Atlantic salmon (Salmo salar) muscle precursor cells differentiate into osteoblasts in vitro: Polyunsaturated fatty acids and hyperthermia influence gene expression and differentiation

https://doi.org/10.1016/j.bbalip.2009.10.001Get rights and content

Abstract

The formation and mineralisation of bone are two critical processes in fast-growing Atlantic salmon (Salmo salar). The mechanisms of these processes, however, have not been described in detail. Thus, in vitro systems that allow the study of factors that influence bone formation in farmed Atlantic salmon are highly warranted. We describe here a method by which unspecialised primary cells from salmon white muscle can differentiate to osteoblasts in vitro. We have subsequently used the differentiated cells as a model system to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions, namely polyunsaturated fatty acids, PUFAs, and temperature. Muscle precursor cells changed their morphology from triangular or spindle-shaped cells to polygonal or cubical cells after 3 weeks in osteogenic medium. In addition, gene expression studies showed that marker genes for osteoblastogenesis; alp, col1a1, osteocalcin, bmp2 and bmp4 increased after 3 weeks of incubation in osteogenic media showing that these cells have differentiated to osteoblasts at this stage. Adding CLA or DHA to the osteoblast media resulted in a reduced PGE2 production and increased expression of osteocalcin. Further, temperature studies showed that differentiating osteoblasts are highly sensitive to increased incubation temperature at early stages of differentiation. Our studies show that unspecialised precursor cells isolated from salmon muscle tissue can be caused to differentiate to osteoblasts in vitro. Furthermore, this model system appears to be suitable for the study of osteoblast biology in vitro.

Introduction

Osteoblasts are mononucleated bone-forming cells that originate locally from mesenchymal progenitor cells. Mesenchymal progenitor cells not only form cells of the osteoblast lineage (osteoblasts, osteocytes and bone-lining cells), they also give rise to adipocytes, myocytes and chondrocytes [1], [2]. Several studies have shown that these cells have a certain degree of developmental plasticity (modulation) and that differentiated cells also have a certain degree of plasticity (trans-differentiation) [3], [4]. In vitro culture systems that allow the examination of the cells' capacity for modulation and trans-differentiation are well‑established for several mammalian species [5] and for the advanced marine teleost species Sea bream (Sparus aurata) [6]. Undifferentiated mammalian muscle satellite cells are able to differentiate in vitro into one of several cell types, among them osteoblasts [7]. It is not known whether mesenchymal skeletal progenitor cells from Atlantic salmon (Salmo salar) have a capacity for modulation or trans-differentiation. However, we have shown that primary precursor cells located in the adipose and muscle tissue of Atlantic salmon can proliferate and differentiate into mature adipocytes and muscle cells, respectively, in vitro [8], [9].

Specific markers that can confirm the identity of a certain cell type in vitro are required in order to determine whether unspecialised precursor cells can successfully mature into specialised cells. Osteoblasts express several phenotypic markers, both collagenous (e.g. collagen1a) and non-collagenous (e.g. alp and osteocalcin) bone matrix proteins [10], [11]. Osteoblast differentiation is specialized and strictly regulated by a number of transcription factors and signalling molecules. Two members of the bone morphogenetic protein (BMP) family, bmp2 and bmp4, are involved in bone and cartilage development [12], [13]. Among the downstream targets of BMPs are runx2 and other osteoblast-related transcription factors such as osterix [14], [15]. These osteoblast-related factors directly activate a number of osteoblast markers such as col1a, osteopontin and osteocalcin [3], [10]. In comparison, sox9 is an essential transcription factor of chondrocyte differentiation and cartilage formation in vertebrates [16]. Sox9 directly regulates the expression of col2a, the gene that encodes the major cartilage matrix protein expressed in chondro-progenitor cells and also expressed at high levels in chondrocytes [17], [18]. Further, four muscle-specific bHLH factors (MRFs); mrf4, myogenin, Myf-5, and MyoD are expressed early in embryogenesis, at a time when muscle lineage decisions are established. MyoD and myf5 are required for myogenic determination, whereas myogenin is a downstream transcription factor involved in differentiation [19], [20]. Mrf4 has a more complex role and is considered as both a determination and differentiation factor [21]. In addition, developing and mature muscle cells express the structural protein mlc, which may be used as a marker for cells turning into myoblasts [22], [23]. PPARγ plays an important role in controlling differentiation programs of multipotent mesenchymal progenitor cells that favor adipogenesis over osteogenesis [24], [25]. Teleosts have a single pparγ gene homologous to the mammalian pparγ [26]. Recent results from our group have shown that two alternatively transcription variants of pparγ, named pparγ-long and pparγ-short, are present in Atlantic salmon and that pparγ-short is significantly up-regulated during adipocyte differentiation [27].

Due to the growing need of replacing fish oils in commercial fish feeds, the use of vegetable oils has increased during the latest years. These vegetable oils typically have a high content of n-6 PUFAs that lead to decreased n-3/n-6 PUFA ratios in both the fish diet and the fish tissue [28], [29], [30], [31], [32], [33]. Dietary PUFAs are important regulators of many cellular functions in mammals, including those related to bone cell formation. Low n-3/n-6 ratios of PUFAs reduce bone formation and cause greater bone resorption activity in mammals [34], [35], [36]. Bioactive fatty acids such as CLA affect bone biology and may have similar effects as those of n-3 PUFAs [37], [38]. CLA, like n-3 PUFAs, reduces the production of PGE2 in rats, but different mechanisms may operate from those in fish [37]. PGE2 is a potent agent regulating bone formation [39], [40] and has been shown to inhibit bone formation at high concentrations in vitro [41]. Recent studies on European sea bass larvae have shown that the concentrations of dietary PUFAs, particularly dietary EPA and DHA, are related to vertebral malformations [42], [43], but it remains to be determined whether the n-3/n-6 PUFA ratios in fish diets affect bone mineral density.

Use of elevated water temperature during early development to reduce production time is a major factor affecting the prevalence of skeletal abnormalities in farmed fish, [44], [45], [46]. In a recent study, we found that by increasing the rearing temperature of salmon from 12 °C to 16 °C the expression of most skeletal genes examined were affected ([47], submitted). Genes that code for extracellular matrix constituents, such as osteocalcin and osteonectin, were in general down-regulated and results indicated that osteoblast activity was reduced. Nevertheless, there is limited knowledge of how temperature and other factors affect osteoblast differentiation and further bone development, and how these mechanisms are linked to the development of spinal deformities in salmonids. Thus, the development of an Atlantic salmon osteoblast culture is therefore an important step in order to reveal the specific mechanisms underlying the pathogenesis of skeletal deformities. Moreover, such a salmon osteoblast culture will provide improved opportunities for studying the evolutionary development of bone differentiation and remodelling among vertebrates, since experiments with mammalian cell cultures have, to a great extent, already described the major pathways that are involved in skeletal development and remodelling (review [3], [48]).

The aim of the present study was to establish a method that would cause unspecialised primary cells from salmon muscle to differentiate to osteoblasts in vitro. We used real-time quantitative RT-PCR assays for genes that encode typical osteoblast-related membrane and extracellular matrix molecules (alp, col1a1, osteocalcin, osteonectin); for two of the most potent growth factors involved in the recruitment and differentiation of mesenchymal precursors (bmp2 and bmp4); and for the main transcription factor related to osteogenesis (runx2). We then analyzed how these genes were expressed in differentiating osteoblasts under different culture conditions: (i) in a medium that induced osteogenic differentiation, (ii) in a medium that had been supplemented with fatty acids having different n-3/n-6 PUFA ratios and different concentrations of CLA, and (iii) under different temperature regimens.

Section snippets

Materials

Atlantic salmon (Salmo salar) fry were obtained from Aqua Gen (Sunndalsøra, Norway) and raised on a commercial diet in an experimental unit at the Agricultural University of Norway.

L-15, fetal bovine serum (FBS), antibiotics, antimycotics, HEPES, L-glutamine, collagenase, trypsin, CaCl2, Ca-ionophore (calimycin), glycerol-2-phosphate disodium salt hydrate, l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, (1,25)-dihydroxyvitamin D3, dexamethasone, laminin and thermanox cover slips were

Cell morphology during differentiation

Changes in cell morphology were studied during a differentiation period of 3 weeks. Newly isolated cells from salmon muscle tissue consisted of a heterogeneous population of spindle-shaped myosatellite cells and small, round or triangular cells (Fig. 1A). The myosatellite cells at this stage had relatively high proliferative capacity, with approximately 50% of the cells staining positive for PCNA.

Few cells had the characteristic elongated appearance of muscle cells after a few days in the

Discussion

Osteoblast cultures have been established for several mammals: human, rat and sheep [5], and for gilthead sea bream [6]. We show here that precursor cells isolated from Atlantic salmon muscle tissue can differentiate to osteoblasts in vitro when incubated in an osteogenic medium.

Isolated salmon muscle precursor cells were stimulated to osteogenic differentiation by an incubation medium containing CaCl2, β-glycerophosphate, l-ascorbic acid, dexamethasone and 1,25-dihydroxyvitamin D3. These

Conclusions

This is the first report that shows that unspecialised precursor cells isolated from salmon muscle tissue are able to differentiate to osteoblasts-like cells in vitro. These cells appear to be a suitable model system for the study of osteoblast biology in vitro. Their morphology has several osteoblast-like characteristics, and alkaline phosphatase is present in the cells. Furthermore, the cells express several osteoblast-related markers such as osteocalcin, col1a1, bmp2 and bmp4. The production

Acknowledgments

The authors are grateful to Inger Ø. Kristiansen, Målfrid Bjerke and Hege Munck for technical assistance during the project. We thank Harald Støkken for his skilful work in the aquarium division. The Norwegian Research Council supported the work.

References (88)

  • B.A. Watkins et al.

    Dietary ratio of (n-6)/(n-3) polyunsaturated fatty acids alters the fatty acid composition of bone compartments and biomarkers of bone formation in rats

    J. Nutr.

    (2000)
  • B.A. Watkins et al.

    Modulatory effect of omega-3 polyunsaturated fatty acids on osteoblast function and bone metabolism

    Prostaglandins Leukot. Essent. Fat. Acids

    (2003)
  • W.S.S. Jee et al.

    Prostaglandin-E2 enhances cortical bone mass and activates intracortical bone remodeling in intact and ovariectomized female rats

    Bone

    (1990)
  • S. Mori et al.

    Effects of Prostaglandin-E2 on production of new cancellous bone in the axial skeleton of ovariectomized rats

    Bone

    (1990)
  • L.G. Raisz et al.

    Inhibition of bone collagen-synthesis by prostaglandin-E2 in organ-culture

    Prostaglandins

    (1974)
  • H. Takle et al.

    The effect of heat and cold exposure on HSP70 expression and development of deformities during embryogenesis of Atlantic salmon (Salmo salar)

    Aquaculture

    (2005)
  • G. Karsenty et al.

    Reaching a genetic and molecular understanding of skeletal development

    Dev. Cell

    (2002)
  • O.H. Lowry et al.

    The determination of serum protein concentration with a gradient tube

    J. Biol. Chem.

    (1945)
  • J. Folch et al.

    A simple method for the isolation and purification of total lipids from animal tissues

    J. Biol. Chem.

    (1957)
  • M. Hoshi et al.

    2,3-Erythro-dihydroxyhexacosanoic acid and homologs—isolation from yeast cerebrin phosphate and determination of their structures

    J. Lipid Res.

    (1973)
  • R. Ogawa et al.

    Osteogenic and chondrogenic differentiation by adipose-derived stem cells harvested from GFP transgenic mice

    Biochem. Biophys. Res. Commun.

    (2004)
  • C.A. Gersbach et al.

    Runx2/Cbfa1 stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype

    Exp. Cell Res.

    (2004)
  • L.C. Gerstenfeld et al.

    Expression of differentiated function by mineralizing cultures of chicken osteoblasts

    Dev. Biol.

    (1987)
  • M.P. Mark et al.

    Developmental expression of 44-kDa bone phosphoprotein (osteopontin) and bone gamma-carboxyglutamic acid (gla)-containing protein (osteocalcin) in calcifying tissues of rat

    Differentiation

    (1988)
  • B. de Crombrugghe et al.

    Transcriptional mechanisms of chondrocyte differentiation

    Matrix Biology

    (2000)
  • B.A. Watkins et al.

    Protective actions of soy isoflavones and n-3 PUFAs on bone mass in ovariectomize rats

    J. Nutr. Biochem.

    (2005)
  • S. Cusack et al.

    The effect of conjugated linoleic acid on the viability and metabolism of human osteoblast-like cells

    Prostaglandins Leukot. Essent. Fat. Acids

    (2005)
  • G.M. Berge et al.

    Conjugated linoleic acid in diets for juvenile Atlantic salmon (Salmo salar); effects on fish performance, proximate composition, fatty acid and mineral content

    Aquaculture

    (2004)
  • G. Koumoundouros et al.

    The effect of rearing conditions on development of saddleback syndrome and caudal fin deformities in Dentex dentex (L.)

    Aquaculture

    (2001)
  • J.G. Breen et al.

    Heat shock during rat embryo development in vitro results in decreased mitosis and abundant cell death

    Reprod. Toxicol.

    (1999)
  • Y. Mochida et al.

    Decorin modulates matrix mineralization in vitro

    Biochem. Biophys. Res. Commun.

    (2003)
  • J.D. Termine et al.

    Osteonectin, a bone-specific protein linking mineral to collagen

    Cell

    (1981)
  • T.A. Owen et al.

    Pleiotropic effects of vitamin-D on osteoblast gene-expression are related to the proliferative and differentiated state of the bone cell phenotype—dependency upon basal levels of gene-expression, duration of exposure, and bone-matrix competence in normal rat osteoblast cultures

    Endocrinology

    (1991)
  • M.F. Pittenger et al.

    Multilineage potential of adult human mesenchymal stem cells

    Science

    (1999)
  • J.E. Aubin

    Bone stem cells

    J. Cell. Biochem.

    (1998)
  • K.H. Wlodarski et al.

    Metaplasia of chondrocytes into osteoblasts

    Folia Biol. Krakow

    (2006)
  • A.R. Pombinho et al.

    Development of two bone-derived cell lines from the marine teleost Sparus aurata; evidence for extracellular matrix mineralization and cell-type-specific expression of matrix Gla protein and osteocalcin

    Cell Tissue Res.

    (2004)
  • M.R. Wada et al.

    Generation of different fates from multipotent muscle stem cells

    Development

    (2002)
  • A. Vegusdal et al.

    An in vitro method for studying the proliferation and differentiation of Atlantic salmon preadipocytes

    Lipids

    (2003)
  • A. Vegusdal et al.

    beta-oxidation, esterification, and secretion of radiolabeled fatty acids in cultivated Atlantic salmon skeletal muscle cells

    Lipids

    (2004)
  • T.A. Franz-Odendaal et al.

    Buried alive: how osteoblasts become osteocytes

    Dev. Dyn.

    (2006)
  • T. Ikeda et al.

    In situ hybridization of bone-matrix proteins in undecalcified adult-rat bone sections

    J. Histochem. Cytochem.

    (1992)
  • T. Kobayashi et al.

    BMP signaling stimulates cellular differentiation at multiple steps during cartilage development

    Proc. Natl. Acad. Sci. U. S. A.

    (2005)
  • B.L.M. Hogan

    Bone morphogenetic proteins: multifunctional regulators of vertebrate development

    Genes Dev.

    (1996)
  • Cited by (0)

    View full text