A growing number of studies have been showing that dietary probiotics can exert beneficial health effects in both humans and animals. We previously demonstrated that dietary supplementation with Lactobacillus rhamnosus - a component of the human gut microflora - enhances reproduction, larval development, and the biomineralization process in Danio rerio (zebrafish). The aim of this study was to identify the pathways affected by L. rhamnosus during zebrafish larval development. Our morphological and histochemical findings show that L. rhamnosus accelerates bone deposition through stimulation of the expression of key genes involved in ossification, e.g. runt-related transcription factor 2 (runx2), Sp7 transcription factor (sp7), matrix Gla protein (mgp), and bone gamma-carboxyglutamate (gla) protein (bglap) as well as through inhibition of sclerostin (sost), a bone formation inhibitor. Western blot analysis of mitogen-activated protein kinase 1 and 3-(Mapk1 and Mapk3), which are involved in osteoblast and osteocyte differentiation, documented an increase in Mapk1 16 days post fertilization (dpf) and of Mapk3 23 dpf in individuals receiving L. rhamnosus supplementation. Interestingly, a reduction of sost detected in the same individuals suggests that the probiotic may help treat bone disorders.
References
[1]
Rawls F, Samuel BS, Gordon JI (2004) Gnotobiotic zebrafish reveal evolutionarily conserved responses to the gut microbiota. Proc Natl Acad Sci USA 101: 4596–4601.
[2]
Dimitroglou A, Merrifield DL, Carnevali O, Picchietti S, Avella MA, et al.. (2011) Microbial manipulations to improve fish health and production–a Mediterranean perspective. Fish Shell Immunol 30: :1–16 Review.
[3]
Oliva-Teles A (2012) Nutrition and health of aquaculture fish. J Fish Dis35: :83–108. Review.
[4]
Picchietti S, Mazzini M, Taddei AR, Renna R, Fausto AM, et al. (2007) Effects of administration of probiotic strains on GALT of larval gilthead seabream: Immunohistochemical and ultrastructural studies. Fish Shell Immunol 22: 57–67.
[5]
Picchietti S, Fausto AM, Randelli E, Carnevali O, Taddei AR, et al. (2009) Early treatment with Lactobacillus delbrueckii strain induces an increase in intestinal T-cells and granulocytes and modulates immune-related genes of larval Dicentrarchus labrax (L.). Fish Shell Immunol 26: 368–376.
[6]
Abelli L, Randelli E, Carnevali O, Picchietti S (2009) Stimulation of gut immune system by early administration of probiotic strains in Dicentrarchus labrax and Sparus aurata.. Ann NY Acad Sci 1163: 340–342.
[7]
Pérez-Sánchez T, Balcázar JL, Merrifield DL, Carnevali O, Gioacchini G, et al. (2011) Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection. Fish Shell Immunol 3: 196–201.
[8]
Standen BT, Rawling MD, Davies SJ, Castex M, Foey A, et al. (2013) Probiotic Pediococcus acidilactici modulates both localised intestinal- and peripheral-immunity in tilapia (Oreochromis niloticus). Fish Shellfish Immunol 35(4): 1097–1104.
[9]
Rollo A, Sulpizio R, Nardi M, Silvi S, Orpianesi C, et al. (2006) Live microbial feed supplement in aquaculture for improvement of stress tolerance. Fish Physiol Biochem 32: 167–177.
[10]
Lauzon HL, Gudmundsdottir S, Steinarsson A, Oddgeirsson M, Petursdottir SK, et al. (2010) Effects of bacterial treatment at early stages of Atlantic cod (Gadus morhua L.) on larval survival and development. J Appl Microbiol 108: 624–632.
[11]
Avella MA, Olivotto I, Silvi S, Place AR, Carnevali O (2010) Effect of dietary probiotics on clownfish: a molecular approach to define how lactic acid bacteria modulate development in a marine fish. Am J Physiol Regul Integr Comp Physiol 298: R359–371.
[12]
Avella MA, Place A, Du SJ, Williams E, Silvi S, et al. (2012) Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems. PLoS One 7: e45572.
[13]
Palermo FA, Mosconi G, Avella MA, Carnevali O, Verdenelli MC, et al. (2011) Modulation of cortisol levels, endocannabinoid receptor 1A, proopiomelanocortin and thyroid hormone receptor alpha mRNA expressions by probiotics during sole (Solea solea) larval development. Gen Comp Endocrinol 171: 293–300.
[14]
Gioacchini G, Maradonna F, Lombardo F, Bizzaro D, Olivotto I, et al. (2010) Increase of fecundity by probiotic administration in zebrafish (Danio rerio). Reproduction 140: 953–959.
[15]
Gioacchini G, Giorgini E, Merrifield DL, Hardiman G, Borini A, et al. (2012) Probiotics can induce follicle maturational competence: the Danio rerio case. Biol Reprod 86: 65.
[16]
Gioacchini G, Dalla Valle L, Benato F, Fimia GM, Nardacci R, et al. (2013) Interplay between autophagy and apoptosis in the development of Danio rerio follicles and the effects of a probiotic. Reprod Fertil Dev 25(8): 1115–1125.
[17]
Giorgini E, Conti C, Ferraris P, Sabbatini S, Tosi G, et al. (2010) Effects of Lactobacillus rhamnosus on zebrafish oocyte maturation: an FTIR imaging and biochemical analysis. Anal Bioanal Chem 398: 3063–3072.
[18]
Lombardo F, Gioacchini G, Carnevali O (2011) Probiotic-based Nutritional Effects on Killifish reproduction. Fish Aquacult J FAJ-33
[19]
Witten PE, Huysseune A (2009) A comparative view on mechanisms and functions of skeletalremodelling in teleost fish, with special emphasis on osteoclasts and their function. Biol Rev 84: 315–346.
[20]
Witten PE, Huysseune A (2010) The unobtrusivemajority: mononucleated bone resorbing cells inteleost fish and mammals.J Appl Ichthyol. 26(2): 225–229.
[21]
Witten PE, Hansen A, Hall BK (2001) Features of mono- and multinucleated bone resorbing cells of the zebrafish Danio rerio and their contribution to skeletal development, remodeling, and growth. J Morphol 250: 197–207.
[22]
Hall BK, Witten PE (2007) Plasticity of and transitions between skeletal tissues in vertebrate evolution and development. In: Anderson JS, Sues HD editors. Major Transitions in Vertebrate Evolution.Bloomington, IN, Indiana University Press. pp. 13–56.
[23]
Apschner AA, Schulte-Merker S, Witten PE (2011) Not all bones are created equal - using zebrafish and other teleost species in osteogenesis research. Methods Cell Biol 105: 239–255.
[24]
Witten PE, Huysseune A, Hall BK (2010) A practical approach for the identification of the many cartilaginous tissues in teleost fish. J Appl Ichthyol 26(2): 257–262.
[25]
Benjamin M (1988) Mucochondroid (mucous connective) tissue in the head of teleosts. Anat Embryol178: 461–474.
[26]
Benjamin M (1990) The cranial cartilages of teleosts and their classification. J Anat 169: 153–172.
[27]
Bensimon-Brito A, Cancela ML, Huysseune A, Witten PE (2010) The zebrafish (Danio rerio) caudal complex – a model to study vertebral body fusion. J Appl Ichthyo l26(2): 235–238.
[28]
Lian JB, Stein GS (2003) The temporal and spatial subnuclear organization of skeletal gene regulatory machinery: integrating multiple levels of transcriptional control. Calcif Tissue Int 72: :631–637.
[29]
Lian JB, Stein GS (2003) Runx2/Cbfa1: a multifunctional regulator of bone formation. Curr Pharm Des 9: :2677–2685. Review.
[30]
Flores MV, Tsang VW, Hu W, Kalev-Zylinska M, Postlethwait J, et al. (2004) Duplicate zebrafish runx2 orthologues are expressed in developing skeletal elements. Gene Expr Patterns 4(5): 573–581.
[31]
van der Meulen T, Kranenbarg S, Schipper H, Samallo J, van Leeuwen JL, et al. (2005) Identification and characterisation of two runx2 homologues in zebrafish with different expression patterns. Biochim Biophys Acta 1729(2): 105–117.
[32]
Flores MV, Lam EY, Crosier P, Crosier K (2006) A hierarchy of Runx transcription factors modulate the onset of chondrogenesis in craniofacial endochondral bones in zebrafish. Dev Dyn 235(11): 3166–3176.
[33]
Yang DC, Tsai CC, Liao YF, Fu HC, Tsay HJ, et al. (2011) Twist controls skeletal development and dorsoventral patterning by regulating runx2in zebrafish. PLoS One 6(11): e27324.
[34]
Pinto JP, Concei？？o NM, Viegas CSB, Leite RB, Hurst LD, et al. (2005) Identification of a New pebp2A2 Isoform From Zebrafishrunx2 Capable of Inducing Osteocalcin Gene Expression In Vitro. J Bone Min Res 20(8): 1440–1453.
[35]
Li N, Felber K, Elks P, Croucher P, Roehl HH (2009) Tracking gene expression during zebrafish osteoblast differentiation. Dev Dyn 238(2): 459–466.
[36]
Ducy P, Desbois C, Boyce B, Pinero G, Story B, et al. (1996) Increased bone formation in osteocalcin-deficient mice. Nature 382: 448–452.
[37]
Karsenty G (2000) The central regulation of bone remodeling. Trends Endocrinol Metab 11: :437–439. Review.
[38]
Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, et al. (2002) The novel zinc finger-containintranscription factor osterix is required for osteoblast differentiation and bone formation. Cell 108: 17–29.
[39]
Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, et al. (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89: 755–764.
[40]
Delmas PD, Christiansen C, Mann KG, Price PA (1990) Bone Gla protein (osteocalcin) assay standardization report. J Bone Miner Res 5: 5–11.
[41]
Newman B, Gigout LI, Sudre L, Grant ME, Wallis GA (2001) Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival. J Cell Biol 154: 659–666.
[42]
Gavaia PJ, Simes DC, Ortiz-Delgado JB, Viegas CS, Pinto JP, et al. (2006) Osteocalcin and matrix Gla protein in zebrafish (Danio rerio) and Senegal sole (Solea senegalensis): comparative gene and protein expression during larval development through adulthood. Gene Expr Patterns 6: 637–652.
[43]
Kross？y C, Ornsrud R, Wargelius A (2009) Differential gene expression of bgp and mgp in trabecular and compact bone of Atlantic salmon (Salmo salar L.) vertebrae. J Anat 215(6): 663–672.
[44]
Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, et al. (1997) Spontaneous calcification of arteries and cartilage in mice lacking matrix Gla protein. Nature 386: 78–81.
[45]
McNulty MS, Bedell VM, Greenwood TM, Craig TA, Ekker SC, et al. (2012) Expression of sclerostin in the developing zebrafish (Danio rerio) brain and skeleton. Gene Expr Patterns 12: 228–235.
[46]
Li X, Ominsky MS, Warmington KS, Morony S, Gong J, et al. (2009) Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res 24: 578–588.
[47]
Xiao G, Gopalakrishnan R, Jiang D, Reith E, Benson MD, et al. (2002) Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells. J Bone Miner Res 17: 101–110.
[48]
Geest CR, Coffer PJ (2009) MAPK signaling pathways in the regulation of hematopoiesis. J Leuk Biol 86: 237–250.
[49]
Vantaggiato C, Formentini I, Bondanza A, Bonini C, Naldini L, et al. (2006) ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially. J Biol 5: 14.
[50]
Matsushita T, Chan YY, Kawanami A, Balmes G, Landreth GE, et al. (2009) Extracellular signal-regulated kinase 1 (ERK1) and ERK2 play essential roles in osteoblast differentiation and in supporting osteoclastogenesis. Mol Cell Biol 29: 5843–5857.
[51]
Walker MB, Kimmel CB (2007) A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotech Histochem 82: 23–28.
[52]
Maniatis T, Fritsch EF, Sambrook J (1989) Molecular Cloning: a Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory. Cold Spring Harbor, NewYork.
[53]
Pearse AGE (1985) Histochemistry: Theoretical and Applied. Churchill Livingstone. Edinburgh.pp. 1512
[54]
Maradonna F, Carnevali O (2007) Vitellogenin, zona radiata protein, cathepsin D and heat shock protein 70 as biomarkers of exposure to xenobiotics. Biomarkers 12: 240–255.
[55]
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.
[56]
Carnevali O, Avella MA, Gioacchini G (2013) Effects of probiotic administration on zebrafish development and reproduction. Gen Comp Endocrinol 188: 297–302.
[57]
Lamari F, Castex M, Larcher T, Ledevin M, Mazurais D, et al. (2013) Comparison of the effects of the dietary addition of two lactic acid bacteria on the development and conformation of sea bass larvae, Dicentrarchus labrax, and the influence on associated microbiota. Aquaculture 376: 137–145.
[58]
Lucero CM, Vega OA, Osorio MM, Tapia JC, Antonelli M, et al. (2013) The cancer-related transcription factor Runx2 modulates cell proliferation in humanosteosarcoma cell lines. J Cell Physiol 228(4): 714–723.
[59]
Mundlos S, Otto F, Mundlos C, Mulliken JB, Aylsworth AS, et al. (1997) Mutations involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell 30 89(5): 773–779.
[60]
Nishimura R, Hata K, Matsubara T, Wakabayashi M, Yoneda T (2012) Regulation of bone and cartilage development by network between BMP signalling and transcription factors. J Biochem 151(3): 247–254.
[61]
Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA (2003) MSX2 promotes osteo-genesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem 278: 45969–45977.
[62]
Hale JE, Fraser JD, Price PA (1988) The identification of matrix Gla protein in cartilage. J Biol Chem 263: 5820–5824.
[63]
Price PA, Faus SA, Williamson MK (2000) Warfarin-induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol 20: 317–327.
[64]
Pinto JP, Concei？？o N, Gavaia PJ, Cancela ML (2003) Matrix Gla protein gene expression and protein accumulation colocalize with cartilage distribution during development of the teleost fish Sparus aurata. Bone 32: 201–210.
[65]
Liu F, Malaval L, Aubin JE (2003) Global amplification polymerase chain reaction reveals novel transitional stages during osteoprogenitor differentiation. J Cell Sci 116: 1787–1796.
[66]
Lewiecki EM (2011) Sclerostin: a novel target for intervention in the treatment of osteoporosis. Discov Med 12: 263–273.
[67]
McCabe LR, Irwin R, Schaefer L, Britton RA (2013) Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice. J Cell Physiol 228(8): 1793–1798.
[68]
Wortzel I, Seger R (2011) The ERK Cascade: Distinct Functions within Various Subcellular Organelles. Genes Cancer 2: 195–209.
[69]
Murakami S, Balmes G, McKinney S, Zhang Z, Givol D, et al. (2004) Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype. Genes Dev 18: 290–305.
[70]
Matsushita T, Wilcox WR, Chan YY, Kawanami A, Bükülmez H, et al. (2009) FGFR3 promotes synchondrosis closure and fusion of ossification centers through the MAPK pathway. Hum Mol Genet 18: 227–240P.
[71]
Bentires-Alj M, Kontaridis MI, Neel BG (2006) Stops along the RAS pathway in human genetic disease. Nat Med 12: 283–285.
[72]
Aoki Y, Niihori T, Narumi Y, Kure S, Matsubara Y (2008) The RAS/MAPK syndromes: novel roles of the RAS pathway in human genetic disorders. Hum Mutat 29: 992–1006.
[73]
Jun JH, Yoon WJ, Seo SB, Woo KM, Kim GS, et al. (2010) BMP2- activated Erk/MAP kinase stabilizes Runx2 by increasing p300 levels and histone acetyltransferase activity. J Biol Chem 285: 36410–36419.
[74]
Cho YH, Gu YM, Oh b JW, Lee KY (2011) Osterix is regulated by Erk1/2 during osteoblast differentiation. Biochem Biophys Res Comm 415: 472–478.
