MiR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs depending on distinct activation and roles of Wnt and BMP signaling pathway

MiR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs

To investigate the function of miR-26a in osteogenic differentiation of BMSCs and ADSCs, we cultured BMSCs and ADSCs in a medium containing ascorbic acid, β-glycerophosphate and dexamethasone, widely used in in vitro osteogenesis model.²⁸ After osteogenic induction, BMSCs and ADSCs highly expressed Alp (alkaline phosphatase, a marker of early osteogenic differentiation) and Ocn (osteocalcin, a critical marker of mature osteoblasts), and formed mineralized nodules (Supplementary Figure S1D–G). Notably, the expression of miR-26a was significantly increased during osteogenic differentiation of both BMSCs and ADSCs (Figures 1a and b).

To investigate the role of miR-26a in BMSC and ADSC differentiation, we performed gain- and loss-of-function assay. Synthesized pre-miR-26a or anti-miR-26a was transfected to efficiently overexpress or knock down miR-26a separately (Supplementary Figure S2). In BMSCs, overexpression of miR-26a increased ALP activity, Alp mRNA and Runx2 mRNA expression at day 7, and mineralized nodule formation and Ocn expression at day 14 (Figures 1c–e), mirroring what was seen in knockdown of miR-26a. In contrast, overexpression of miR-26a significantly inhibited osteogenic differentiation of ADSCs, whereas knockdown of miR-26a promoted ADSC osteogenic differentiation (Figures 1f–h).

To further confirm the function of miR-26a in vivo, we transplanted MSCs transfected with pre-miR-26a, anti-miR-26a or negative control subcutaneously into immunocompromised mice. H&E staining of implants showed that BMSC-overexpressed miR-26a (BMSC/pre-miR-26a) formed more bone tissue, whereas BMSC/anti-miR-26a formed less than control. Conversely, overexpression of miR-26a inhibited the in vivo bone formation of ADSCs, whereas knockdown of miR-26a promoted ADSC bone formation (Figures 1i and j). Consistent with previous studies,^{10, 11} our results indicated that miR-26 promotes BMSC osteogenic differentiation but inhibits ADSC osteogenic differentiation.

MiR-26a targets on both GSK3β and Smad1

To investigate the molecular basis of miR-26a, we employed three miRNA target prediction databases (TargetScan (http://www.targetscan.org/mmu_50/), PicTar (http://pictar.mdc-berlin.de/) and TargetRank (http://genes.mit.edu/targetrank/)) to predict the target mRNA. Among the predicted mRNAs that could regulate osteogenesis, Smad1 (Figure 2a) and GSK3β (Figure 2c) have been experimentally certified.^{11, 29, 30, 31} To confirm the direct binding of miR-26a on Smad1 and GSK3β mRNAs, we performed luciferase activity assay by co-transfecting pMIR reporter containing the binding sites of Smad1 or GSK3β 3′ UTR with pre-miR-26a or anti-miR-26a. Confirmative with the in silico prediction, overexpression of miR-26a inhibited the luciferase activity of reporters of Smad1 and GSK3β, whereas knockdown of miR-26a increased the luciferase activity of both reporters (Figures 2b and d). Gain- and loss-of-function assays further confirmed that miR-26a decreased both Smad1 and GSK3β protein accumulation in BMSCs (Figure 2f) and ADSCs (Figure 2h). Real-time reverse transcription-PCR (RT-PCR) showed that the mRNA levels of Smad1 and GSK3β were not significantly changed after overexpression or knockdown of miR-26a (Figures 2e and g), indicating that miR-26a functions through post-transcriptional regulation.

MiR-26a regulates the osteogenic differentiation of BMSCs and ADSCs through different targets

As Smad1 is the positive regulator but GSK3β is the negative regulator of BMSC osteogenesis,^{32, 33} we moved forward to explore which was the major target of miR-26a by knocking down GSK3β or Smad1 using specific siRNA (Supplementary Figure S3). After knocking down of GSK3β, miR-26a no longer obviously affected the osteogenic differentiation of BMSCs. However, miR-26a still potently promoted BMSC osteogenic differentiation after knocking down of Smad1 (Figures 3a–c). On the contrary, knockdown of Smad1 prohibited the function of miR-26a on osteogenic differentiation of ADSCs, but miR-26a still inhibited osteogenic differentiation of ADSCs after knocking down of GSK3β (Figures 3d–f). Taken together, these results indicated that miR-26a functions majorly by inhibiting GSK3β to promote osteogenesis of BMSCs, and restraining Smad1 to suppress ADSC differentiation.

GSK3β and Smad1 have distinct importance in osteogenic differentiation of BMSCs and ADSCs

The observation that miR-26a targeted on both GSK3β and Smad1 but functioned majorly by regulating different targets in BMSCs and ADSCs raised a question. One possible answer is that GSK3β and Smad1 have different importance in the regulation of BMSC and ADSC differentiation. To verify this hypothesis, we compared the function of GSK3β and Smad1 between BMSCs and ADSCs through loss-of-function assay. As expected, GSK3β siRNA significantly promoted osteogenic differentiation of BMSCs (Figures 4a–c), whereas it caused no significant changes in ADSCs (Figures 4d–f). Smad1 siRNA significantly decreased osteogenic differentiation of ADSCs (Figures 4d–f), but only slightly affected the differentiation of BMSCs (Figures 4a–c).

We moved to the next question of why GSK3β and Smad1 have distinct importance in osteogenic differentiation of different MSCs. The effectivity of one signaling molecule is largely dependent on the activation status of the signaling pathway. Real-time RT-PCR showed that the expression of Wnt ligands was increased over 10-fold during the early stage of BMSC osteogenic differentiation, and the expression of Wnt receptor and downstream genes was also significantly increased till day 7 (Figure 5a). Comparatively, only a few BMP pathway genes were slightly increased during BMSC differentiation (Figure 5b). In contrast, the expression of BMP pathway ligands, receptor and downstream regulators was significantly enhanced during osteogenic differentiation of ADSCs (Figure 5d), and only parts of the Wnt pathway genes were modestly increased (Figure 5c). During osteogenic differentiation process, BMSCs expressed more GSK3β but less Smad1 than ADSCs (Figure 5e).

Moreover, western blot analysis showed that active β-catenin protein accumulation, a marker of Wnt pathway activation, was increased >10-fold during the middle stage of osteogenic differentiation of BMSCs (Figure 5f). Conversely, phosphorylated Smad1 (pSmad1) protein accumulation, a marker of BMP pathway activation, was slightly decreased at day 7 of BMSC osteogenic differentiation (Figure 5f). On the contrary, pSmad1 protein level was significantly increased whereas active β-catenin protein accumulation was decreased during osteogenic differentiation of ADSCs (Figure 5g).

To confirm the signaling activation status in vivo, we performed immunofluorescence assay in MSCs transplants harvested after 4 weeks. Consistent with the in vitro results, active β-catenin protein level in BMSCs was significantly higher than that in ADSCs, whereas pSmad1 protein accumulation in BMSCs was lower than that in ADSCs (Figures 5h and i). Taken together, these results indicated that Wnt signaling is dominantly activated during osteogenic differentiation of BMSCs, whereas BMP signaling is more active during ADSC differentiation.

BMSCs and ADSCs depend on different signaling pathways to promote osteogenic differentiation

The differential activation of Wnt and BMP signaling suggested that BMSCs and ADSCs apply different signaling pathways to promote osteogenic differentiation. To certify the notion, we supplied Dorsomorphin to inhibit BMP pathway and Dickkopf-1 (DKK1) to inhibit Wnt pathway. Although both Dorsomorphin and DKK1 suppressed osteogenic differentiation of BMSCs, DKK1 was more effective than Dorsomorphin (Figures 6a–c). In ADSCs, Dorsomorphin significantly suppressed the osteogenic differentiation (Figures 6d–f). Interestingly, in contrast with its role in BMSC differentiation, DKK1 did not affect ADSC osteogenic differentiation (Figures 6d–f).

We next performed gain-of-function assay using recombinant Wnt3a to activate Wnt signaling and BMP2 to activate BMP signaling. Both BMP2 and Wnt3a enhanced the mineralized nodule formation and osteogenic markes expression in BMSCs, and Wnt3a was more effective than BMP2 (Figures 6g–i). In ADSCs, BMP2 markedly promoted the osteogenic differentiation of ADSCs, whereas Wnt3a slightly inhibited the osteogenic differentiation of ADSCs (Figures 6j–l). Taken together, these results indicated that Wnt pathway activation is indispensable for BMSC osteogenic differentiation, whereas BMP signaling activation is more important to promote ADSC osteogenic differentiation.

MiR-26a regulates different signaling pathways in BMSCs and ADSCs

Our results above showed that differential activation of Wnt and BMP pathways determined the distinct importance of GSK3β and Smad1 in BMSC and ADSC differentiation. To further verify the notion, we checked the effects of miR-26a on activation of Wnt and BMP pathways in different MSCs after 14 days of osteogenic induction. Overexpression of miR-26a increased active β-catenin expression, whereas knockdown of miR-26a decreased active β-catenin expression in BMSCs (Figure 7a). MiR-26a also inhibited pSmad1 protein slightly in BMSCs. As pSmad1 protein rarely expressed in BMSCs after osteogenic differentiation, the absolute quantity change of pSmad1 was much lower than that of active β-catenin (Figure 7a). Immunofluorescence assay of in vivo transplants revealed that miR-26a significantly inhibited the expression of GSK3β (Supplementary Figure S4A and B) and increased the level of active β-catenin (Figures 7c and d). As pSmad1 expression in BMSC implants was scarce, we did not detect the effect of miR-26a on pSmad1 level in vivo (Figures 7c and d).

In ADSCs, miR-26a markedly effected pSmad1 protein expression (Figure 7b). Although miR-26a slightly inhibited GSK3β, miR-26a knockdown or overexpression did not significantly affect the active β-catenin protein level in ADSCs. Immunofluorescence assay also confirmed that miR-26a inhibited the activation of Smad1 (Supplementary Figure S4C and D) and pSmad1 (Figures 7e and f) in vivo. We did not detect the regulation of miR-26a on active β-catenin of ADSC transplants (Figures 7e and f).

In conclusion, we found that BMP and Wnt signaling pathways were differentially activated and played distinct role in osteogenic differentiation of BMSCs and ADSCs. The different gene regulation program determined the opposite function of miR-26a in BMSC and ADSC osteogenic differentiation. Our findings uncovered that tissue-specific MSCs applied differential signaling regulation programs for cell-fate commitment, suggesting the complexity in tissue-specific stem cell research and application.

Animals

C57BL/6J mice and BALB/c nude mice were obtained from the Laboratory Animal Research Centre of the Fourth Military Medical University (Xi'an, China). All procedures that involved animals were approved by the animal use and care committee of the Fourth Military Medical University (License Number: 2014 KQ-005). All mice were housed under specific pathogen-free conditions (22 °C, 12-h light/12-h dark cycle, and 50–55% humidity) with free access to food pellets and tap water.

Materials

Recombinant mouse BMP2 (R&D, Minneapolis, MN, USA), recombinant mouse Wnt3a (R&D), recombinant mouse Dkk1 (PeproTech, Rocky Hill, NJ, USA) and dorsomorphin (Santa Cruz Biotechnology, Santa Cruz, TX, USA) were obtained commercially.

Cell culture and identification

The 4- to 6-week-old C57BL/6J mice were used for cell culture. BMSCs were isolated and cultured as previously described.⁵⁴ Mouse were killed and the hindlimbs were aseptically removed and bones were dissected free of soft tissues. Marrow cavities of femur and tibia were flushed with culture medium as α-MEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS, 1% penicillin and streptomycin and 2-mercaptoethanol (2-ME). 1.5 × 10⁷ cells were seeded in 10 cm tissue culture flasks and incubated in humidified atmosphere of 5% CO₂ at 37 °C. Nonadherent cells were removed by frequent medium change during 72 h. The remaining adherent colonies were cultured for 14 days until confluent, and passaged after digestion with 0.25% trypsin for 3 min.

ADSCs were isolated from scraps of subcutaneous adipose tissues as previously described.⁵⁵ In brief, the adipose tissues were minced and incubated in 1% collagenase type I for 1.5 h in a 37 °C water bath shaker. The digested solution was filtered through a 200-μM and a 100-μm cell strainer to separate undigested tissue fragments. Adipocytes and aqueous supernatant were separated by centrifugation at 800 × g for 5 min. Then, the deposit was resuspended in culture medium and plated in 10 cm tissue culture flasks and maintained in humid incubator at 37 °C and 5% CO₂. Hematopoietic lineage cells attached to Petri dish after 1 h were removed, and the nonadherent cells containing ADSC populations were fed with α-MEM supplemented with 10% FBS, 1% penicillin and streptomycin until colony forming and reaching 70–80% confluence.

Cells at passage 3 were used for all experiments. Both BMSCs and ADSCs highly expressed MSC markers (Sca-1, CD90, CD106) and did not express hematopoietic cell marker (CD34), and had self-renewal and multipotent differentiation potential (Supplementary Figures S1A–K).

Osteogenic differentiation and mineralization assay

After the cells reached 70–80% confluence, differentiation medium containing α-MEM, 10% FBS, 100 μg/ml ascorbic acid, 2 mM β-glycerophosphate and 10 nM dexamethasone was used to induce osteogenic differentiation of both MSCs. The medium was changed every 3 days. After induction for 7 days, ALP staining was performed with the BCIP/NBT Alkaline Phosphatase Color Development Kit (Beyotime Co., Shanghai, China) following the standard protocol. After 14 days of osteogenic differentiation, 1% alizarin red (Sigma, St. Louis, MO, USA) was used to detect calcium accumulation according to the manufacturer's suggested protocol. Then, alizarin red was incubated with 100 mM cetylpyridinium chloride (Sigma) for 30 min at room temperature and quantified by spectrophotometer at 540 nm. The final calcium levels in each group were normalized with the total protein concentration obtained from the duplicate plates.

Real-time RT-PCR of mRNA and miRNA

Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer's standard instructions. For reverse transcription of mRNA, random-primed cDNA was synthesized from 2 μg of total RNA using a PrimeScript RT Reagent Kit (TaKaRa, Dalian, China). Real-time RT-PCR analysis was performed using the SYBR Premix Ex Taq II Kit (TaKaRa) and detected on the ABI Prism 7500 HT Sequence Detection System (Applied Bio-Systems, Foster City, CA, USA). β-Actin and U6 were used as loading controls for quantitation of mRNA and microRNA. The optimized microRNA-specific primers for miR-26a and the endogenous control U6 were commercially obtained (RiboBio, Guangzhou, China). Primers of PCR are listed in Supplementary Table S1.

Western blot analysis

Cells were harvested in RIPA lysis buffer (Beyotime Co.). Whole-cell protein extracts were quantified by the BCA assay, separated on 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk powder in PBST for 3 h. The membranes were probed overnight with the primary antibodies and then 4 h with peroxidase-conjugated secondary antibodies (Boster, Wuhan, China). Antibodies used in this study included the mouse β-Actin, rabbit Smad1, rabbit phospho-Smad1, mouse GSK3β, rabbit phospho-GSK3β (Cell Signaling, Beverly, MA, USA), rabbit β-catenin (Abcam, Cambridge, MA, USA) and mouse active β-catenin (Millipore). The blots were visualized using an ECL Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's recommended instructions. To measure the protein abundance, gray value of the blots in scanned images was measured with ImageJ Plus software (National Institutes of Health, Bethesda, MD, USA). Gray value of each target protein was normalized to that of β-Actin before comparison.

The in vivo bone formation assay

BALB/c nude mice, 6 weeks old, were used as hosts for implantation. BMSCs and ADSCs were first transfected with miR-26a precursors, inhibitors or negative controls as described before, and then cultured in osteogenic differentiation medium for 3 days. Approximately 4 × 10⁶ cells were mixed with 40 mg hydroxyaptite-tricalcium phosphate (HA-TCP) powders (Sigma-Aldrich, St. Louis, MO, USA) and subcutaneously implanted into the dorsal surface of immunocompromised mice. Implants were harvested after 4 or 8 weeks for immunofluorescence and histological assay separately.

H&E staining of MSC implants

MSC implants separated from surrounding fibrous capsule were fixed in 4% neutral buffered formalin (Sigma) overnight and decalcified with EDTA (10%) (Sigma) for 2 weeks. Then, the implants were embedded in paraffin and sectioned at 5 mm. H&E staining was performed according to standard protocols. Photos were taken under magnification of × 40 on four consecutive microscopic fields of the sections of implants. Osteoid formation in transplants was semiquantified as osteoid area per total area in H&E staining photos with Image Pro software (Media Cybernetics, Silver Spring, MD, USA).

Immunofluorescence assay of MSC implants

Decalcified MSC implants were dehydrated in 30% sucrose solutions until submerged in bottom. Samples were embedded into optimal cutting temperature compound (OCT) for frozen sections, and analyzed by immunofluorescence assay following the standard protocol. Antibodies used in this study including goat Smad1 (Santa Cruz Biotechnology), mouse active β-catenin (Millipore), rabbit phospho-Smad1 and rabbit GSK3β (Cell Signaling). The samples were treated with fluorescence-labeled secondary antibodies and the nuclei were stained with 100 ng/ml of 4',6-diamidno-2-phenylinde (DAPI) (Beyotime Co.). All samples were examined under a confocal microscope (Olympus Optical, Tokyo, Japan).

Transfection of miRNA precursors and inhibitors

Cells were seeded into 12-well or 6-well plates and transfection was performed shortly before or at 70% confluence. The microRNA precursors, inhibitors and negative controls of miR-26a were commercially purchased (Ambion, Austin, TX, USA). The cells were transfected with pre-miR-26a and negative control at a final concentration of 50 nM, and anti-miR-26a of 100 nM. Cell suspensions were overlaid onto the transfection complexes and incubated at 37 °C and 5% CO₂ for 24 h until further study.

Transfection of siRNA

Cells were seeded into 12-well or 6-well plates and transfected at 40–60% confluence. The Smad1 siRNA sequence (5′-GAACUGAAGCCUCUGGAAU-3′, 3′-CUUGACUUCGGAGACCUUA-5′), Gsk3β siRNA sequence (5′-GGUAUAUCAAGCCAAACUU-3′, 3′-CCAUAUAGUUCGGUUUGAA-5′) and scramble RNA were commercially synthesized (RiboBio). The cells were transfected with miRNAs together with Smad1 siRNA or Gsk3β siRNA by Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions, and transected with scramble RNA as a negative control at the same time. Final concentration of microRNA precursors, negative control and siRNA were 50 nM, and the concentration of inhibitor was 100 nM. The cells were incubated at 37 °C in the incubator for 24 h before further study.

Luciferase reporter assay

To construct a vector of mouse Smad1 and GSK3β, the Smad1 oligonucleotide sequence were amplified using primers (forward: 5′- GGCTCCTTCGTCAGGTCTCCA-3′ reverse: 5′-ACGGATGAAATAGGATTGTGGG-3′) with Hindlll and Spel sites at their extremities to insert the pMIR-Report vector (Ambion). GSK3β oligonucleotide sequences were amplified using primers (forward: 5′- CCACCATCCTTATCCCTCCAC-3′ reverse: 5′-GGAGGAGCAGAGCATTAAACACA-3′). HEK293T cells were seeded in a 96-well plate at 50 to 60% confluence. After 24 h, cells were co-transfected with reporter constructs as pMIR-Report (pMIR-Cont), pMIR-Smad1 or pMIR-GSK3β plasmids (125ng), and microRNA controls, precursors or inhibitors (20 pmol). All transfections were conducted using Lipofectamine 2000 (Invitrogen). Luciferase assays were performed 48 h after transfection using the Dual Luciferase Reporter Assay System (Promega, Madison, WI, USA). Firefly luciferase activity was normalized to Renilla luciferase activity for each transfected well. Each transfected well was assayed in triplicate.

Statistical analysis

Data are presented as mean±S.D. Comparisons were analyzed using Student's two-tailed t-test or one-way ANOVA for experiments with more than three groups. All experiments were repeated at least three times, and representative experiments are shown. Differences were considered to be significant when P<0.05.