Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology
Retinoic acid induces two osteocalcin isoforms and inhibits markers of osteoclast activity in Atlantic cod (Gadus morhua) ex vivo cultured craniofacial tissues
Introduction
It is well established that vitamin A and its main active metabolite, retinoic acid (RA), is essential for development including bone formation (Ross et al., 2000). Retinoic acid mediates gene expression mainly through ligand activated RA receptors (RARs α, β, and γ) and its dimerising partner retinoic x receptor (RXRs α, β, and γ) (Ross et al., 2000). These proteins are members of the steroid/thyroid hormone receptor super family which activates specific responsive elements in the promoter region of target genes. Several members of the cytochrome P450 monooxygenase super family, including cytochrome P450 26A (cyp26a), are involved in clearance of RA from the body (Thatcher et al., 2010) and is induced by RA through the RA responsive element in the cyp26a gene promoter region (Loudig et al., 2000). In vitro studies on mammalian model systems have reported conflicting results regarding the action of RA on bone development. RA have been shown to both induce (Oliva et al., 1993, Song et al., 2005, James et al., 2010, Zhang et al., 2010) and inhibit (Ohishi et al., 1995) osteoblastogenesis. Early studies investigating the effect of vitamin A and RA in mammals demonstrated that both excess and deficient doses can result in severe developmental abnormalities including craniofacial deformities (Wilson et al., 1953, Yip et al., 1980, Gardner et al., 1998). The impact of excess and deficient levels of vitamin A has also been studied in connection with bone deformities in intensively reared teleosts larvae including Japanese flounder (Paralichthys olivaceus) (Takeuchi et al., 1998), European sea bass (Dicentrarchus labrax) (Villeneuve et al., 2005, Villeneuve et al., 2006, Mazurais et al., 2009, Darias et al., 2010, Georga et al., 2011), Senegalese sole (Solea senegalensis) (Fernández et al., 2009, Fernandez and Gisbert, 2010, Fernandez et al., 2011) and Atlantic salmon (Salmo salar) (Ornsrud et al., 2004). The occurrence of abnormalities caused by both too high and too low doses of vitamin A demonstrates the importance of balanced vitamin A enrichment in feed for early developmental stages of fish. Although the underlying mechanisms by which RA modulate expression of genes involved in bone remodeling is not clear, these studies indicate that RA affects the tightly controlled balance between bone resorption and bone formation which is crucial for forming and maintaining healthy bone structures. Bone remodeling is determined by the activity of osteoblasts for bone formation and osteoclasts for bone resorption. Bone matrix consists of both collagenous and non-collagenous matrix proteins. Osteocalcin (OC) (also called bone gla protein), matrix gla protein (MGlaP), osteonectin/secreted protein, acidic, cysteine-rich (SPARC) and periostin (POSTN) are all non-collagenous matrix proteins with high affinity to bone and dental tissue. POSTN have recently been recognized as a member of the vitamin K-dependent Ca2 +, gamma-carboxyglutamic acid (Gla) protein family together with OC and MGlaP (Price, 1989, Price et al., 2002, Nishimoto et al., 2003, Coutu et al., 2008). The Gla proteins have affinity to calcium and hydroxyapatite in bone and dental tissue. Osteocalcin is the most abundant non-collagenous matrix proteins and has been widely used as a biomarker for bone development and osteoblast activity in humans (Lee et al., 2000) and in fish (Fernandez et al., 2011, Darias et al., 2010, Gavaia et al., 2006, Inohaya et al., 2007, Mazurais et al., 2008, Pombinho et al., 2004). Although OC has been extensively studied, particularly in mammals, the exact function of this protein has yet to be elucidated. Studies on mice have shown an accelerated rate of mineralization in OC knock out strains compared to the wild type (Ducy et al., 1996). However a more sensitive mineralization assay revealed a possible function in bone maturation (Boskey et al., 1998). Other involvements have been proposed including osteoclast maturation (Ishida and Amano, 2004) and more recently in energy homeostasis (Lee et al., 2007). The bone resorbing action of osteoclasts is mediated by several different collagenases and proteases including matrix metalloproteinase 9 (MMP9) and cathepsin K (CTSK), of which CTSK is considered to be the main contributor (Troen, 2006). In addition, bone metabolism is also dependent on recruitment of osteoblasts and osteoclasts through differentiation of precursor cells. This involves a range of different transcription factors of which runt-related transcription factor 2 (RUNX2) and osterix are believed to be the key factors determining commitment to the osteoblast linage (Lian et al., 2006) while receptor activator of nuclear factor kappa-β ligand (RANKL) and osteoprotegerin (OPG) regulate osteoclast differentiation (Khosla, 2001).
Cultivation of marine fish species has shown that the occurring craniofacial deformities may depend on early nutritional status (Lall and Lewis-McCrea, 2007). This has also been shown for Atlantic cod (Gadus morhua), where larvae given natural zooplankton perform much better than larvae fed commercial diets (enriched rotifers) (Hamre et al., 2008, Busch et al., 2010). Rotifers are particularly low in carotenoids (Hamre et al., 2008) and this could lead to vitamin A deficiency. In the present study, we used Atlantic cod cranial tissue cultures to investigate RAs ability to modulate transcription of genes involved in bone formation and resorption.
Section snippets
Animals and tissue culture
Atlantic cod were collected from a commercial cod hatchery (Havlandet Marin Yngel AS, Florø, Norway) at 25 and 65 days post hatch (dph), see Fig. 1 for details on developmental stages). The fish was stored in insulated boxes with aeration until sampling 1 h after arrival to the lab. Heads from 25 dph Atlantic cod larvae (length 6–6.5 mm, n = 60) and lower jaw from 65 dph Atlantic cod juveniles (mean length 17 ± 1.3 mm, n = 360) were excised immediately after the animals were killed by decapitation. Heads
Expression of transcripts involved in RA metabolism
The differential expression of cyp26a was used as a positive control of RA exposure. This gene was up-regulated in Atlantic cod heads (pilot) and jaw explants after both 14 and 24 h of exposure to RA (p < 0.05, Mann Whitney U Test) and was the most highly induced gene in the present study (Fig. 2). The fold change was raging from 867 to 2632 in a dose dependant manner.
Expression of transcripts related to osteoblast differentiation and activity
Exposure of 25 dph cod head explants to RA resulted in a significant down-regulation of osterix mRNA levels compared to control. The
Discussion
Transcription of both oc1 and oc2 was induced in Atlantic cod lower jaw cultures following RA treatment. These results indicate that RA increases the activity of mature osteoblasts in Atlantic cod. Induction of oc expression by retinoids has not only been shown in vitro in mammals (Oliva et al., 1993, Schrader et al., 1993, Jaaskelainen et al., 2003, Zhang et al., 2010), but also in vivo for gilthead seabream (Sparus aurata) following high levels of vitamin A in the diet (Fernandez et al., 2011
Acknowledgment
Øystein Sæle is thanked for his gracious donation of the pictures of stained bone in Atlantic cod heads used in this work. Hui-Shan Tung and Synnøve Winterthun is thanked for excellent technical help. Havlandet AS is thanked for supporting our research and for providing us with live fish. The Norwegian Cod Genome Sequencing Consortium (www.codgenome.no) is thanked for making their sequencing data available. This study was financed by the Norwegian Research Council (project number NFR 185177/S40
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