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Calcineurin Plays an Important Role in the Shell Formation of Pearl Oyster (Pinctada fucata)

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Abstract

Calcineurin (CN) is a multifunctional protein involved in many important physiological processes in mammalians, but the function of CN in mollusks is still largely unknown. In the present study, through the shell regeneration system, the changes of enzymatic activity of CN were determined in the process of shell regeneration in pearl oyster Pinctada fucata. CN was activated immediately and continuously in the shell regeneration process. The speed of shell regeneration was measured and the ultrastructure of inner shell surface was observed by scanning electron microscopy after inhibiting CN by intramuscular injection of immunosuppresant cyclosporine A (CsA). The results showed that the speed of shell regeneration was delayed and the morphology of calcite and aragonite in the inner shell surface became abnormal when CN was inhibited by CsA. Meanwhile, RT-PCR analysis revealed that the expression of P. fucata BMP-2 in mantle tissue decreased with CsA injection. In vitro secretion level of proteoglycans (PGs) in primary cultures of mantle cells was also decreased when mantle cells were exposed to CsA. Taken together, our results, for the first time, show that CN is involved in the shell formation through regulating the expression of Pf-BMP-2 in mantle tissue, which controls the secretion of PGs/GAGs of the mantle epithelial cells.

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References

  • Addadi L, Moradian J, Shay E, Maroudas NG, Weiner S (1987) A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation: relevance to biomineralization. Proc Natl Acad Sci USA 84:2732–2736

    Article  CAS  PubMed  Google Scholar 

  • Almeida MJ, Pereira L, Milet C, Haigle J, Barbosa M, Lopez E (2001) Comparative effects of nacre water-soluble matrix and dexamethasone on the alkaline phosphatase activity of MRC-5 fibroblasts. J Biomed Mater Res 57:306–312

    Article  CAS  PubMed  Google Scholar 

  • Aramburu J, Rao A, Klee CB (2000) Calcineurin: from structure to function. Curr Top Cell Regul 36:237–295

    Article  CAS  PubMed  Google Scholar 

  • Aramburu J, Heitman J, Crabtree GR (2004) Calcineurin: a central controller of signalling in eukaryotes. EMBO Rep 5:343–348

    Article  CAS  PubMed  Google Scholar 

  • Awumey EM, Moonga BS, Sodam BR, Koval AP, Adebanjo OA, Kumegawa M, Zaidi M, Epstein S (1999) Molecular and functional evidence for calcineurin-A alpha and beta isoforms in the osteoclast: novel insights into cyclosporin A action on bone resorption. Biochem Biophys Res Commun 254:248–252

    Article  CAS  PubMed  Google Scholar 

  • Bjornsson S (1993) Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue. Anal Biochem 210:282–291

    Article  CAS  PubMed  Google Scholar 

  • Clipstone NA, Crabtree GR (1992) Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature 357:695–697

    Article  CAS  PubMed  Google Scholar 

  • Csanky E, Asztalos L, Vasko A, Szucs I, Devenyi K, Szilasi M, Balla J (2007) Successful renal transplantation following lung transplantation: a survey of the first Hungarian case. Orv Hetil 148:2147–2151

    Article  PubMed  Google Scholar 

  • de la Pompa JL, Timmerman LA, Takimoto H, Yoshida H, Elia AJ, Samper E, Potter J, Wakeham A, Marengere L, Langille BL, Crabtree GR, Mak TW (1998) Role of the NF-ATc transcription factor in morphogenesis of cardiac valves and septum. Nature 392:182–186

    Article  PubMed  Google Scholar 

  • Delling U, Tureckova J, Lim HW, De Windt LJ, Rotwein P, Molkentin JD (2000) A calcineurin-NFATc3-dependent pathway regulates skeletal muscle differentiation and slow myosin heavy-chain expression. Mol Cell Biol 20:6600–6611

    Article  CAS  PubMed  Google Scholar 

  • Diedrichs H, Hagemeister J, Chi M, Boelck B, Muller-Ehmsen J, Schneider CA (2007) Activation of the calcineurin/NFAT signalling cascade starts early in human hypertrophic myocardium. J Int Med Res 35:803–818

    CAS  PubMed  Google Scholar 

  • Doege KJ, Sasaki M, Kimura T, Yamada Y (1991) Complete coding sequence and deduced primary structure of the human cartilage large aggregating proteoglycan, aggrecan. Human-specific repeats, and additional alternatively spliced forms. J Biol Chem 266:894–902

    CAS  PubMed  Google Scholar 

  • Fritz M, Belcher AM, Radmacher M, Walters DA, Hansma PK, Stucky GD, Morse DE, Mann S (1994) Flat pearls from biofabrication of organized composites on inorganic substrates. Nature 371:49–51

    Article  CAS  Google Scholar 

  • Gong N, Ma Z, Li Q, Li Q, Yan Z, Xie L, Zhang R (2008a) Characterization of calcium deposition and shell matrix protein secretion in primary mantle tissue culture from the marine pearl oyster Pinctada fucata. Mar Biotechnol (NY) 10:457–465

    Article  CAS  Google Scholar 

  • Gong N, Shangguan J, Liu X, Yan Z, Ma Z, Xie L, Zhang R (2008b) Immunolocalization of matrix proteins in nacre lamellae and their in vivo effects on aragonitic tablet growth. J Struct Biol 164:33–40

    Article  CAS  PubMed  Google Scholar 

  • Graef IA, Chen F, Chen L, Kuo A, Crabtree GR (2001) Signals transduced by Ca(2+)/calcineurin and NFATc3/c4 pattern the developing vasculature. Cell 105:863–875

    Article  CAS  PubMed  Google Scholar 

  • Har-el R, Tanzer ML (1993) Extracellular matrix. 3: Evolution of the extracellular matrix in invertebrates. FASEB J 7:1115–1123

    CAS  PubMed  Google Scholar 

  • Hardingham TE, Fosang AJ (1992) Proteoglycans: many forms and many functions. FASEB J 6:861–870

    CAS  PubMed  Google Scholar 

  • Hascall VC, Calabro A, Midura RJ, Yanagishita M (1994) Isolation and characterization of proteoglycans. Methods Enzymol 230:390–417

    Article  CAS  PubMed  Google Scholar 

  • Kong Y, Gu J, Yan Z, Li C, Gong N, Zhu F, Li D, Zhang Y, Zheng G, Wang H, Xie L, Zhang R (2009) Cloning and characterization of Prisilkin-39, a novel matrix protein serving a dual role in the prismatic layer formation from the oyster Pinctada fucata. J Biol Chem 284:10841–10854

    Article  CAS  PubMed  Google Scholar 

  • Kuroda Y, Hisatsune C, Nakamura T, Matsuo K, Mikoshiba K (2008) Osteoblasts induce Ca2+ oscillation-independent NFATc1 activation during osteoclastogenesis. Proc Natl Acad Sci USA 105:8643–8648

    Article  CAS  PubMed  Google Scholar 

  • Li S, Xie L, Zhang C, Zhang Y, Gu M, Zhang R (2004) Cloning and expression of a pivotal calcium metabolism regulator: calmodulin involved in shell formation from pearl oyster (Pinctada fucata). Comp Biochem Physiol B Biochem Mol Biol 138:235–243

    Article  PubMed  Google Scholar 

  • Li C, Huang J, Li S, Fan W, Hu Y, Wang Q, Zhu F, Xie L, Zhang R (2009) Cloning, characterization and immunolocalization of two subunits of calcineurin from pearl oyster (Pinctada fucata). Comp Biochem Physiol B Biochem Mol Biol 153:43–53

    Article  PubMed  Google Scholar 

  • Liao H, Mutvei H, Hammarstrom L, Wurtz T, Li J (2002) Tissue responses to nacreous implants in rat femur: an in situ hybridization and histochemical study. Biomaterials 23:2693–2701

    Article  CAS  PubMed  Google Scholar 

  • Loosanoff VL, Nomejko CA (1955) Growth of oysters with damaged shell-edges. Biol Bull (Woods Hole, Mass) 151–159

  • Lowenstam HA, Weiner S (1989) On biomineralization. Oxford University Press, New York

    Google Scholar 

  • Ma Z, Huang J, Sun J, Wang G, Li C, Xie L, Zhang R (2007) A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata. J Biol Chem 282:23253–23263

    Article  CAS  PubMed  Google Scholar 

  • Machii A, Wada KT (1989) Some marine invertebrates tissue culture. In: Mitsuhashi J (ed) Invertebrate cell systems applications. CRC, Boca Raton, pp 225–233

    Google Scholar 

  • Mann S (2001) Biomineralization: principles and concepts in bioinorganic materials chemistry. Oxford University Press, Oxford

    Google Scholar 

  • Marin F, Luquet G (2004) Molluscan shell proteins. C R Palevol 3:469–492

    Article  Google Scholar 

  • Marin F, Luquet G, Marie B, Medakovic D (2008) Molluscan shell proteins: primary structure, origin, and evolution. Curr Top Dev Biol 80:209–276

    Article  CAS  PubMed  Google Scholar 

  • Marxen JC, Becker W (2000) Calcium binding constituents of the organic shell matrix from the freshwater snail Biomphalaria glabrata. Comp Biochem Physiol B Biochem Mol Biol 127:235–242

    Article  CAS  PubMed  Google Scholar 

  • Meenakshi VR, Blackwelder PL, Wilbur KM (1973) An ultrastructural study of shell regeneration in Mytilus edulis (Mollusca:Bivalve). J Zool 171:475–484

    Article  Google Scholar 

  • Meenakshi VR, Blackwelder PL, Hare PE, Wilbur KM, Watabe N (1975) Studies on shell regeneration. I. Matrix and mineral composition of the normal and regenerated shell of Pomacea paludosa. Comp Biochem Physiol A Comp Physiol 50:347–351

    Article  CAS  PubMed  Google Scholar 

  • Miyashita T, Hanashita T, Toriyama M, Takagi R, Akashika T, Higashikubo N (2008) Gene cloning and biochemical characterization of the BMP-2 of Pinctada fucata. Biosci Biotechnol Biochem 72:37–47

    Article  CAS  PubMed  Google Scholar 

  • Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Robbins J, Grant SR, Olson EN (1998) A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93:215–228

    Article  CAS  PubMed  Google Scholar 

  • Mount AS, Wheeler AP, Paradkar RP, Snider D (2004) Hemocyte-mediated shell mineralization in the eastern oyster. Science 304:297–300

    Article  CAS  PubMed  Google Scholar 

  • Movsowitz C, Epstein S, Fallon M, Ismail F, Thomas S (1988) Cyclosporin-A in vivo produces severe osteopenia in the rat: effect of dose and duration of administration. Endocrinology 123:2571–2577

    Article  CAS  PubMed  Google Scholar 

  • Navasa M, Monegal A, Guanabens N, Peris P, Rimola A, Munoz-Gomez J, Visa J, Rodes J (1994) Bone fractures in liver transplant patients. Br J Rheumatol 33:52–55

    Article  CAS  PubMed  Google Scholar 

  • Poncet J, Serpentini A, Thiebot B, Villers C, Bocquet J, Boucaud-Camou E, Lebel J (2000) In vitro synthesis of proteoglycans and collagen in primary cultures of mantle cells from the nacreous mollusk, Haliotis tuberculata: a new model for study of molluscan extracellular matrix. Mar Biotechnol (NY) 2:387–398

    CAS  Google Scholar 

  • Raman R, Sasisekharan V, Sasisekharan R (2005) Structural insights into biological roles of protein-glycosaminoglycan interactions. Chem Biol 12:267–277

    Article  CAS  PubMed  Google Scholar 

  • Rao A, Luo C, Hogan PG (1997) Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol 15:707–747

    Article  CAS  PubMed  Google Scholar 

  • Reddi AH (2000) Morphogenetic messages are in the extracellular matrix: biotechnology from bench to bedside. Biochem Soc Trans 28:345–349

    Article  CAS  PubMed  Google Scholar 

  • Rousseau M, Pereira-Mouries L, Almeida MJ, Milet C, Lopez E (2003) The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3–E1 mouse pre-osteoblasts. Comp Biochem Physiol B Biochem Mol Biol 135:1–7

    PubMed  Google Scholar 

  • Rusnak F, Mertz P (2000) Calcineurin: form and function. Physiol Rev 80:1483–1521

    CAS  PubMed  Google Scholar 

  • Simkiss K (1965) The organic matrix of the oyster shell. Comp Biochem Physiol 16:427–435

    Article  CAS  PubMed  Google Scholar 

  • Simkiss K, Wilbur KM (1989) Biomineralization—cell biology and mineral deposition. Academic, San Diego

    Google Scholar 

  • Stewart MC, Saunders KM, Burton-Wurster N, Macleod JN (2000) Phenotypic stability of articular chondrocytes in vitro: the effects of culture models, bone morphogenetic protein 2, and serum supplementation. J Bone Miner Res 15:166–174

    Article  CAS  PubMed  Google Scholar 

  • Sud D, Doumenc D, Lopez E, Milet C (2001) Role of water-soluble matrix fraction, extracted from the nacre of Pinctada maxima, in the regulation of cell activity in abalone mantle cell culture (Haliotis tuberculata). Tissue Cell 33:154–160

    Article  CAS  PubMed  Google Scholar 

  • Takayanagi H (2007) The role of NFAT in osteoclast formation. Ann N Y Acad Sci 1116:227–237

    Article  CAS  PubMed  Google Scholar 

  • Timmermans LP (1973) Mantle activity following shell injury to the pond snail, Lymnaea stagnalis L. Malacologia 14:53–61

    Google Scholar 

  • Tomita M, Reinhold MI, Molkentin JD, Naski MC (2002) Calcineurin and NFAT4 induce chondrogenesis. J Biol Chem 277:42214–42218

    Article  CAS  PubMed  Google Scholar 

  • Watabe N (1965) Studies on shell formation. XI. Crystal–matrix relationships in the inner layers of mollusk shells. J Ultrastruct Res 12:351–370

    Article  CAS  PubMed  Google Scholar 

  • Watabe N (1983) Shell repair. In: Saleuddin ASM, Wilbur KM (eds) The physiology of Mollusca vol. IV. Academic, New York, pp 289–316

    Google Scholar 

  • Watabe N, Blackwelder PL (1980) Ultrastructural and calcium localization in the mantle epithelium of the freshwater gastropod Pomacea paludosa during shell regeneration. In: Omori M, Watabe N (eds) The mechanisms of biomineralization in animals and plants. Tokai University Press, Tokyo, pp 131–144

    Google Scholar 

  • Watabe N, Wilbur KM (1960) Influence of the organic matrix on crystal type in molluscs. Nature 188:334–334

    Article  Google Scholar 

  • Weiss IM, Tuross N, Addadi L, Weiner S (2002) Mollusc larval shell formation: amorphous calcium carbonate is a precursor phase for aragonite. J Exp Zool 293:478–491

    Article  CAS  PubMed  Google Scholar 

  • Westbroek P, Marin F (1998) A marriage of bone and nacre. Nature 392:861–862

    Article  CAS  PubMed  Google Scholar 

  • Winslow MM, Pan M, Starbuck M, Gallo EM, Deng L, Karsenty G, Crabtree GR (2006) Calcineurin/NFAT signaling in osteoblasts regulates bone mass. Dev Cell 10:771–782

    Article  CAS  PubMed  Google Scholar 

  • Worms D, Weiner S (1986) Mollusk shell organic matrix: Fourier transform infrared study of the acidic macromolecules. J Exp Zool 237:11–20

    Article  CAS  Google Scholar 

  • Wozney JM (1998) The bone morphogenetic protein family: multifunctional cellular regulators in the embryo and adult. Eur J Oral Sci 106(Suppl 1):160–166

    CAS  PubMed  Google Scholar 

  • Yamanaka Y, Abu-Amer W, Foglia D, Otero J, Clohisy JC, Abu-Amer Y (2008) NFAT2 is an essential mediator of orthopedic particle-induced osteoclastogenesis. J Orthop Res 26:1577–1584

    Article  CAS  PubMed  Google Scholar 

  • Yan Z, Ma Z, Zheng G, Feng Q, Wang H, Xie L, Zhang R (2008) The inner-shell film: an immediate structure participating in pearl oyster shell formation. Chembiochem 9:1093–1099

    Article  CAS  PubMed  Google Scholar 

  • Yoo SA, Park BH, Park GS, Koh HS, Lee MS, Ryu SH, Miyazawa K, Park SH, Cho CS, Kim WU (2006) Calcineurin is expressed and plays a critical role in inflammatory arthritis. J Immunol 177:2681–2690

    CAS  PubMed  Google Scholar 

  • Zhang C, Zhang R (2006) Matrix proteins in the outer shells of molluscs. Mar Biotechnol (NY) 8:572–586

    Article  Google Scholar 

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Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (2006AA09Z441 and 2006AA09Z413), the National Natural Science Foundation of China (no. 30530600 and no. 30221003), and the grant J0630647.

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Authors and Affiliations

  1. Institute of Marine Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, 100084, China

    Changzhong Li, Yilin Hu, Jian Liang, Yawei Kong, Jing Huang, Qiaoli Feng, Shuo Li, Guiyou Zhang, Liping Xie, Liping Xie, Rongqing Zhang & Rongqing Zhang

  2. Protein Science Laboratory of the Ministry of Education, Tsinghua University, Beijing, 100084, China

    Guiyou Zhang, Liping Xie & Rongqing Zhang

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  1. Changzhong Li
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  2. Yilin Hu
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Correspondence to Liping Xie or Rongqing Zhang.

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Li, C., Hu, Y., Liang, J. et al. Calcineurin Plays an Important Role in the Shell Formation of Pearl Oyster (Pinctada fucata). Mar Biotechnol 12, 100–110 (2010). https://doi.org/10.1007/s10126-009-9204-3

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