Sphingosine-1-phosphate hinders the osteogenic differentiation of dental pulp stem cells in association with AKT signaling pathways

Abstract

Sphingosine-1-phosphate (S1P) is an important lipid mediator that regulates a diverse range of intracellular cell signaling pathways that are relevant to tissue engineering and regenerative medicine. However, the precise function of S1P in dental pulp stem cells (DPSCs) and its osteogenic differentiation remains unclear. We here investigated the function of S1P/S1P receptor (S1PR)-mediated cellular signaling in the osteogenic differentiation of DPSCs and clarified the fundamental signaling pathway. Our results showed that S1P-treated DPSCs exhibited a low rate of differentiation toward the osteogenic phenotype in association with a marked reduction in osteogenesis-related gene expression and AKT activation. Of note, both S1PR1/S1PR3 and S1PR2 agonists significantly downregulated the expression of osteogenic genes and suppressed AKT activation, resulting in an attenuated osteogenic capacity of DPSCs. Most importantly, an AKT activator completely abrogated the S1P-mediated downregulation of osteoblastic markers and partially prevented S1P-mediated attenuation effects during osteogenesis. Intriguingly, the pro-inflammatory TNF-α cytokine promoted the infiltration of macrophages toward DPSCs and induced S1P production in both DPSCs and macrophages. Our findings indicate that the elevation of S1P under inflammatory conditions suppresses the osteogenic capacity of the DPSCs responsible for regenerative endodontics.

References

[1] Fernandes G, Yang S. Application of platelet-rich plasma with stem cells in bone and periodontal tissue engineering. Bone Res., 2016, 4: 16036.

[2] Gibon E, Lu L, Goodman SB. Aging, inflammation, stem cells, and bone healing. Stem Cell Res. Ther., 2016, 7: 44.

[3] Chen XY, . Systematic comparison of biologically active foreign ions-codoped calcium phosphate microparticles on osteogenic differentiation in rat osteoporotic and normal mesenchymal stem cells. Oncotarget, 2017, 8: 36578-36590.

[4] Camargo WA, . Diabetes mellitus and bone regeneration: a systematic review and meta-analysis of animal studies. Tissue Eng. Part B Rev., 2017, 23: 471-479.

[5] Bergenholtz G. Pathogenic mechanisms in pulpal disease. J. Endod., 1990, 16: 98-101.

[6] Cooper PR, . Inflammation-regeneration interplay in the dentine-pulp complex. J. Dent., 2010, 38: 687-697.

[7] Veerayutthwilai O, Byers MR, Pham TT, Darveau RP, Dale BA. Differential regulation of immune responses by odontoblasts. Oral Microbiol. Immunol., 2007, 22: 5-13.

[8] Laino G, . A new population of human adult dental pulp stem cells: a useful source of living autologous fibrous bone tissue (LAB). J. Bone Miner. Res., 2005, 20: 1394-1402.

[9] Pierdomenico L, . Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp. Transplantation, 2005, 80: 836-842.

[10] Bluteau G, Luder HU, De Bari C, Mitsiadis TA. Stem cells for tooth engineering. Eur. Cells Mater., 2008, 16: 1-9.

[11] Woloszyk A, . Influence of the mechanical environment on the engineering of mineralised tissues using human dental pulp stem cells and silk fibroin scaffolds. PloS ONE, 2014, 9: e111010.

[12] Soares DG, . Simvastatin and nanofibrous poly(l-lactic acid) scaffolds to promote the odontogenic potential of dental pulp cells in an inflammatory environment. Acta Biomater., 2018, 68: 190-203.

[13] Kim SG, Malek M, Sigurdsson A, Lin LM, Kahler B. Regenerative endodontics: a comprehensive review. Int. Endod. J., 2018, 51: 1367-1388.

[14] d’Aquino R, . Human mandible bone defect repair by the grafting of dental pulp stem/progenitor cells and collagen sponge biocomplexes. Eur. Cells Mater., 2009, 18: 75-83.

[15] Papaccio G, . Long-term cryopreservation of dental pulp stem cells (SBP-DPSCs) and their differentiated osteoblasts: a cell source for tissue repair. J. Cell. Physiol., 2006, 208: 319-325.

[16] Yasui T, . Isolation of dental pulp stem cells with high osteogenic potential. Inflamm. Regen., 2017, 37

[17] Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc. Natl Acad. Sci. USA, 2000, 97: 13625-13630.

[18] Wasserman AH, Venkatesan M, Aguirre A. Bioactive lipid signaling in cardiovascular disease, development, and regeneration. Cells, 2020, 9: 1391.

[19] Musso G, Cassader M, Paschetta E, Gambino R. Bioactive lipid species and metabolic pathways in progression and resolution of nonalcoholic steatohepatitis. Gastroenterology, 2018, 155: 282-302.e288.

[20] Das UN. Bioactive lipids in age-related disorders. Adv. Exp. Med. Biol., 2020, 1260: 33-83.

[21] Spiegel S, Milstien S. Sphingosine 1-phosphate, a key cell signaling molecule. J. Biol. Chem., 2002, 277: 25851-25854.

[22] Obinata H, Hla T. Sphingosine 1-phosphate and inflammation. Int. Immunol., 2019, 31: 617-625.

[23] Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat. Rev. Mol. Cell Biol., 2003, 4: 397-407.

[24] Nincheri P, . Sphingosine 1-phosphate induces differentiation of adipose tissue-derived mesenchymal stem cells towards smooth muscle cells. Cell. Mol. Life Sci., 2009, 66: 1741-1754.

[25] Anliker B, Chun J. Lysophospholipid G protein-coupled receptors. J. Biol. Chem., 2004, 279: 20555-20558.

[26] Matsuzaki E, . Sphingosine-1-phosphate promotes the nuclear translocation of β-catenin and thereby induces osteoprotegerin gene expression in osteoblast-like cell lines. Bone, 2013, 55: 315-324.

[27] Sato C, Iwasaki T, Kitano S, Tsunemi S, Sano H. Sphingosine 1-phosphate receptor activation enhances BMP-2-induced osteoblast differentiation. Biochem. Biophys. Res. Commun., 2012, 423: 200-205.

[28] Petrie Aronin CE, . The enhancement of bone allograft incorporation by the local delivery of the sphingosine 1-phosphate receptor targeted drug FTY720. Biomaterials, 2010, 31: 6417-6424.

[29] Petrie Aronin CE, . FTY720 promotes local microvascular network formation and regeneration of cranial bone defects. Tissue Eng. Part A, 2010, 16: 1801-1809.

[30] Lee DE, . The sphingosine-1-phosphate receptor 1 binding molecule FTY720 inhibits osteoclast formation in rats with ligature-induced periodontitis. J. Periodontal Res., 2017, 52: 33-41.

[31] Mukherjee A, Rotwein P. Akt promotes BMP2-mediated osteoblast differentiation and bone development. J. Cell Sci., 2009, 122: 716-726.

[32] Franceschi RT, Ge C. Control of the osteoblast lineage by mitogen-activated protein kinase signaling. Curr. Mol. Biol. Rep., 2017, 3: 122-132.

[33] Cekici A, Kantarci A, Hasturk H, Van Dyke TE. Inflammatory and immune pathways in the pathogenesis of periodontal disease. Periodontology 2000, 2014, 64: 57-80.

[34] Luo G, Li F, Li X, Wang ZG, Zhang B. TNF‑α and RANKL promote osteoclastogenesis by upregulating RANK via the NF‑κB pathway. Mol. Med. Rep., 2018, 17: 6605-6611.

[35] Graves DT, Li J, Cochran DL. Inflammation and uncoupling as mechanisms of periodontal bone loss. J. Dent. Res., 2011, 90: 143-153.

[36] Hammad SM, . Dual and distinct roles for sphingosine kinase 1 and sphingosine 1 phosphate in the response to inflammatory stimuli in RAW macrophages. Prostaglandins Other Lipid Mediat, 2008, 85: 107-114.

[37] Nishida D, . RANKL/OPG ratio regulates odontoclastogenesis in damaged dental pulp. Sci. Rep., 2021, 11

[38] Hamdan, N., Bhagirath, A. Y. & Batista, E. L., Jr Sphingosine kinase activity and sphingosine-1-phosphate in the inflamed human periodontium. Oral Dis. https://doi.org/10.1111/odi.13995 (2021).

[39] Higashi K, . Sphingosine-1-phosphate/S1PR2-mediated signaling triggers Smad1/5/8 phosphorylation and thereby induces Runx2 expression in osteoblasts. Bone, 2016, 93: 1-11.

[40] Brizuela L, . Osteoblast-derived sphingosine 1-phosphate to induce proliferation and confer resistance to therapeutics to bone metastasis-derived prostate cancer cells. Mol. Oncol., 2014, 8: 1181-1195.

[41] Ishii M, . Sphingosine-1-phosphate mobilizes osteoclast precursors and regulates bone homeostasis. Nature, 2009, 458: 524-528.

[42] Ishii M, Kikuta J. Sphingosine-1-phosphate signaling controlling osteoclasts and bone homeostasis. Biochim. Biophys. Acta, 2013, 1831: 223-227.

[43] Matsuzaki E, Minakami M, Matsumoto N, Anan H. Dental regenerative therapy targeting sphingosine-1-phosphate (S1P) signaling pathway in endodontics. Jpn. Dent. Sci. Rev., 2020, 56: 127-134.

[44] Kluk MJ, Hla T. Signaling of sphingosine-1-phosphate via the S1P/EDG-family of G-protein-coupled receptors. Biochim. Biophys. Acta, 2002, 1582: 72-80.

[45] Chi H. Sphingosine-1-phosphate and immune regulation: trafficking and beyond. Trends Pharmacol. Sci., 2011, 32: 16-24.

[46] Brinkmann V, . Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat. Rev. Drug Discov., 2010, 9: 883-897.

[47] Grassi S, . Sphingosine 1-phosphate receptors and metabolic enzymes as druggable targets for brain diseases. Front. Pharmacol., 2019, 10: 807.

[48] Ishii M, Kikuta J, Shimazu Y, Meier-Schellersheim M, Germain RN. Chemorepulsion by blood S1P regulates osteoclast precursor mobilization and bone remodeling in vivo. J. Exp. Med., 2010, 207: 2793-2798.

[49] Kikuta J, . Sphingosine-1-phosphate-mediated osteoclast precursor monocyte migration is a critical point of control in antibone-resorptive action of active vitamin D. Proc. Natl Acad. Sci. USA, 2013, 110: 7009-7013.

[50] Weske S, . Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss. Nat. Med., 2018, 24: 667-678.

[51] Weske S, . Agonist-induced activation of the S1P receptor 2 constitutes a novel osteoanabolic therapy for the treatment of osteoporosis in mice. Bone, 2019, 125: 1-7.

[52] Igarashi J, Michel T. Sphingosine 1-phosphate and isoform-specific activation of phosphoinositide 3-kinase beta. Evidence for divergence and convergence of receptor-regulated endothelial nitric-oxide synthase signaling pathways. J. Biol. Chem., 2001, 276: 36281-36288.

[53] Chang EJ, . The JNK-dependent CaMK pathway restrains the reversion of committed cells during osteoclast differentiation. J. Cell Sci., 2008, 121: 2555-2564.

Funding

National Research Foundation of Korea (NRF)(2018R1A5A2020732, 2018R1A2B2001867, 2017M3A9E4047243)