The Japanese Society of Developmental Biologists
Develop. Growth Differ. (2015) 57, 264–273
doi: 10.1111/dgd.12207
Original Article
miR-29a modulates tumor necrosis factor-a-induced osteogenic inhibition by targeting Wnt antagonists Caixia Li, Pingping Zhang and Jieruo Gu* Department of Rheumatology, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510630, Guangdong, China
We previously found that miR-29a was significantly downregulated in Ankylosing spondylitis (AS) patients, a chronic inflammatory disease associated with bone metabolic disorder, however, the underlying mechanism remains unclear. In this study, we demonstrated that miR-29a regulates tumor necrosis factor-a (TNF-a) mediated bone loss mainly by targeting DKK1 and GSK3b, thus activating the Wnt/b-catenin pathway. Our findings may provide new insight into the pathogenesis of the bone metabolism disorder in inflammation environment and provide promising therapeutic target. Key words: miR-29a, TNF-a, Wnt antagonists.
Introduction Bone formation and skeletal development relies on the balance between osteoblasts-induced bone formation and osteoclasts-mediated bone resorption, which appear to be regulated by many factors. Growing evidence shows that bone homeostasis is interrupted in an inflammation environment (Zwerina et al. 2006; Charatcharoenwitthaya et al. 2007; Yago et al. 2007). Tumor necrosis factor-a (TNF-a), which is produced by macrophage cells, has been considered a key pro-inflammatory cytokine. Many reports showed that TNF-a accounts for inflammation-related bone loss. It can inhibit BMP-induced osteoblastogenesis by activating the SAPK/JNK pathway (Mukai et al. 2007). Moreover, TNF-a promotes the formation of osteoclasts and the bone resorption by inducing RANKL expression (Kobayashi et al. 2000), in addition, TNF-a can suppress bone formation by inducing the Wnt antagonists DKK-1 and sclerostin (Diarra et al. 2007). However, no consensus on the molecular mechanism of TNF-a-inhibited osteoblastogenesis was reached. MicroRNAs (miRNAs), which modulate gene expression post-transcriptionally by binding to the 30 -untranslational region (30 -UTR) of targets mRNAs,
*Author to whom all correspondence should be addressed. Email: [email protected] Received 19 January 2015; revised 28 February 2015; accepted 28 February 2015. ª 2015 Japanese Society of Developmental Biologists
have emerged as important regulators in diverse biological processes (Ambros 2004; Wienholds & Plasterk 2005; Gangaraju & Lin 2009). Recent studies have suggested that miR-29a plays a critical role in multiple physiological and pathological processes, such as apoptosis, and carcinogenesis, cell proliferation and osteoblast differentiation (Mott et al. 2007; Kapinas et al. 2009; Xu et al. 2009). miR-29a positively regulated osteoblast differentiation mainly through targeting the canonical Wnt pathway (Kapinas et al. 2009), it could protect against glucocorticoid-induced bone loss and fragility in rats (Wang et al. 2013a,b). miR-29a expression was regulated by TNF-a in vitro (Roderburg et al. 2011). Moreover, in our previous study, miR-29a was identified most differentially expressed in Ankylosing spondylitis (AS) patients (data not shown), a chronic inflammatory disease associated with bone metabolic disorder, furthermore, Enbrel treatment, which acts as a blockade of TNF-a, could have dramatically upregulated miR-29a. These findings, together with the published studies showing that miRNAs are involved in osteogenic differentiation regulated by TNF-a (Wu et al. 2012; Yang et al. 2013), indicating that miR-29a may be highly related to TNF-a-mediated bone metabolic abnormalities. In this study, TNF-a impaired osteogenesis in hFOB cells associated with decreased miR-29a level. Neutralization of TNF-a or inhibition of nuclear factor-kB (NF-kB) pathway both increased miR-29a expression. Furthermore, overexpression of miR-29a partially attenuated TNF-a mediated impaired osteogenic differentiation.
miR-29a modulates TNF-a-induced osteogenic inhibition
Finally, we demonstrated that miR-29a affected bone metabolism in hFOB cells by targeting antagonists of Wnt/b-catenin signaling.
265
FuGENE 6 (Roche) (FuGENE/DNA ratio 3:1) as previously described. Quantitative real time PCR
Materials and methods Cell culture and osteogenic differentiation The human embryonic kidney cell line (HEK293T) and the conditionally immortalized human fetal osteoblastic 1.19 cell line (hFOB) both were obtained from the Institute of Sciences Cell Bank of China. HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin. The hFOB cells were cultured at 33.4°C for proliferation in medium consisting of 1:1 DMEM/Ham’s F-12 medium without phenol red, supplemented with 10% fetal bovine serum, and 0.3 g/L G418 (all from Gibco). For osteoblastic differentiation, the cells were maintained at 39.4°C in the growth medium supplemented with 0.1 g/L ascorbic acid, 108 mol/L menb-glycerol phosphate, adione, 5 9 103 mol/L 7 10 mol/L 1-25(OH)2-D3 (all from Sigma) (Harris et al. 1995). Chemical Recombinant TNF-a and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma. miR-29a mimic, inhibitor, small interfering RNA (siRNA) and negative control were synthesized by Genepharma. Transfection of oligonucleotides The hFOB cells were cultured overnight and then were transfected with miR-29a mimic (sense, 50 -UAGCAC CAUCUGAAAUCGGUUA-30 ; antisense, 50 -ACCGAUUU CAGAUGGUGCUAUU-30 ) (50 nmol/L), inhibitor (50 -UAACCGAUUUCAGAUGGUGCUA) (100 nmol/L), siGSK3b (sense, 50 -CUCAAGAACUGUCAAGUAAdTdT-30 ; antisense, 50 -UUACUUGACAGUUCUUGAGdTdT-30 ), and siDKK1 (sense, 50 -GAUGGGUAUUCCAGAAGAATT-30 ; antisense, 50 -UUCUUCUGGAAUACCCAUCTT-30 ) (100 nmol/L), negative control (50 -CAGUACUUUUGUGUAGUACAA-30 ) (100 nmol/L) respectively, using X-tremeGENE siRNA transfection reagent (Roche) according to the manufacturer’s instructions. Forty-eight hours later, the transfection efficiency was measured by using 50 FAM labeled oligonucleotides. Co-transfection of the pmirGLO dual-luciferase miRNA target vector (pmirGLO) (Promega) or recombinant plasmid and miR-29a mimic were administrated with
Total RNA was isolated from cells using TRIzol reagent (Invitrogen). Nuclear RNA was isolated by Nuclear or Cytoplasmic RNA Purification Kit (Thermo Fisher Scientific), according to the manufacturer’s protocols. Then the RNA was reversely transcribed to cDNA with the cDNA synthesis kit (TaKaRa) using 1 lg RNA as template. Amplification and detection of genes were performed by quantitative real-time polymerase chain reaction (qRT-PCR) using a standard SYBR Green PCR kit (TaKaRa) in an Applied Biosystems 7500 Sequence Detection System, following the manufacturer’s protocols. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the reference for mRNAs. Specific primers used for alkaline phosphatase (ALPL) were (forward: 50 -CATGCTGAGTGACACAGACAAGAA-30 ) and (reverse: 50 -ACAGCAGACTGCGCCTGGTA-30 ); for osteocalcin (BGLAP) were (forward: 50 -GGACCATCTTTCTG CTCA-30 ) and (reverse: 50 -CGGAGTCTGTTCACTACCT TA-30 ); for b-catenin were (forward: 50 -ACTAAGCAGGA AGGGATGGA-30 ) and (reverse: 50 -ATGACGAAGAGCA CAGATGG-30 ); for GSK3b were (forward: 50 -TGGCAG CAAGGTAACCACAG-30 ) and (reverse: 50 -CGGTTCTTA AATCGCTTGTCCTG-30 ); for DKK1 were (forward: 50 -AG CACCTTGGATGGGTATTC-30 ) and (reverse: 50 -GAGG CACAGTCTGATGACCG-30 ). Specific primers for GAPDH were (forward: 50 -GCACCGTCAAGGCTGAGAAC-30 ) and (reverse: 50 -TGGTGAAGACGCCAGTGGA-30 ). All-in-one TM First-Strand cDNA Synthesis Kit and the Hairpin-it miRNA qPCR Quantitation Kit (GeneCopoeia) were used to quantify miR-29a levels, according to the manufacturer’s instructions. Specific primers for miR-29a were (forward: 50 -CGAAATAGCACCATCTGAAATC-30 ) and (reverse: 50 ATCCATGAGAGATCCCTACCG-30 ). U6 was used as the control for miR-29a. Specific primers for U6 were (forward: 50 -CTCGCTTCGGCAGCACA-30 ) and (reverse: 50 -TCCTC CATTAGGAACTCTCACAC-30 ). Each sample was measured in triplicate, and the relative expression levels were calculated by 2DDCt . ALPL activity and Alizarin red staining Osteogenic differentiation was determined by ALPL activity and alizarin red staining. ALPL activity and ALPL staining were performed by the ALP assay kit (Abnova) and BCIP/NBT (5-bromo-4-chloro3-indolyl-phosphate/4-nitroblue tetrazolium chloride) alkaline phosphatase color development kit (Beyotime), respectively. Calcium accumulation was detected by 2% Alizarin red staining (Cyagen ª 2015 Japanese Society of Developmental Biologists
266
Biosciences), instructions.
C. Li et al.
according
to
the
manufacturer’s
Results TNF-a inhibits osteogenic differentiation
miRNA target prediction miR-29a target genes were predicted by the databases TargetScan (http://www.TargetScan.org), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid). Luciferase assay The 30 -UTR of the candidate target genes were amplified from human genomic DNA and the PCR products were cloned into the SacI and SalI restriction sites of the pmirGLO dual-luciferase miRNA target vector (Promega) (named “pmirGLO-wt-30 UTR”), then the recombinant wild type plasmid was used as a template to generate mutant plasmid (named “pmirGLOmut-30 UTR”) with Site-Directed mutagenesis (SBS Genetech) following the manufacturer’s instructions. For analysis of Wnt signaling activity, hFOB cells were transfected with pRL-TK (5 ng) (Promega) and TOPFlash or FOPFlash (200 ng) (Millipore) using FuGENE 6 (Roche) as described above. The luciferase activity levels were measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions, the values were normalized to firefly luciferase. Western blotting Different groups of hFOB cells were lysed in RIPA lysis buffer (keyGEN). Different proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore). After being blocked 2 h at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4°C and matched secondary antibody at room temperature for 2 h. The antibodies used were as follows: rabbit anti-GSK3b monoclonal antibody, rabbit anti-DKK1 monoclonal antibody, rabbit antiNon-phospho (Active) b-catenin monoclonal antibody, rabbit anti-b-actin monoclonal antibody (Cell Signaling Technology) and matched horse radish peroxidase (HRP)-conjugated secondary antibody (Asbio Technology). Statistical analysis All data are expressed as means standard deviation (SD) of at least three independent experiments. Comparisons were analyzed by using Student’s t-test, P-value
Develop. Growth Differ. (2015) 57, 264–273
doi: 10.1111/dgd.12207
Original Article
miR-29a modulates tumor necrosis factor-a-induced osteogenic inhibition by targeting Wnt antagonists Caixia Li, Pingping Zhang and Jieruo Gu* Department of Rheumatology, Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510630, Guangdong, China
We previously found that miR-29a was significantly downregulated in Ankylosing spondylitis (AS) patients, a chronic inflammatory disease associated with bone metabolic disorder, however, the underlying mechanism remains unclear. In this study, we demonstrated that miR-29a regulates tumor necrosis factor-a (TNF-a) mediated bone loss mainly by targeting DKK1 and GSK3b, thus activating the Wnt/b-catenin pathway. Our findings may provide new insight into the pathogenesis of the bone metabolism disorder in inflammation environment and provide promising therapeutic target. Key words: miR-29a, TNF-a, Wnt antagonists.
Introduction Bone formation and skeletal development relies on the balance between osteoblasts-induced bone formation and osteoclasts-mediated bone resorption, which appear to be regulated by many factors. Growing evidence shows that bone homeostasis is interrupted in an inflammation environment (Zwerina et al. 2006; Charatcharoenwitthaya et al. 2007; Yago et al. 2007). Tumor necrosis factor-a (TNF-a), which is produced by macrophage cells, has been considered a key pro-inflammatory cytokine. Many reports showed that TNF-a accounts for inflammation-related bone loss. It can inhibit BMP-induced osteoblastogenesis by activating the SAPK/JNK pathway (Mukai et al. 2007). Moreover, TNF-a promotes the formation of osteoclasts and the bone resorption by inducing RANKL expression (Kobayashi et al. 2000), in addition, TNF-a can suppress bone formation by inducing the Wnt antagonists DKK-1 and sclerostin (Diarra et al. 2007). However, no consensus on the molecular mechanism of TNF-a-inhibited osteoblastogenesis was reached. MicroRNAs (miRNAs), which modulate gene expression post-transcriptionally by binding to the 30 -untranslational region (30 -UTR) of targets mRNAs,
*Author to whom all correspondence should be addressed. Email: [email protected] Received 19 January 2015; revised 28 February 2015; accepted 28 February 2015. ª 2015 Japanese Society of Developmental Biologists
have emerged as important regulators in diverse biological processes (Ambros 2004; Wienholds & Plasterk 2005; Gangaraju & Lin 2009). Recent studies have suggested that miR-29a plays a critical role in multiple physiological and pathological processes, such as apoptosis, and carcinogenesis, cell proliferation and osteoblast differentiation (Mott et al. 2007; Kapinas et al. 2009; Xu et al. 2009). miR-29a positively regulated osteoblast differentiation mainly through targeting the canonical Wnt pathway (Kapinas et al. 2009), it could protect against glucocorticoid-induced bone loss and fragility in rats (Wang et al. 2013a,b). miR-29a expression was regulated by TNF-a in vitro (Roderburg et al. 2011). Moreover, in our previous study, miR-29a was identified most differentially expressed in Ankylosing spondylitis (AS) patients (data not shown), a chronic inflammatory disease associated with bone metabolic disorder, furthermore, Enbrel treatment, which acts as a blockade of TNF-a, could have dramatically upregulated miR-29a. These findings, together with the published studies showing that miRNAs are involved in osteogenic differentiation regulated by TNF-a (Wu et al. 2012; Yang et al. 2013), indicating that miR-29a may be highly related to TNF-a-mediated bone metabolic abnormalities. In this study, TNF-a impaired osteogenesis in hFOB cells associated with decreased miR-29a level. Neutralization of TNF-a or inhibition of nuclear factor-kB (NF-kB) pathway both increased miR-29a expression. Furthermore, overexpression of miR-29a partially attenuated TNF-a mediated impaired osteogenic differentiation.
miR-29a modulates TNF-a-induced osteogenic inhibition
Finally, we demonstrated that miR-29a affected bone metabolism in hFOB cells by targeting antagonists of Wnt/b-catenin signaling.
265
FuGENE 6 (Roche) (FuGENE/DNA ratio 3:1) as previously described. Quantitative real time PCR
Materials and methods Cell culture and osteogenic differentiation The human embryonic kidney cell line (HEK293T) and the conditionally immortalized human fetal osteoblastic 1.19 cell line (hFOB) both were obtained from the Institute of Sciences Cell Bank of China. HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco) containing 10% fetal bovine serum, 100 U/mL penicillin and 100 lg/mL streptomycin. The hFOB cells were cultured at 33.4°C for proliferation in medium consisting of 1:1 DMEM/Ham’s F-12 medium without phenol red, supplemented with 10% fetal bovine serum, and 0.3 g/L G418 (all from Gibco). For osteoblastic differentiation, the cells were maintained at 39.4°C in the growth medium supplemented with 0.1 g/L ascorbic acid, 108 mol/L menb-glycerol phosphate, adione, 5 9 103 mol/L 7 10 mol/L 1-25(OH)2-D3 (all from Sigma) (Harris et al. 1995). Chemical Recombinant TNF-a and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma. miR-29a mimic, inhibitor, small interfering RNA (siRNA) and negative control were synthesized by Genepharma. Transfection of oligonucleotides The hFOB cells were cultured overnight and then were transfected with miR-29a mimic (sense, 50 -UAGCAC CAUCUGAAAUCGGUUA-30 ; antisense, 50 -ACCGAUUU CAGAUGGUGCUAUU-30 ) (50 nmol/L), inhibitor (50 -UAACCGAUUUCAGAUGGUGCUA) (100 nmol/L), siGSK3b (sense, 50 -CUCAAGAACUGUCAAGUAAdTdT-30 ; antisense, 50 -UUACUUGACAGUUCUUGAGdTdT-30 ), and siDKK1 (sense, 50 -GAUGGGUAUUCCAGAAGAATT-30 ; antisense, 50 -UUCUUCUGGAAUACCCAUCTT-30 ) (100 nmol/L), negative control (50 -CAGUACUUUUGUGUAGUACAA-30 ) (100 nmol/L) respectively, using X-tremeGENE siRNA transfection reagent (Roche) according to the manufacturer’s instructions. Forty-eight hours later, the transfection efficiency was measured by using 50 FAM labeled oligonucleotides. Co-transfection of the pmirGLO dual-luciferase miRNA target vector (pmirGLO) (Promega) or recombinant plasmid and miR-29a mimic were administrated with
Total RNA was isolated from cells using TRIzol reagent (Invitrogen). Nuclear RNA was isolated by Nuclear or Cytoplasmic RNA Purification Kit (Thermo Fisher Scientific), according to the manufacturer’s protocols. Then the RNA was reversely transcribed to cDNA with the cDNA synthesis kit (TaKaRa) using 1 lg RNA as template. Amplification and detection of genes were performed by quantitative real-time polymerase chain reaction (qRT-PCR) using a standard SYBR Green PCR kit (TaKaRa) in an Applied Biosystems 7500 Sequence Detection System, following the manufacturer’s protocols. GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was used as the reference for mRNAs. Specific primers used for alkaline phosphatase (ALPL) were (forward: 50 -CATGCTGAGTGACACAGACAAGAA-30 ) and (reverse: 50 -ACAGCAGACTGCGCCTGGTA-30 ); for osteocalcin (BGLAP) were (forward: 50 -GGACCATCTTTCTG CTCA-30 ) and (reverse: 50 -CGGAGTCTGTTCACTACCT TA-30 ); for b-catenin were (forward: 50 -ACTAAGCAGGA AGGGATGGA-30 ) and (reverse: 50 -ATGACGAAGAGCA CAGATGG-30 ); for GSK3b were (forward: 50 -TGGCAG CAAGGTAACCACAG-30 ) and (reverse: 50 -CGGTTCTTA AATCGCTTGTCCTG-30 ); for DKK1 were (forward: 50 -AG CACCTTGGATGGGTATTC-30 ) and (reverse: 50 -GAGG CACAGTCTGATGACCG-30 ). Specific primers for GAPDH were (forward: 50 -GCACCGTCAAGGCTGAGAAC-30 ) and (reverse: 50 -TGGTGAAGACGCCAGTGGA-30 ). All-in-one TM First-Strand cDNA Synthesis Kit and the Hairpin-it miRNA qPCR Quantitation Kit (GeneCopoeia) were used to quantify miR-29a levels, according to the manufacturer’s instructions. Specific primers for miR-29a were (forward: 50 -CGAAATAGCACCATCTGAAATC-30 ) and (reverse: 50 ATCCATGAGAGATCCCTACCG-30 ). U6 was used as the control for miR-29a. Specific primers for U6 were (forward: 50 -CTCGCTTCGGCAGCACA-30 ) and (reverse: 50 -TCCTC CATTAGGAACTCTCACAC-30 ). Each sample was measured in triplicate, and the relative expression levels were calculated by 2DDCt . ALPL activity and Alizarin red staining Osteogenic differentiation was determined by ALPL activity and alizarin red staining. ALPL activity and ALPL staining were performed by the ALP assay kit (Abnova) and BCIP/NBT (5-bromo-4-chloro3-indolyl-phosphate/4-nitroblue tetrazolium chloride) alkaline phosphatase color development kit (Beyotime), respectively. Calcium accumulation was detected by 2% Alizarin red staining (Cyagen ª 2015 Japanese Society of Developmental Biologists
266
Biosciences), instructions.
C. Li et al.
according
to
the
manufacturer’s
Results TNF-a inhibits osteogenic differentiation
miRNA target prediction miR-29a target genes were predicted by the databases TargetScan (http://www.TargetScan.org), RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid). Luciferase assay The 30 -UTR of the candidate target genes were amplified from human genomic DNA and the PCR products were cloned into the SacI and SalI restriction sites of the pmirGLO dual-luciferase miRNA target vector (Promega) (named “pmirGLO-wt-30 UTR”), then the recombinant wild type plasmid was used as a template to generate mutant plasmid (named “pmirGLOmut-30 UTR”) with Site-Directed mutagenesis (SBS Genetech) following the manufacturer’s instructions. For analysis of Wnt signaling activity, hFOB cells were transfected with pRL-TK (5 ng) (Promega) and TOPFlash or FOPFlash (200 ng) (Millipore) using FuGENE 6 (Roche) as described above. The luciferase activity levels were measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions, the values were normalized to firefly luciferase. Western blotting Different groups of hFOB cells were lysed in RIPA lysis buffer (keyGEN). Different proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore). After being blocked 2 h at room temperature, the PVDF membranes were incubated with primary antibodies overnight at 4°C and matched secondary antibody at room temperature for 2 h. The antibodies used were as follows: rabbit anti-GSK3b monoclonal antibody, rabbit anti-DKK1 monoclonal antibody, rabbit antiNon-phospho (Active) b-catenin monoclonal antibody, rabbit anti-b-actin monoclonal antibody (Cell Signaling Technology) and matched horse radish peroxidase (HRP)-conjugated secondary antibody (Asbio Technology). Statistical analysis All data are expressed as means standard deviation (SD) of at least three independent experiments. Comparisons were analyzed by using Student’s t-test, P-value
