreted protein acidic and rich in cysteine (SPARC), also known as basement-membrane protein 40 or osteonectin, is a 43-kDa glycoprotein classified as a matricellular protein (1,2). SPARC is the most abundant non-collagenous protein in the bone matrix and demonstrates high affinity for calcium, type I collagen and hydroxyapatite, affinities associated with their mineralization (1). Furthermore, SPARC has been reported to localize in non-mineralized tissues, which makes its distribution ubiquitous (3,4). SPARC not only binds to collagen fibrils (types I, II, III and V) but also to basement collagen type IV (5,6) and vascular endothelial growth factor A (7). Phenotypically, mice with Sparc gene knock out (KO) are characterized by cataracts, decreases in collagens and bone mineral density, and increases in levels of adipose tissue (8–10). Bone marrow stromal-derived cells from Sparc KO mice have been reported to demonstrate reduced ability to form mineralized nodules and reduced osteocalcin (OCN) mRNA expression levels, and enhanced adipocyte development through the upregulation of adipsin and CCAAT/enhancer-binding protein δ (C/EBPδ) expression levels (11). Sparc KO cells have also been reported to show reduced collagen type I and transforming growth factor-β1 mRNA and protein expression levels (12). Taken together, these findings indicated that SPARC may commit cells to the osteoblast lineage by inhibiting their differentiation into adipocytes.
SPARC has also been reported to localize to nuclei (13,14) and the endoplasmic reticulum (ER) (15). Intracellularly, SPARC has been reported to be associated with tubulin (16) and osteopontin (OPN) (17), and to accumulate in nasopharyngeal angiofibromas (18). Moreover, SPARC was not secreted into the culture medium but was reported to have been detected in lysates of chronic myelogenous leukemia cells (19). Bioinformatics analysis studies have reported that SPARC binds to xeroderma pigmentosum group C complex subunit and zinc finger protein 579 intracellularly (20). Therefore, although SPARC has been reported to be localized intracellularly, its intracellular activity associated with osteoblastogenesis/adipogenesis remains unclear.
Adipogenesis by mesenchymal stem cells is induced by several transcription factors, such as peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα. C/EBPβ and C/EBPδ are factors that induce PPARγ- and C/EBPα-expressing preadipocytes, which suggests that PPARγ and C/EBPα serve roles during the late stages of adipogenic differentiation (21). However, activator protein-1 (AP-1) is an important transcription factor involved in early stage commitment to the adipocyte lineage (21).
AP-1 is a homo- or hetero-dimeric transcription factor consisting of basic region leucine zipper domain proteins, such as Fos and Jun, that translocates into the nucleus. Although the Fos proteins, such as c-Fos, Fra-1, Fra-2, FosB and the short isoform of FosB (ΔFosB), heterodimerize only with their counterpart Jun proteins, such as c-Jun, JunB and JunD, the Jun proteins homodimerize or heterodimerize with Fos proteins. The Fos/Jun and Jun/Jun dimers bind to TPA-response element (TRE; 5′-TGACTCA-3′) (22). c-Fos has been reported to induce the production of adipocyte P2/fatty acid-binding protein 4 (FABP4), which serves an important role in adipogenesis (23). Mutations in the c-FOS gene have been reported to predict the phenotypic development of congenital generalized lipodystrophy and Berardinelli-Seip congenital lipodystrophy, rare genetic syndromes characterized by the absence of adipose tissue (24), which suggests that c-Fos is important for adipogenesis. The present study evaluated whether SPARC interacted with c-Fos to commit to the osteoblastic lineage by the inhibition of adipogenesis.
Materials and methods
Reagents
RPMI 1640 medium and simvastatin were purchased from MilliporeSigma; α-minimum essential medium (α-MEM) was purchased from MP Biomedicals; and ascorbic acid, β-glycerophosphate alizarin red-S, Oil red O and ethidium bromide were purchased from FUJIFILM Wako Pure Chemical Corporation. Fetal bovine serum (FBS) was purchased from Hyclone (Cytiva). Guanidinium thiocyanate, phenol, chloroform and Bacto Yeast Extract were purchased from Nacalai Tesque, Inc. Prime STAR GXL DNA Polymerase and Xfect Transfection Reagent, SYBR Premix Ex Taq II, In-Fusion® HD Cloning Kit and Brevibacillus expression system II were purchased from Takara Bio, Inc.; and Blocking Regent N102 was from NOF Corporation. Immobilon-P polyvinylidene fluoride (PVDF) membranes and the chemiluminescent reagent Luminata™ Forte Western HRP substrate were purchased from MilliporeSigma; and Hybond-C nitrocellulose membranes and Hybond-N+ nylon membranes were purchased from GE Healthcare. Radioimmunoprecipitation assay (RIPA) Buffer, Dulbecco's modified Eagle medium (DMEM), Bacto™ Soytone, SuperScript IV Reverse Transcriptase and a High-Capacity cDNA Reverse Transcription Kit (cat. no. 4387406) were purchased from Thermo Fisher Scientific, Inc. Prime STAR GXL DNA Polymerase and Dual-Luciferase® Reporter Assay System were purchased from Promega Corporation; and avidin-linked HRP and DCÔ protein assay kits were from Bio-Rad Laboratories, Inc.
Antibodies
Anti-SPARC polyclonal antibody (pAb) (1:1,000; cat. no. ab55847) was purchased from Abcam, anti-c-Jun (G-4) monoclonal antibody (mAb) (1:1,000; cat. no. sc-74543), and anti-c-Fos (H-125) mAb (1:1,000; cat. no. sc-7202) were purchased from Santa Cruz Biotechnology, Inc. Anti-β actin pAb (1:1,000; cat. no. GTX109639) and anti-GAPDH pAb (1:1,000; cat. no. GTX100118) were purchased from GeneTex, Inc.; anti-Lamin-A pAb (1:1,000; cat. no. 3267) was purchased from BioVision, Inc. Anti-FLAG M2 mAb (1:1,000; cat. no. F1804) was purchased from MilliporeSigma; and biotin-conjugated anti-rabbit IgG pAb (1:10,000; cat. no. 111-065-144) and biotin-conjugated anti-mouse IgG pAb (1:10,000; cat. no. 115-065-003) were purchased from Jackson ImmunoResearch Laboratories.
Vectors
p3×FLAG-CMV-9 and p3×FLAG-CMV-10 vectors were purchased from MilliporeSigma. The pAP1(1)-Luc vector was purchased from Affymetrix, Inc.; pGL4.75[hRluc CMV] was from Promega Corporation; and pNCMO2 was from Takara Bio, Inc.
Sequence datasets
Sequence data for cDNA and proteins were downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov). Accession numbers for the datasets used were as follows: Human SPARC (NM_003118.4 and NP_003109.1); mouse SPARC (NM_009242.5); mouse c-Fos, cDNA(NM_010234.2 and NP_034364); mouse c-Jun (NM_010591.2 and NP_034721.1).
Cells and cell culture
The mouse bone marrow stromal ST2 cell line was purchased from the RIKEN BioResource Center (25,26). The human oral cancer Ca9-22 cell line was a gift from Dr Kimiharu Hirose (Ohu University School of Dentistry, Koriyama, Japan) and was originally obtained from Japanese Collection of Research Bioresources Cell Bank (27). ST2 cells were cultured in RPMI 1640 supplemented with 10% FBS and used for osteoblastic and adipocyte differentiation experiments (28). Ca9-22 cells were cultured in DMEM supplemented with 10% FBS and used for cloning of the SPARC gene. All cultures were maintained at 37°C under a humidified atmosphere containing 5% CO2 (unless otherwise noted).
The ST2 cell clone with low SPARC production (Fig. 1A) was selected using the limiting dilution method. Briefly, ST2 cells were inoculated into a 96-well culture plate at a density of 1 cell/well in RPMI 1640 supplemented with 10% FBS; this culture medium was used throughout cell cloning. Immediately after inoculation, an inverted phase-contrast microscope was used to demonstrate that there was 1 cell/well. When the colony formed 2 weeks after seeding, cell culture vessels were scaled up stepwise when they reached 70–80% confluence after culturing for ~10 days: i.e., one well of a 96-well plate, one well of a 24-well culture plate, one well of a 6-well plate, and three wells of a 6-well plate. When cells reached 70–80% confluence in three wells in a 6-well plate, with cells in one well used for RNA extraction, those in the other used for cell lysate preparation and those remaining frozen at −80°C as the stock. SPARC mRNA and protein expression levels were assessed using reverse transcription-quantitative PCR (RT-qPCR) and western blotting, respectively. Finally, the clone where SPARC mRNA was not detected after 30 cycles of RT-qPCR was used for subsequent experiments in the present study, these cells were subsequently referred to as SPARC-low ST2 cells.
For osteoblastic differentiation, 70% confluent ST2 cells were incubated with α-MEM supplemented with 10% FBS, 50 µg/ml ascorbic acid and 10 mM β-glycerophosphate for 3 weeks. In some cases, 10−6 M simvastatin was also added and cultured for 3 weeks. Calcified deposits were assessed by staining with 40 mM alizarin red-S (pH, 4.2) at room temperature for 10 min. After washing with distilled water five times, images of the culture plates were captured using a digital camera and the red-stained areas were quantified using ImageJ software version 1.5.3 (National Institutes of Health) (26). For adipocyte differentiation, 70% confluent ST2 cells were further incubated with RPMI 1640 medium supplemented with 5 mg/ml insulin, 1 mM dexamethasone and 0.5 mM 1-methyl-3-isobutylxanthine (IBMX) for 2 weeks. Triacylglycerol (TAG) deposits were assessed by staining with Oil Red O at room temperature for 10 min. After washing with 60% isopropanol three times, Oil Red O-stained images of culture plates were captured using a digital camera and high-power images were also captured by an inverted light microscope (objective magnification, ×20), and Oil Red O was eluted using 100% dimethyl sulfoxide at room temperature for 5 min and quantified using spectrophotometry at 531 nm (25).
Construction of expression vectors for SPARC and its mutant and transfection
Total RNA was extracted from Ca9-22 cells using the standard acid guanidinium-phenol-chloroform (AGPC) extraction method (29). RNA was reverse transcribed using SuperScript IV Reverse Transcriptase at 55°C for 10 min followed by 80°C for 10 min for inactivation of the enzyme and cDNA was amplified using Prime STAR GXL DNA Polymerase and specific primers (Table I). The qPCR thermocycling protocol consisted of denaturation at 98°C for 10 sec, followed by 30 cycles of annealing at 60°C for 15 sec and extension at 68°C for 60 sec. The resulting fragments were cloned into the vectors p3×FLAG-CMV-9 and p3×FLAG-CMV-10. The amino acid sequence of human SPARC (Fig. 2B) consisted of amino acid residues (AA) 1–17 as the signal peptide and AA 18–303 as the mature protein. The expression vectors were constructed using the In-Fusion® HD Cloning Kit as follows: SPARC (AA 18–303) with wild-type signal peptide (AA 1–17), SPARC (AA 18–303) with preprotrypsin signal peptide, and SPARC (AA 18–303) without signal peptide (Fig. 2C). To confirm the existence of the ectopically expressed SPARC in the conditioned medium (CM), a SPARC-expression vector in which the FLAG sequence was inserted between wild-type signal peptide (AA 1–17) and mature SPARC (AA 18–303) was also generated.
Transfection of vectors into SPARC-low ST2 cells was proceeded using Xfect Transfection Reagent according to the manufacturers' protocol. Briefly, 5 µg plasmids were mixed with 1.5 µl Xfect polymer and incubated at room temperature for 10 min, followed by the mixture being added to the cultures in a 6-well culture plate. After incubation of the cells in a CO2 incubator for 4 h, the culture medium was refreshed and the cells were further incubated for 24 h for AP-1 measurements and 48 h for preparation of CM.
Preparation of SPARC-present/absent CM
SPARC-low ST2 cells were transfected with mock or Flag-tagged SPARC expression vector as aforementioned. Post-transfection, the medium was refreshed with RPMI 1640 supplemented with 10% FBS, and the cells were incubated for a further 48 h. The CM was collected and clarified by centrifugation at 1,000 × g for 10 min to obtain the following: SPARC (+) CM and SPARC (−) CM.
Serum-free SPARC (+) or (−) CM was prepared from the parallel cultures and subjected to western blot analysis. Briefly, after the transfected cells were cultured for 24 h in RPMI 1640 supplemented with 10% FBS, they were cultured for a further 24 h in serum-free RPMI 1640. The serum-free CM was collected and clarified by centrifugation at 1,000 × g at room temperature for 10 min. The supernatant was analyzed by western blotting.
RT-PCR and RT-qPCR
Total RNA was extracted from parental ST2 cells, SPARC-low ST2 cells and transfected cells using the (AGPC) extraction method. RNA was reverse transcribed at 55°C for 10 min, followed by 80°C for 10 min for inactivation of the enzyme, using SuperScript IV Reverse Transcriptase with Oligo dT for PCR and at 37°C for 60 min, followed by 95°C for 5 min for inactivation of the enzyme, using a High-Capacity cDNA Reverse Transcription Kit for qPCR.
For RT-PCR, the resultant cDNA was amplified using Prime STAR GXL DNA Polymerase using specific primers (Table I). The thermocycling protocol consisted of 98°C for 30 sec, followed by 30 cycles for Sparc or 19 cycles for Actb of denaturation at 98°C for 10 sec, annealing at 60°C for 15 sec and extension at 68°C for 60 sec. The PCR products were separated using 1% agarose gel electrophoresis with 10 mM sodium borate medium and visualized using 0.5 µg/ml ethidium bromide staining at room temperature for 20 min.
For quantification of mRNA expression, the resultant cDNA was amplified using SYBR Premix Ex Taq II in a TP-870 Thermal Cycler Dice Real Time System (Takara Bio, Inc.) with specific primers (Table II). Two-step thermal cycling after an initial denaturation phase at 95°C for 30 sec was used in the present study as follows: 45 cycles of denaturation at 95°C for 5 sec and, annealing and extension at 60°C for 30 sec. The mRNA expression levels of each target gene were normalized relative to the mRNA expression levels of Actb in the same samples. The data were assessed using the 2−ΔΔCq method (30).
AP-1 activity
AP-1 activity was assessed using a luciferase reporter assay. Briefly, SPARC-low ST2 cells were seeded at a density of 1×105 cells/well in 24-well culture plates, incubated overnight in a CO2 incubator and co-transfected at 37°C for 4 h with the AP1(1) Luc vector and the pGL4.75 vector using Xfect Transfection Reagent. After removal of the transfection reagent, the cells were incubated for an additional 24 h at 37°C and luminescence intensity was assessed using a TriStar2 LB942 plate reader (Titertek-Berthold) using a dual reporter assay kit (31). The data was normalized to co-transfected Renilla luciferase activity.
Western blotting
Whole cell lysates of parental ST2 cells, SPARC-low ST2 cells and transfected cells were prepared using RIPA buffer. Nuclear fractions were extracted as described previously (32). Briefly, 1×106 cells were lysed using 400 µl Buffer A [10 mM HEPES-NaOH, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 1 mM DTT and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] and incubated on ice for 20 min. The suspensions were centrifuged at 21,000 × g for 10 sec, and the supernatants were collected as post-nuclear fractions. The precipitates were resuspended in 50 µl Buffer C [20 mM HEPES-NaOH, pH 7.9; 100 mM KCl; 1 mM MgCl2; 0.2 mM CaCl2; 0.2 mM EDTA; 10% (v/v) glycerol; 1 mM DTT and 0.2 mM PMSF] and incubated on ice for 20 min, followed by centrifugation of the suspensions at 21,000 × g for 2 min, with the resultant supernatants collected as nuclear extracts. Following reduction with DTT, 10 µg protein aliquots were separated by SDS-PAGE on 10% gels, followed by transfer of the proteins to PVDF membranes. The membranes were blocked by incubation with TBS-T (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% Tween-20) containing 20% blocking reagent N102 at room temperature for 1 h, followed by overnight incubation with the aforementioned primary antibodies at 4°C overnight and incubation with the aforementioned biotinylated secondary antibodies for 1 h at room temperature. The blots were subsequently incubated with streptavidin-conjugated HRP for 20 min at room temperature, and the signals were detected using Luminata™ Forte Western HRP luminescent substrate (33).
Preparation of recombinant proteins using Brevibacillus brevis
Total RNA, which was extracted from Ca9-22 cells for SPARC and parental ST2 cells for c-Fos and c-Jun, according to the AGPC extraction method, was reverse transcribed at 55°C for 10 min using SuperScript IV Reverse Transcriptase with Oligo dT. cDNA was amplified using the Prime STAR GXL DNA Polymerase kit, as follows: Initial denaturation at 98°C for 30 sec, followed by 30 cycles of denaturation at 98°C for 10 sec, annealing at 60°C for 15 sec and extension at 68°C for 3.5 min (SPARC) or 60 sec (c-Fos and c-Jun). The specific primers are shown in Table I. The resultant cDNAs were cloned into the vector pNCMO2 with 4×His sequences using an In-Fusion® HD Cloning Kit according to the manufacturer's protocol. The details of the cloned genes were as follows: SPARC [human, NM_003118.4 (nt 116–973 corresponding to AA 18 to 303)]; c-Fos [mouse, NM_010234.2 (nt 140–1282 corresponding to AA 1 to 330)]; c-Jun [mouse, NM_010591.2 (nt 952–1956 corresponding to AA 1 to 334)]. These constructs were transfected into Brevibacillus brevis using the Brevibacillus expression system II. Each protein-expressing strain was cultured in 2SY medium (20.0 g/l glucose, 40.0 g/l Bacto Soytone, 5.0 g/l Bacto Yeast Extract, 0.15 g/l CaCl2·2H2O) at 37°C overnight and the culture supernatants were subjected to Ni Sepharose excel (cat. no. 17-3712-01; GE Healthcare) chromatography to purify these proteins according to the manufacturer's protocol.
Electrophoretic mobility shift assay (EMSA)
EMSA was performed using oligo DNA with a biotinylated 5′-end. Briefly, the following sequences: DNA oligonucleotide containing TRE sequence forward (F), 5′-TGAGTCAATGAGTCAGCTGACTCATTGACTCA-3′ and reverse (R), 5′-GGTGAGTCAATGAGTCAGCTGACTCATTGACTCA-3′; and DNA oligonucleotide containing mutant TRE sequence F, 5′-TGTGACAATGTGACAGCTGTCACATTGTCACA-3′ and R, 5′-GGTGTGACAATGTGACAGCTGTCACATTGTCACA-3′, were annealed and used as probes (underlined bases indicate mutation positions of the TRE element). Recombinant proteins (2 µg), which were prepared using the Brevibacillus expression system II as aforementioned, were incubated with probes (2 fmol) in buffer containing 10 mM Tris (pH 7.5), 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 100 ng Poly(dI/dC), 4% glycerol and 0.05% NP-40 for 15 min at room temperature, followed by electrophoresis on undenatured 4% PAGE. The nucleic acids were subsequently transferred to Hybond-N+ membranes and detected using avidin-conjugated HRP and HRP luminescent substrate (34).
Far western blotting
To evaluate the direct binding of SPARC and AP-1 components, far western blotting was performed using recombinant proteins: The subjected protein is blotted on the membrane and then the candidate protein of the counterpart is overplayed. Finally, the yielded complex on the membrane is detected. This assay is considered suitable to assess the direct binding of proteins and is comparable with other assays, such as the immunoprecipitation assay, which are also able to detect indirect binding complex. Briefly, recombinant proteins (0.5, 1.0 and 2.0 µg) from Brevibacillus brevis were spotted onto Hybond-C membranes. Membranes were blocked using 5% skim milk at room temperature for 1 h, then 1.2 µg/cm2 of each recombinant protein (c-Fos, c-Jun and SPARC) was overlaid and incubated at room temperature for 1 h. They were blocked again using 10% N102 blocking solution in TBST. After washing with TBST, the primary antibodies (anti-SPARC, anti-c-Fos and anti-c-Jun) were used to detect their respective proteins, according to the aforementioned method described for western blotting.
Protein assay
Protein concentrations were determined prior to western blotting, EMSA and far western blotting according to the Lowry protein assay method using DC™ protein assay kits with bovine serum albumin as the standard, according to the manufacturer's protocol.
Statistical analysis
All experiments were performed in triplicate to ensure that ≥2 independent experiments yielded similar results. Results were presented as the mean ± standard error of the mean (n=3). Comparisons between two independent groups were evaluated using unpaired Student's t-test using Microsoft Excel version 2210 (Microsoft Corporation) and multiple comparisons of ≥3 groups were evaluated using one-way ANOVA followed by Scheffé's F-test (https://astatsa.com/OneWay_Anova_with_TukeyHSD/). P<0.05 was considered to indicate a statistically significant difference.
Results
Phenotype of SPARC-low ST2 cells
A clone without obvious expression of SPARC was selected from parental ST2 cells and was thereafter named SPARC-low ST2 cells. The mRNA and protein expression levels of SPARC in the parental and SPARC-low ST2 cells were shown in Fig. 1A. SPARC-low ST2 cells did not express Sparc mRNA nor secrete SPARC into the culture medium, although a non-specific signal showing the almost same molecular weight as SPARC was detected from the lysate (Fig. 1A). Culture of these SPARC-low ST2 cells in osteogenic differentiation medium containing ascorbic acid and β-glycerophosphate reduced calcification when compared with parental ST2 cells (Fig. 1B), which is in agreement with previous reports (11,35). Simvastatin has been reported to be a strong promoter of calcification in the presence of ascorbic acid and β-glycerophosphate (36), an effect which was significantly lower in SPARC-low ST2 cells than in the parental cell line (Fig. 1B). RT-qPCR demonstrated that the mRNA expression levels of osteoblastic differentiation-associated genes, such as alkaline phosphatase, transforming growth factor β2 and type I collagen were significantly lower in SPARC-low ST2 cells compared with those in parental ST2 cells. However, Runx2 and osteocalcin mRNA expression levels were not significantly altered in SPARC-low ST2 cells compared with those in parental ST2 cells (Fig. 1C). Whether there were changes in the rate of adipogenic differentiation in SPARC-low ST2 cells was then assessed using adipogenic differentiation medium containing insulin, IBMX and dexamethasone. This medium induced TAG deposition in parental ST2 cells; however, accumulation was significantly higher in SPARC-low ST2 cells compared with that in parental ST2 cells (Fig. 1D). The mRNA expression levels of adipogenesis-related genes, such as adipsin and glucose transporter 4 were significantly higher in SPARC-low ST2 cells compared with those in the parental ST2 cells. In addition, the mRNA expression levels of Pparg2 and Fabp4 were induced by adipogenic differentiation medium; however, the induced mRNA expression levels were, unexpectedly, significantly lower in SPARC-low ST2 cells compared with those in parental ST2 cells (Fig. 1E). Fabp4 KO preadipocytes have previously been reported to differentiate to adipogenic cells more than Fabp4 WT cells (37), which suggested that the contribution of FABP4 to adipogenesis was dependent on the differentiation state of the cells (preadipocytes vs. bone marrow stromal cells). Phenotypically, SPARC-low ST2 cells resemble the cells derived from Sparc KO mice (8–10). The 3T3-L1 cell line has been used for adipogenic differentiation or as an adipocyte model (38); however, it has not been reported to differentiate into osteoblasts. The MC3T3-E1 cell line has been used to assess osteoblastic differentiation (26), but has rarely been reported to differentiate into adipogenic cells. However, ST2 cells have the phenotypic ability to differentiate into either cell type depending on the culturing conditions. These findings indicated that ST2 cells were a more appropriate model to test the effects of SPARC than 3T3-L1 and MC3T3-E1 cells.
Extracellular SPARC incorporated into cells reduces AP-1 activity
The intracellular function of SPARC was initially evaluated by assessing its presence in the nuclear and post-nuclear fractions of ST2 cell lysates, and in their CM. SPARC was not detected in the nucleus but was demonstrated in the post-nuclear fractions of both parental ST2 cells and SPARC-rescued SPARC-low ST2 cells (Fig. 2A) and their CM (Fig. 3). The amino acid sequence of human SPARC is highly conserved among animal species (92% homology with mouse origin) (Fig. 2B); therefore, the SPARC expression vector was constructed using human cDNA.
The effects of Sparc expression on AP-1 activity were evaluated using the TRE-driven luciferase expression vector. AP-1 activity was significantly higher in SPARC-low ST2 cells compared with its activity in parental ST2 cells. However, the induction of SPARC expression using a signal peptide derived from SPARC or preprotrypsin significantly reduced the activity levels of AP-1 in SPARC-low ST2 cells compared with in the SPARC-low ST2 cells (Fig. 2C); however, this reduction was not demonstrated when SPARC was expressed without the signal peptide. These data suggested that the intracellular localization of SPARC depended on its signal peptide sequence. Although it was initially hypothesized that the intracellular localization of SPARC was due to the SPARC WT signal peptide, the effect of replacement of the SPARC WT signal peptide with preprotrypsin signal peptide was not significant and SPARC lacking signal peptide demonstrated no AP-1 inhibitory activity, which suggested that AP-1 inhibition occurred due to incorporation of the secreted SPARC rather than intracellularly expressed SPARC.
When SPARC-low ST2 cells and their parental cells were cultured in CM from SPARC-rescued SPARC-low ST2 cells [SPARC (+) CM], AP-1 activity was significantly lower compared with that in cells cultured in CM from mock-transfected SPARC-low ST2 cells [SPARC (−) CM] (Fig. 3). Furthermore, the increased AP-1 activity in SPARC-low ST2 cells was markedly reduced by SPARC (+) CM treatment to a similar level to that shown in control/SPARC (−) CM-treated parental ST2 cells. These results suggested that SPARC was secreted into the extracellular space and incorporated into the cytosol, where it interfered with the AP-1 complex/consensus sequence.
SPARC binds c-Fos
To evaluate whether SPARC reduced AP-1 transcription activity by interfering with its dimerization, the recombinant peptides c-Fos, c-Jun and SPARC were expressed in Brevibacillus brevis, and EMSA was performed using these peptides and a double-strand oligonucleotide containing the TRE sequence or non-consensus sequence (mutant TRE) as the negative control (Fig. 4). The complex electrophoretically migrated slower than the free DNA oligo by the addition of both c-Fos and c-Jun; however, the presence of SPARC prevented those mobility shifts. Furthermore, mutation of the TRE sequence markedly abrogated complex formation. As strong signals were not detected using the immunoprecipitation assay in preliminary experiments to assess protein-protein interaction (data not shown), far western blotting was performed to assess whether SPARC directly bound c-Fos or c-Jun to prevent the dimerization of AP-1 components. In the experiments in which the SPARC-blotted membrane was overlaid with c-Fos and the experiments in which the c-Fos-blotted membrane was overlaid with SPARC, specific and direct binding of SPARC to c-Fos was detected (Fig. 5). However, both SPARC-blotted membrane overlaid with c-Jun and c-Jun-blotted membrane overlaid with SPARC did not show any specific binding.
Discussion
The matricellular proteins include SPARC, thrombospondins, tenascins and CCNs, such as cysteine-rich 61 and connective tissue growth factor (39). Also included are members of the small integrin-binding ligand N-linked glycoprotein family, such as OPN, periostin, bone sialoprotein, dentin sialophosphoprotein, matrix extracellular phosphoglycoprotein and dentin matrix protein-1. These proteins function at the cell surface, interacting with cell surface receptors, growth factors, proteinases and other extracellular matrix proteins. Some of these proteins have been reported to have intracellular functions. For example, OPN has been reported to interact with the intracellular domain of CD44 (40) and thrombospondin can bind to the ER luminal domain of activating transcription factor 6α (41). SPARC has been reported to be an intracellular protein (13–17,20); however, its function is not completely understood. The present study demonstrated that intracellular SPARC bound to c-Fos.
Phenotypically, Sparc KO mice have been reported to be characterized by impaired bone formation and enhanced adipose tissue formation (11). However, c-Fos KO mice have been reported to be characterized by osteopetrosis, a condition in which bone mass is abnormally high due to defective bone resorption; however, bone-forming activity remains unclear (42,43). As c-Fos has been reported to be activated in osteoclast precursors and to be required for osteoclast differentiation (44), Thus, c-Fos is thought to serve an important role in bone resorption through differentiation/activation of osteoclasts directly to pre-osteoclasts and/or indirectly through osteoblasts. However, to the best of our knowledge, the effects of c-Fos on adipogenesis have not been previously reported. Transgenic mice carrying Fra-1, a c-Fos-related protein and the counterpart of c-Jun, have been reported to have increased bone formation (45). Mouse adipose-derived stromal cells with higher Fosl1 transgene expression have been reported to have reduced adipogenesis (46). Fosl2-transgenic mice have been reported to have an osteosclerotic phenotype due to excess osteoblastogenesis, whereas Fosl2 KO in mice has been reported to impair osteoblastogenesis and enhance an adipogenic phenotype (47). Similarly, mice transgenic for ΔFosB have been reported to have increased osteosclerosis and reduced adipogenesis through two independent cell-autonomous mechanisms (48). Fra-1 and Fra-2 have been reported to inhibit PPARγ expression levels, whereas c-Fos may enhance PPARγ expression in nonalcoholic fatty liver disease (49). As Jun family members have been hypothesized to contribute less to bone formation than Fos proteins (50), Fos proteins, with the exception of c-Fos, have been hypothesized to serve an important role in the induction of cells of the osteoblastic lineage concomitant with a decrease in adipogenesis. The phenotype of Sparc KO mice is highly similar to that of Fosl2 KO mice, including low bone density and an abundance of adipose tissue (8,10,51). These findings suggested that SPARC may be involved in the activation of AP-1, via Fos proteins such as Fra-1, Fra-2 and ΔFosB. That is, SPARC may bind to c-Fos and function as a decoy counterpart of c-Fos to reduce dimerization, thus increasing the ability of other Fos proteins to dimerize with Jun, inhibiting adipogenesis and promoting osteoblastogenesis.
This hypothesis was supported by previous studies, which reported that Fra-2 suppressed PPARγ2 in adipocytes (51) and increased bone formation (45,52). The present study demonstrated that unstimulated basal AP-1 activity was markedly higher in SPARC-low ST2 cells compared with that in parental ST2 cells. Because most basal AP-1 activity was attributed to c-Fos, AP-1 activity may be higher in SPARC-low ST2 cells due to effects on c-Fos. That is, SPARC may inhibit the apparent basal activity of AP-1 by binding to c-Fos, suggesting that SPARC could inhibit c-Fos-containing AP-1 formation, enabling Jun proteins to dimerize with other Fos proteins, thus promoting osteoblastogenesis. The DNA-binding specificity of Fra-1/c-Jun heterodimers was previously reported to be indistinguishable from that of c-Fos/c-Jun heterodimers (53); however, a further study reported that their binding specificities were clearly distinguishable concerning PPARγ (49). The discrepant AP-1 activity changes (AP-1 activity was higher, whereas the mRNA expression levels of the AP-1 target gene PPARγ2 were lower in SPARC-low ST2 cells compared with in parental ST2 cells) demonstrated in the present study may also have been due to bio-physiological conditions that limited response to the transiently introduced vector that determined promoter activity and its inability to be integrated into the genome (54).
In the present study, it was demonstrated that secreted SPARC functioned intracellularly as a decoy counterpart of c-Fos, after its incorporation from the extracellular space. The un-secreted form (absence of signal peptide) demonstrated no AP-1-inhibiting activity. Furthermore, the far western blotting assay using recombinant proteins, which were not in their glycosylated forms, demonstrated that SPARC bound c-Fos. Because SPARC is a highly glycosylated protein, it is still possible that glycosylation of SPARC may affect AP-1 activity. Further studies are required to elucidate the inhibitory mechanism.
Sparc KO mice were initially established using a Mf1/129Sv/Ev outbred mixed background by targeting exon 6 (8). Phenotypically, these mice were characterized by obvious cataract formation and adipose tissue accumulation, but their skeletal size was not changed. Other Sparc KO mice were established with different backgrounds and strategies. For example, 129SV/C57BL/6 Sparc KO mice targeting exon 4 were characterized by cataract formation, adipose tissue accumulation and osteopenia (9,10). Osteopenia was also reported in Sparc KO 129Sv/Ev inbred mice targeting exon 6, whereas symptoms of osteopenia were not reported in Mf1/129Sv/Ev outbred mice with Sparc KO because osteopenia was hidden by high degrees of individual variability (55). Consequently, Sparc deficiency has been reported to exert skeletal effects that are not as severe as adipose tissue accumulation (9). Notably, the ability of SPARC to inhibit adipogenesis was not hidden by high degrees of individual variability, which suggested that SPARC strongly inhibited adipogenesis but did not strongly induce osteoblastogenesis. This inhibition of adipogenesis suggested that SPARC was involved in the commitment of bone marrow stromal cells to differentiation into osteoblasts.
SPARC levels (both mRNA and protein) have been reported to be closely related to obesity and diabetes mellitus. Weight loss resulting from a very low-calorie diet has been reported to cause a concomitant 33% reduction of SPARC gene expression in adipose tissue, whereas weight gain through consumption of a fast-food diet increased SPARC gene expression in adipose tissue by 30% (56). SPARC expression has also been reported to be correlated with leptin-independent fat mass and insulin resistance (56), associations supported by the high SPARC expression in adipose tissue of leptin receptor-deficient mice (57). SPARC mRNA expression levels in both abdominal and visceral adipose tissue have been reported to be correlated with body mass index, and serum SPARC concentration has been shown to be associated with fasting insulin concentration and the homeostasis model assessment of insulin resistance score (58).
The aforementioned clinical observations seemingly oppose the Sparc KO phenotype being the accumulation of adipose tissue; however, SPARC no doubt serves an important role in the regulation of adipogenesis, as well as in bone marrow stromal cell differentiation, committing cells to the osteoblast lineage through inhibition of adipogenesis as a decoy counterpart of c-Fos. Our findings suggest providing novel insights into regenerative medicine.
Acknowledgements
The present study was based on a PhD thesis (Tomoya Hatori), which was published by Ohu University Press in 2020.
Funding
The present study was supported by JSPS KAKENHI (grant nos. JP17K11885, JP18K09642 and JP19K10074.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
TH and YK wrote the manuscript. TH, TM and AS performed the experiments. TH, KT and YK analyzed the data. TM, KT and YK designed the experiments. TH, TM and YK confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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