TNF-α-Induced SOX5 Upregulation Is Involved in the Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells Through KLF4 Signal Pathway원문보기
Xu, Lijun
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Zheng, Lili
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Wang, Zhifang
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Li, Chong
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Li, Shan
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Xia, Xuedi
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Zhang, Pengyan
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Li, Li
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
,
Zhang, Lixia
(Department of Endocrinology, The First Affiliated Hospital of Zhengzhou University)
Postmenopausal osteoporosis (PMOP) is a common systemic skeletal disease characterized by reduced bone mass and microarchitecture deterioration. Although differentially expressed SOX5 has been found in bone marrow from ovariectomized mice, its role in osteogenic differentiation in human mesenchymal ...
Postmenopausal osteoporosis (PMOP) is a common systemic skeletal disease characterized by reduced bone mass and microarchitecture deterioration. Although differentially expressed SOX5 has been found in bone marrow from ovariectomized mice, its role in osteogenic differentiation in human mesenchymal stem cells (hMSCs) from bone marrow in PMOP remains unknown. In this study, we investigated the biological function of SOX5 and explore its molecular mechanism in hMSCs from patients with PMOP. Our findings showed that the mRNA and protein expression levels of SOX5 were upregulated in hMSCs isolated from bone marrow samples of PMOP patients. We also found that SOX5 overexpression decreased the alkaline phosphatase (ALP) activity and the gene expression of osteoblast markers including Collagen I, Runx2 and Osterix, which were increased by SOX5 knockdown using RNA interference. Furthermore, $TNF-{\alpha}$ notably upregulated the SOX5 mRNA expression level, and SOX5 knockdown reversed the effect of $TNF-{\alpha}$ on osteogenic differentiation of hMSCs. In addition, SOX5 overexpression increased Kruppel-like factor 4 (KLF4) gene expression, which was decreased by SOX5 silencing. KLF4 knockdown abrogated the suppressive effect of SOX5 overexpression on osteogenic differentiation of hMSCs. Taken together, our results indicated that $TNF-{\alpha}$-induced SOX5 upregulation inhibited osteogenic differentiation of hMSCs through KLF4 signal pathway, suggesting that SOX5 might be a novel therapeutic target for PMOP treatment.
Postmenopausal osteoporosis (PMOP) is a common systemic skeletal disease characterized by reduced bone mass and microarchitecture deterioration. Although differentially expressed SOX5 has been found in bone marrow from ovariectomized mice, its role in osteogenic differentiation in human mesenchymal stem cells (hMSCs) from bone marrow in PMOP remains unknown. In this study, we investigated the biological function of SOX5 and explore its molecular mechanism in hMSCs from patients with PMOP. Our findings showed that the mRNA and protein expression levels of SOX5 were upregulated in hMSCs isolated from bone marrow samples of PMOP patients. We also found that SOX5 overexpression decreased the alkaline phosphatase (ALP) activity and the gene expression of osteoblast markers including Collagen I, Runx2 and Osterix, which were increased by SOX5 knockdown using RNA interference. Furthermore, $TNF-{\alpha}$ notably upregulated the SOX5 mRNA expression level, and SOX5 knockdown reversed the effect of $TNF-{\alpha}$ on osteogenic differentiation of hMSCs. In addition, SOX5 overexpression increased Kruppel-like factor 4 (KLF4) gene expression, which was decreased by SOX5 silencing. KLF4 knockdown abrogated the suppressive effect of SOX5 overexpression on osteogenic differentiation of hMSCs. Taken together, our results indicated that $TNF-{\alpha}$-induced SOX5 upregulation inhibited osteogenic differentiation of hMSCs through KLF4 signal pathway, suggesting that SOX5 might be a novel therapeutic target for PMOP treatment.
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문제 정의
We further found that SOX5 overexpression inhibited osteogenic differentiation of hMSCs. These findings provide an evidence for the potential role of SOX5 in regulating osteogenic differentiation of hMSCs in PMOP.
가설 설정
Fig. 2. SOX5 overexpression inhibited osteogenic differentiation of hMSCs. SOX5 mRNA and protein expression were examined by qRTPCR (A) and western blotting (B) in cells cultured with OM for 0 days (control) and 14 days (OM).
Interestingly, in PMOP, estrogen deficiency upregulates production of TNF-α, we found that TNF-α induced SOX5 gene expression during osteogenic differentiation of hMSCs in vitro. Whether estrogen deficiency could directly affect the SOX5 gene expression? This question is still further investigated. In addition, to further confirm the biological function of SOX5 in estrogen deficiency-induced osteoporosis, the effect of SOX5 on osteogenic differentiation and bone formation in the OVX rat model would be also required for research.
제안 방법
In this study, we detected SOX5 gene expression in hMSCs of bone marrow samples from patients with PMOP and from healthy premenopausal women. We found that the mRNA and protein expression levels of SOX5 were notably upregulated in PMOP group compared with the healthy control.
To further investigate the molecular mechanism of SOX5 mediating osteogenic differentiation of hMSCs, the gene expression of KLF4 was detected. The data of qRT-PCR and Western blotting showed that the mRNA and protein expression levels of KLF4 were notably upregulated during osteogenic differentiation of hMSCs (Figs.
데이터처리
All data in figures were provided as means ± SD. Statistical comparisons were made between two groups with a t-test. P <0.
이론/모형
Then, the OM medium was removed, and cells were lysed using a cell lysis buffer followed by centrifugation at 12,000 g force for 10 min. Besides, the protein concentration for each sample was determined with the Bradford method. Next, the resulting supernatants were incubated with p-nitrophenol phosphate at 37℃ for 30 min at room temperature.
Relative fold changes in mRNA were analyzed by using the 2−ΔΔCT method.
성능/효과
In conclusion, this study indicated for the first time that SOX5 gene expression was upregulated in hMSCs isolated from bone marrow samples from patients with PMOP. SOX5 overexpression inhibited osteogenic differentiation of hMSCs through the KLF4 signaling pathway.
Results comfirmed that TNF-α notably upregulates the SOX5 mRNA and protein expression levels in hMSCs with OM treatment.
Therefore, in our study, we examined the KLF4 gene expression using qRT-PCR and Western blotting. Results showed that KLF4 gene expression was notably upregulated during osteogenic differentiation of hMSCs. SOX5 overexpression remarkably increased KLF4 gene expression.
Taken together, our results indicated that TNFαinduced SOX5 upregulation inhibited osteogenic differentiation of hMSCs through the KLF4 signaling pathway.
후속연구
Whether estrogen deficiency could directly affect the SOX5 gene expression? This question is still further investigated. In addition, to further confirm the biological function of SOX5 in estrogen deficiency-induced osteoporosis, the effect of SOX5 on osteogenic differentiation and bone formation in the OVX rat model would be also required for research.
Moreover, whether SOX5 regulates KLF4 expression through direct binding the promoter region of KLF4 or indirect connection with other transcription factors is still not clear and would be required for further research. Interestingly, in PMOP, estrogen deficiency upregulates production of TNF-α, we found that TNF-α induced SOX5 gene expression during osteogenic differentiation of hMSCs in vitro.
참고문헌 (36)
Abdallah B.M. Kassem M. 2008 Human mesenchymal stem cells: from basic biology to clinical applications Gene Ther 15 109 116 17989700
Alm J.J. Heino T.J. Hentunen T.A. Vaananen H.K. Aro H.T. 2012 Transient 100 nM dexamethasone treatment reduces inter-and intraindividual variations in osteoblastic differentiation of bone marrow-derived human mesenchymal stem cells Tissue Eng Part C Methods 18 658 666 22428545
Axelsson A.S. Mahdi T. Nenonen H.A. Singh T. Hanzelmann S. Wendt A. Bagge A. Reinbothe T.M. Millstein J. Yang X. 2017 Sox5 regulates beta-cell phenotype and is reduced in type 2 diabetes Nat Commun 8 15652 28585545
Dy P. Han Y. Lefebvre V. 2008 Generation of mice harboring a Sox5 conditional null allele Genesis 46 294 299 18543318
Feng X. Shi Y. Xu L. Peng Q. Wang F. Wang X. Sun W. Lu Y. Tsao B.P. Zhang M. 2016 Modulation of IL-6 induced RANKL expression in arthritic synovium by a transcription factor SOX5 Sci Rep 6 32001 27550416
Kameda Y. Takahata M. Mikuni S. Shimizu T. Hamano H. Angata T. Hatakeyama S. Kinjo M. Iwasaki N. 2015 Siglec-15 is a potential therapeutic target for postmenopausal osteoporosis Bone 71 217 226 25460183
Kim J.H. Kim K. Youn B.U. Lee J. Kim I. Shin H.I. Akiyama H. Choi Y. Kim N. 2014 Kruppel-like factor 4 attenuates osteoblast formation, function, and cross talk with osteoclasts J Cell Biol 204 1063 1074 24616223
Li W. Liu M. Su Y. Zhou X. Liu Y. Zhang X. 2015 The Janus-faced roles of Kruppel-like factor 4 in oral squamous cell carcinoma cells Oncotarget 6 44480 44494 26517087
Lotters F.J. van den Bergh J.P. de Vries F. Rutten-van Molken M.P. 2016 Current and Future Incidence and Costs of Osteoporosis-Related Fractures in The Netherlands: Combining Claims Data with BMD Measurements Calcif Tissue Int 98 235 243 26746477
Lv H. Sun Y. Zhang Y. 2015 MiR-133 is involved in estrogen deficiency-induced osteoporosis through modulating osteogenic differentiation of mesenchymal stem cells Med Sci Monit 21 1527 1534 26013661
Mariner P.D. Johannesen E. Anseth K.S. 2012 Manipulation of miRNA activity accelerates osteogenic differentiation of hMSCs in engineered 3D scaffolds J Tissue Eng Regen Med 6 314 324 21706778
Nosho K. Yamamoto H. Takahashi T. Mikami M. Taniguchi H. Miyamoto N. Adachi Y. Arimura Y. Itoh F. Imai K. 2007 Genetic and epigenetic profiling in early colorectal tumors and prediction of invasive potential in pT1 (early invasive) colorectal cancers Carcinogenesis 28 1364 1370 17183069
Ohnishi S. Ohnami S. Laub F. Aoki K. Suzuki K. Kanai Y. Haga K. Asaka M. Ramirez F. Yoshida T. 2003 Downregulation and growth inhibitory effect of epithelial-type Kruppel-like transcription factor KLF4, but not KLF5, in bladder cancer Biochem Biophys Res Commun 308 251 256 12901861
Pineda B. Serna E. Laguna-Fernandez A. Noguera I. Panach L. Hermenegildo C. Tarin J.J. Cano A. Garcia-Perez M.A. 2014 Gene expression profile induced by ovariectomy in bone marrow of mice: a functional approach to identify new candidate genes associated to osteoporosis risk in women Bone 65 33 41 24815918
Raehtz S. Bierhalter H. Schoenherr D. Parameswaran N. McCabe L.R. 2017 Estrogen deficiency exacerbates type 1 diabetes-induced bone TNF-alpha expression and osteoporosis in female mice Endocrinology 158 2086 2101 28419209
Rodriguez J.P. Montecinos L. Rios S. Reyes P. Martinez J. 2000 Mesenchymal stem cells from osteoporotic patients produce a type I collagen-deficient extracellular matrix favoring adipogenic differentiation J Cell Biochem 79 557 565 10996846
Sang C. Zhang Y. Chen F. Huang P. Qi J. Wang P. Zhou Q. Kang H. Cao X. Guo L. 2016 Tumor necrosis factor alpha suppresses osteogenic differentiation of MSCs by inhibiting semaphorin 3B via Wnt/beta-catenin signaling in estrogen-deficiency induced osteoporosis Bone 84 78 87 26723579
Sapir-Koren R. Livshits G. 2017 Postmenopausal osteoporosis in rheumatoid arthritis: The estrogen deficiency-immune mechanisms link Bone 103 102 115 28666971
Silva I. Branco J.C. 2012 Denosumab: recent update in postmenopausal osteoporosis Acta Reumatol Port 37 302 313 24126422
Song I. Choi Y.J. Jin Y. Kim J.W. Koh J.T. Ji H.M. Jeong S.Y. Won Y.Y. Kim W. Chung Y.S. 2017 STRA6 as a possible candidate gene for pathogenesis of osteoporosis from RNAseq analysis of human mesenchymal stem cells Mol Med Rep 16 4075 4081 29067460
Tai S.K. Yang M.H. Chang S.Y. Chang Y.C. Li W.Y. Tsai T.L. Wang Y.F. Chu P.Y. Hsieh S.L. 2011 Persistent Kruppel-like factor 4 expression predicts progression and poor prognosis of head and neck squamous cell carcinoma Cancer Sci 102 895 902 21219537
Tiwari A. Loughner C.L. Swamynathan S. Swamynathan S.K. 2017 KLF4 plays an essential role in corneal epithelial homeostasis by promoting epithelial cell fate and suppressing epithelial-mesenchymal transition Invest Ophthalmol Vis Sci 58 2785 2795 28549095
Udalamaththa V.L. Jayasinghe C.D. Udagama P.V. 2016 Potential role of herbal remedies in stem cell therapy: proliferation and differentiation of human mesenchymal stromal cells Stem Cell Res Ther 7 110 27515026
Wang J. Zhao Q. 2017 Expression of CCR3, SOX5 and LC3 in patients with elderly onset rheumatoid arthritis and the clinical significance Exp Ther Med 14 3573 3576 29042950
Wang D. Han S. Wang X. Peng R. Li X. 2015 SOX5 promotes epithelial-mesenchymal transition and cell invasion via regulation of Twist1 in hepatocellular carcinoma Med Oncol 32 461 25572815
Wang C. Meng H. Wang X. Zhao C. Peng J. Wang Y. 2016 Differentiation of bone marrow mesenchymal stem cells in osteoblasts and adipocytes and its role in treatment of osteoporosis Med Sci Monit 22 226 233 26795027
Wegner M. 1999 From head to toes: the multiple facets of Sox proteins Nucleic Acids Res 27 1409 1420 10037800
Wegner M. 2010 All purpose Sox: The many roles of Sox proteins in gene expression Int J Biochem Cell Biol 42 381 390 19631281
Xu X. Jia X. Mo L. Liu C. Zheng L. Yuan Q. Zhou X. 2017 Intestinal microbiota: a potential target for the treatment of postmenopausal osteoporosis Bone Res 5 17046 28983411
Yang N. Wang G. Hu C. Shi Y. Liao L. Shi S. Cai Y. Cheng S. Wang X. Liu Y. 2013 Tumor necrosis factor alpha suppresses the mesenchymal stem cell osteogenesis promoter miR-21 in estrogen deficiency-induced osteoporosis J Bone Miner Res 28 559 573 23074166
Yao W. Guan M. Jia J. Dai W. Lay Y.A. Amugongo S. Liu R. Olivos D. Saunders M. Lam K.S. 2013 Reversing bone loss by directing mesenchymal stem cells to bone Stem Cells 31 2003 2014 23818248
Zhao J.W. Gao Z.L. Mei H. Li Y.L. Wang Y. 2011 Differentiation of human mesenchymal stem cells: the potential mechanism for estrogen-induced preferential osteoblast versus adipocyte differentiation Am J Med Sci 341 460 468 21289500
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