Excessive osteoclast differentiation and/or bone resorptive function causes a gradual loss of bone, leading to the pathogenesis of bone diseases such as osteoporosis (OP). In this study, a sulfated glucuronorhamnoxylan polysaccharide (designated SPS-CF) of the green alga Capsosiphon fulvescens was evaluated for anti-osteoporotic activity using osteoclastic cells differentiated from RAW264.7 macrophages by receptor activator of NF-κB ligand (RANKL) treatment and ovariectomized (OVX) female mice as a postmenopausal OP model. With negligible cytotoxicity, SPS-CF (50 μg/mL) significantly suppressed tartrate-resistant acid phosphatase (TRAP) activity, actin ring formation, and expression of matrix metalloproteinase 9 (MMP-9), cathepsin K, TRAF6, p-Pyk2, c-Cbl, c-Src, gelsolin, carbonic anhydrase II (CA II), and integrin β3, indicating that SPS-CF inhibits the differentiation and bone resorptive function of osteoclasts. Removal of sulfate groups from SPS-CF abolished its anti-osteoclastogenic activities, demonstrating that sulfate groups are critical for its activity. Oral administration of SPS-CF (400 mg/kg/day) to OVX mice significantly augmented the bone mineral density (BMD) and serum osteoprotegerin (OPG)/RANKL ratio. These results demonstrated that SPS-CF exerts significant anti-osteoporotic activity by dampening osteoclastogenesis and bone resorption via downregulation of TRAF6-c-Src-Pyk2-c-Cbl-gelsolin signaling and augmentation of serum OPG/RANKL ratios in OVX mice, suggesting that SPS-CF can be a novel anti-osteoporotic compound for treating postmenopausal OP.

Department of Biotechnology Graduate School The Catholic University of Korea Bucheon Gyeonggi do 14662 Republic of Korea

Department of Carbohydrate Chemistry and Technology University of Chemistry and Technology Prague Technická 5 166 28 Prague 6 Czech Republic

Department of Medical and Biological Sciences The Catholic University of Korea Bucheon Gyeonggi do 14662 Republic of Korea

Institute of Chemistry Centre for Glycomics Slovak Academy of Sciences Dúbravská cesta 9 845 38 Bratislava Slovakia

See more in PubMed

Ballanti P, Minisola S, Pacitti MT, Scarnecchia L, Rosso R, Mazzuoli GF, Bonucci E (1997) Tartrate-resistant acid phosphate activity as osteoclastic marker: sensitivity of cytochemical assessment and serum assay in comparison with standardized osteoclast histomorphometry. Osteoporos Int 7:39–43 PubMed DOI

Bar-Shavit Z (2007) The osteoclast: a multinucleated, hematopoietic-origin, bone-resorbing osteoimmune cell. J Cell Biochem 102:1130–1139 PubMed DOI

Blair HC, Zaidi M (2006) Osteoclastic differentiation and function regulated by old and new pathways. Rev Endocr Metab Disord 7:23–32 PubMed DOI

Boyce BF, Xing L (2008) Functions of RANKL/RANK/OPG in bone modeling and remodelling. Arch Biochem Biophys 473:139–146 PubMed DOI PMC

Carvalhal F, Cristelo RR, Resende D, Pinto MMM, Sousa E, Correia-da-Silva M (2019) Antithrombotics from the sea: polysaccharides and beyond. Mar Drugs 17:170 DOI PMC

Chellaiah M, Kizer N, Silva M, Alvarez U, Kwiatkowski D, Hruska KA (2000) Gelsolin deficiency blocks podosome assembly and produces increased bone mass and strength. J Cell Biol 148:665–678 PubMed DOI PMC

Chen C, Qin Y, Fang JP, Ni XY, Yao J, Wang HY, Ding K (2015) WSS25, a sulfated polysaccharide, inhibits RANKL-induced mouse osteoclast formation by blocking SMAD/ID1 signaling. Acta Pharmacol Sin 36:1053–1064 PubMed DOI PMC

Choi JW, Lee J, Kim SC, You S, Lee CW, Shin J, Park YI (2019) Glucuronorhamnoxylan from Capsosiphon fulvescens inhibits the growth of HT-29 human colon cancer cells in vitro and in vivo via induction of apoptotic cell death. Int J Biol Macromol 124:1060–1068 PubMed DOI

Dinoro J, Maher M, Talebian S, Jafarkhani M, Mehrali M, Orive G, Foroughi J, Lord MS, Dolatshahi-Pirouz A (2019) Sulfated polysaccharide-based scaffolds for orthopaedic tissue engineering. Biomaterials 214:119214 PubMed DOI

Eghbali-Fatourechi G, Khosla S, Sanyal A, Boyle WJ, Lacey DL, Riggs BL (2003) Role of RANK ligand in mediating increased bone resorption in early postmenopausal women. J Clin Invest 111:1221–1230 PubMed DOI PMC

Eriksen EF (2010) Cellular mechanisms of bone remodelling. Rev Endocr Metab Disord 11:219–227 PubMed DOI PMC

Fedorov SN, Ermakova SP, Zvyagintseva TN, Stonik VA (2013) Anticancer and cancer preventive properties of marine polysaccharides: some results and prospects. Mar Drugs 11:4876–4901 PubMed DOI PMC

Feng X, McDonald JM (2011) Disorders of bone remodelling. Annu Rev Pathol 6:121–145 PubMed DOI PMC

Feng X, Teitelbaum SL (2013) Osteoclasts: new insights. Bone Res 1:11–26 PubMed DOI

Filho GPC, de Sousa AFG, Viana RLS, Rocha HAO, de Medeiros SR, Batistuzzo MSMG (2019) Osteogenic activity of non-genotoxic sulfated polysaccharides from the green seaweed Caulerpa sertularioides. Algal Res 42:101546 DOI

Ghosh T, Pujol CA, Damonte EB, Sinha S, Ray B (2009) Sulfated xylomannans from the red seaweed Sebdenia polydactyla: structural features, chemical modification and antiviral activity. Antivir Chem Chemother 19:235–242 PubMed DOI

Han G, Zuo J, Holliday LS (2019) Specialized roles for actin in osteoclasts: unanswered questions and therapeutic opportunities. Biomolecules 9:17 DOI PMC

Ikebuchi Y, Aoki S, Honma M, Hayashi M, Sugamori Y, Khan M, Kariya Y, Kato G, Tabata Y, Penninger JM, Udagawa N, Aoki K, Suzuki H (2018) Coupling of bone resorption and formation by RANKL reverse signalling. Nature 561:195–200 PubMed DOI

Jeong SC, Jeong YT, Lee SM, Kim JH (2015) Immune-modulating activities of polysaccharides extracted from brown algae Hizikia fusiforme. Biosci Biotechnol Biochem 79:1362–1365 PubMed DOI

Jin X, Zhu L, Li X, Jia J, Zhang Y, Sun X, Ma J, Liu Z, Ma X (2017) Low molecular weight fucoidan inhibits the differentiation of osteoclasts and reduces osteoporosis in ovariectomized rats. Mol Med Rep 15:890–898 PubMed DOI

Karamanou K, Espinosa DCR, Fortuna-Costa A, Pavao MSG (2017) Biological function of unique sulfated glycosaminoglycans in primitive chordates. Glycoconj J 34:277–283 PubMed DOI

Khosla S, Bilezikian JP, Dempster DW, Lewiecki EM, Miller PD, Neer RM, Recker RR, Shane E, Shoback D, Potts JT (2012) Benefits and risks of bisphosphonate therapy for osteoporosis. J Clin Endocrinol Metab 97:2272–2282 PubMed DOI

Kim JH, Lee JE, Kim KH, Kang NJ (2018) Beneficial effects of marine algae-derived carbohydrates for skin health. Mar Drugs 16:459 DOI PMC

Kim YW, Baek SH, Lee SH, Kim TH, Kim SY (2014) Fucoidan, a sulfated polysaccharide, inhibits osteoclast differentiation and function by modulating RANKL signalling. Int J Mol Sci 15:18840–18855 PubMed DOI PMC

Kopplin G, Rokstad AM, Mélida H, Bulone V, Skjåk-Bræk G, Aachmann FL (2018) Structural characterization of fucoidan from Laminaria hyperborea: assessment of coagulation and inflammatory properties and their structure–function relationship. ACS Appl Bio Mat 1:1880–1892 DOI

Kuznetsova TA, Andryukov BG, Besednova NN, Zaporozhets TS, Kalinin AV (2020) Marine algae polysaccharides as basis for wound dressings, drug delivery, and tissue engineering: a review. J Mar Sci Eng 8:481–505 DOI

Lee CW, Seo JY, Choi JW, Lee J, Park JW, Lee JY, Hwang KY, Park YS, Park YI (2014) Potential anti-osteoporotic activity of low-molecular weight hyaluronan by attenuation of osteoclast cell differentiation and function in vitro. Biochem Biophys Res Commun 449:438–443 PubMed DOI

Lee EJ, Kim JL, Gong JH, Park SH, Kang YH (2015) Inhibition of osteoclast activation by phloretin through disturbing alphavbeta3 integrin-c-Src pathway. Biomed Res Int 2015:680145

Lee HG, Cowman MK (1994) An agarose gel electrophoretic method for analysis of hyaluronan molecular weight distribution. Anal Biochem 219:278–287 PubMed DOI

Lees RL, Sabharwal VK, Heersche JNM (2001) Resorptive state and cell size influence intracellular pH regulation in rabbit osteoclasts cultured on collagen-hydroxyapatite films. Bone 28:187–194 PubMed DOI

Liu H, Zhang H, Fan H, Tang S, Weng J (2020) The preventive effect of Cuscutae semen polysaccharide on bone loss in the ovariectomized rat model. Biomed Pharmacother 130:110613 PubMed DOI

Liu J, Curtis EM, Cooper C, Harvey NC (2019) State of the art in osteoporosis risk assessment and treatment. J Endocrinol Invest 42:1149–1164 PubMed DOI PMC

Lu SH, Hsia YJ, Shih KC, Chou TC (2019) Fucoidan prevents RANKL-stimulated osteoclastogenesis and LPS-induced inflammatory bone loss via regulation of Akt/GSK3beta/PTEN/NFATc1 signaling pathway and calcineurin activity. Mar Drugs 17:345 DOI PMC

Miller IJ, Blunt JW (1998) Desulfation of algal galactans. Carbohydr Res 309:39–43 DOI

Miyazaki T, Miyauchi S, Tawada A, Anada T, Suzuki O (2010) Effect of chondroitin sulfate-E on the osteoclastic differentiation of RAW264 cells. Dent Mater J 29:403–410 PubMed DOI

N’deh KPU, Yoo H-S, Chung K-H, Lee K-J, Kim D-H, Yoon JA, An JH, (2020) Collagen extract derived from Yeonsan Ogye chicken increases bone microarchitecture by suppressing the RANKL/OPG ratio via the JNK signaling pathway. Nutrients 12:1967 DOI

Na YS, Kim WJ, Kim SM, Park JK, Lee SM, Kim SO, Synytsya A, Park YI (2010) Purification, characterization and immunostimulating activity of water-soluble polysaccharide isolated from Capsosiphon fulvescens. Int Immunopharmacol 10:364–370 PubMed DOI

Nakamura I, Duong LT, Rodan SB, Rodan GA (2007) Involvement of alpha(v)beta3 integrins in osteoclast function. J Bone Miner Metab 25:337–344 PubMed DOI

Ney JT, Fehm T, Juhasz-Boess I, Solomayer EF (2012) RANK, RANKL and OPG expression in breast cancer - influence on osseous metastasis. Geburtshilfe Frauenheilkd 72:385–391 PubMed DOI PMC

Nishida D, Arai A, Zhao L, Yang M, Nakamichi Y, Horibe K, Hosoya A, Kobayashi Y, Udagawa N, Mizoguchi T (2021) RANKL/OPG ratio regulates odontoclastogenesis in damaged dental pulp. Sci Reports 11:4575

Oliveira RCR, Almeida RR, Goncalves TA (2016) A review of plant sulfated polysaccharides and their relations with anticoagulant activities. J Dev Drugs 05:166

Patel S (2012) Therapeutic importance of sulfated polysaccharides from seaweeds updating the recent findings 3. Biotech 2:171–185

Pollari S, Kakonen RS, Mohammad KS, Rissanen JP, Halleen JM, Warri A, Nissinen L, Pihlavisto M, Marjamaki A, Perala M, Guise TA, Kallioniemi O, Kakonen SM (2012) Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis. Mol Cancer Res 10:597–604 PubMed DOI

Purev E, Neff L, Horne WC, Baron R (2009) c-Cbl and Cbl-b act redundantly to protect osteoclasts from apoptosis and to displace HDAC6 from beta-tubulin, stabilizing microtubules and podosomes. Mol Biol Cell 20:4021–4030 PubMed DOI PMC

Queiroz KC, Medeiros VP, Queiroz LS, Abreu LR, Rocha HA, Ferreira CV, Juca MB, Aoyama H, Leite EL (2008) Inhibition of reverse transcriptase activity of HIV by polysaccharides of brown algae. Biomed Pharmacother 62:303–307 PubMed DOI

Ruocco N, Costantini S, Guariniello S, Costantini M (2016) Polysaccharides from the marine environment with pharmacological, cosmeceutical and nutraceutical potential. Molecules 21:551 DOI PMC

Schepetkin I (1997) Osteoclastic bone resorption: normal and pathological. Ann New York Aca Sci 832:170–193 DOI

Seidlova-Wuttke D, Nguyen BT, Wuttke W (2012) Long-term effects of ovariectomy on osteoporosis and obesity in estrogen-receptor-β–deleted mice. Comp Med 62:8–13 PubMed PMC

Shinmyouzu K, Takahashi T, Ariyoshi W, Ichimiya H, Kanzaki S, Nishihara T (2007) Dermatan sulfate inhibits osteoclast formation by binding to receptor activator of NF-kappa B ligand. Biochem Biophys Res Commun 354:447–452 PubMed DOI

Sophocleous A, Idris AI (2014) Rodent Models of Osteoporosis Bonekey Rep 3:614 PubMed

Sozen T, Ozisik L, Basaran NC (2017) An overview and management of osteoporosis. Eur J Rheumatol 4:46–56 PubMed DOI

Sundaram K, Nishimura R, Senn J, Youssef RF, London SD, Reddy SV (2007) RANK ligand signaling modulates the matrix metalloproteinase-9 gene expression during osteoclast differentiation. Exp Cell Res 313:168–178 PubMed DOI

Synytsya A, Choi DJ, Pohl R, Na YS, Capek P, Lattova E, Taubner T, Choi JW, Lee CW, Park JK, Kim WJ, Kim SM, Lee J, Park YI (2015) Structural features and anti-coagulant activity of the sulphated polysaccharide SPS-CF from a green alga Capsosiphon fulvescens. Mar Biotechnol 17:718–735 DOI

Tobeiha M, Moghadasian MH, Amin N, Jafarnejad S (2020) RANKL/RANK/OPG pathway: a mechanism involved in exercise-induced bone remodelling. Biomed Res Int 6910312

Ueno M, Cho K, Isaka S, Nishiguchi T, Yamaguchi K, Kim D, Oda T (2018) Inhibitory effect of sulphated polysaccharide porphyran (isolated from Porphyra yezoensis) on RANKL-induced differentiation of RAW264 7 cells into osteoclasts. Phytother Res 32:452–458 PubMed DOI

Venkatesan J, Anil S, Rao S, Bhatnagar I, Kim SK (2019) Sulfated polysaccharides from macroalgae for bone tissue regeneration. Curr Pharm Des 25:1200–1209 PubMed DOI

Wada T, Nakashima T, Hiroshi N, Penninger JM (2006) RANKL-RANK signaling in osteoclastogenesis and bone disease. Trends Mol Med 12:17–25 PubMed DOI

Walsh MC, Lee J, Choi Y (2015) Tumor necrosis factor receptor-associated factor 6 (TRAF6) regulation of development, function, and homeostasis of the immune system. Immunol Rev 266:72–92 PubMed DOI PMC

Wang Q, Xie Y, Du QS, Wu XJ, Feng X, Mei L, McDonald JM, Xiong WC (2003) Regulation of the formation of osteoclastic actin rings by proline-rich tyrosine kinase 2 interacting with gelsolin. J Cell Biol 160:565–575 PubMed DOI PMC

Zenger S, Hollberg K, Ljusberg J, Norgard M, Ek-Rylander B, Kiviranta R, Andersson G (2007) Proteolytic processing and polarized secretion of tartrate-resistant acid phosphatase is altered in a subpopulation of metaphyseal osteoclasts in cathepsin K-deficient mice. Bone 41:820–832 PubMed DOI