Parathyroid hormone 1 (1–34) acts on the scales and involves calcium metabolism in goldfish
Research Highlights
► Administration of PTH in goldfish scale increased ALP activity followed by TRAP activity in vitro. ► The mRNA expressions of both RANKL and RANK were raised in PTH-treated scales. ► PTH induced hypercalcemia resulted from bone resorption in the scales in vivo. ► The fine structure of osteoclasts in the goldfish scale was found to be almost identical to mammalian osteoclasts. ► In summary, PTH acts on the scales and regulates calcium metabolism in fish.
Introduction
Parathyroid hormone (PTH) increases plasma calcium level in mammals and is secreted from the parathyroid gland in all tetrapods, but not in fish. The first animal to evolve parathyroid glands is the amphibian [1]. Recently, the PTH gene has been discovered in Fugu rubripes, and 80 amino acids of the protein-coding region were determined [2]. Fugu PTH1 as well as human PTH specifically bound to the human parathyroid hormone receptor (PTHR) and promoted cyclic AMP formation in a mammalian cell line [2]. In addition, it was found that zebrafish had two PTHs [3] and that zebrafish PTH1 and zebrafish PTH2 mRNAs and PTH1 protein were detected in the neuromasts of the lateral line and in the central nervous system during embryogenesis [4]. These results suggest that PTH synthesized in the neural tissues has a physiological significant role in teleosts.
The teleost scale is a calcified tissue that contains osteoblasts and osteoclasts [5], [6], [7]. The scales are a functional internal calcium reservoir during periods of increased calcium demand, such as sexual maturation and starvation [5], [7], [8], [9], [10], [11]. In the scale, as in mammalian bone, type I collagen [12], bone γ-carboxyglutamic acid protein [13], osteonectin [14], [15], and hydroxyapatite [16] are present. Thus, the teleost scale has a number of features in common with mammalian membranous bone.
Recently, we developed a new in vitro assay system using fish scale [17], [18], which can be used to detect the activities of scale osteoblasts and osteoclasts simultaneously using the alkaline phosphatase (ALP) and the tartrate-resistant acid phosphatase (TRAP) assay, respectively. This assay is an original assay system to detect the respective enzyme activity from a single scale by transferring each scale to a 96-well microplate. These markers (TRAP and ALP) have been shown to be affected by a number of hormones and other factors in osteoclasts and osteoblasts [19], [20], [21] in mammals. In the scales of carp, de Vrieze et al. [22] also demonstrated that ALP and TRAP are valid markers for osteoblasts and osteoclasts, respectively.
Using this system, we demonstrated that calcitonin suppressed osteoclastic activity in scale osteoclasts as it does in mammalian osteoclasts [17]. Moreover, we were the first to find that melatonin, a major hormonal product of the pineal gland, functioned negatively in both osteoclasts and osteoblasts [18]. The action of melatonin on bone has also subsequently been reported in in vivo studies in the rat [23]. Moreover, we indicated that osteogenesis in the regenerating scale is very similar to that seen in mammalian membranous bone and that regenerating scales have estrogen receptors which respond to estrogen in the same manner as mammalian osteoblasts [24]. Persson et al [25] reported that estrogen specific binding was detected in the scales of rainbow trout. We suggest that this fish scale culture system is useful for the evaluation of the effect of PTH on bone.
In the present study, we examined the effect of fugu PTH1 on goldfish scale osteoblastic and osteoclastic activities and compared the actions of fugu PTH1 with those of human PTH. To confirm the effect of fugu PTH1 on plasma calcium and the scale (osteoblasts and osteoclasts), in vivo experiments were carried out. In teleosts, three types of receptors for PTH have been identified [26] and it has been reported that zebrafish PTH1 binds to both PTH1R and PTH3R [3]. In order to confirm that PTH's action on the scales of goldfish was via the PTH receptors, we established their presence in the scales by reverse transcription-polymerase chain reaction (RT-PCR) with primers based on the conserved regions for both mammalian and zebrafish PTHR sequences. In addition, expression analyses of both the receptor activator of NF-κB (RANK) and the receptor activator of the NF-κB ligand (RANKL) in the fugu PTH1-treated scales were performed because the RANK–RANKL pathway is necessary for osteoclast differentiation [27], [28], [29]. Moreover, the induction of mononucleated osteoclasts to multinucleated osteoclasts was investigated using goldfish scales autotransplanted to muscle.
Section snippets
Animals
A previous study [17] indicated that the sensitivity for calcemic hormones was higher in mature female than in mature male teleosts. Therefore, female goldfish (Carassius auratus) (30–40 g) were purchased and used for all of the in vitro experiments and mRNA expression analyses. To examine the effect of fugu PTH1 on the calcium metabolism, immature goldfish (4–6 g), in which the endogenous effects of sex steroids are negligible, were used for the in vivo study.
All experimental procedures were
Effects of fugu PTH1 (1–34) and human PTH (1–34) on ALP and TRAP activities in the cultured scales of goldfish
Fugu PTH1 (100 pg/ml–10 ng/ml) significantly increased ALP activity in 6 h incubations (Fig. 1A). After 18 h of incubation, the ALP activity remained significantly increased only at 10 ng/ml (Fig. 1B).
ALP activity was also increased by human PTH with 6 h of incubation; however, by two-way ANOVA analysis, there was a significantly greater (P < 0.05) response with fugu PTH1 than with human PTH at 6 h of incubation (Fig. 1A). The EC50 values of fugu PTH1 and human PTH were 18 and 76 (pg/ml), respectively.
Discussion
Using an in vitro assay system with goldfish scales, we have shown that fugu PTH1 (1–34) increased both ALP and TRAP activities in the scales. The in vitro data were supported by our in vivo findings, in which hypercalcemia was induced by fugu PTH and was associated with a decline of scale calcium content, consistent with mobilization of scale calcium. In addition, we have shown that the ratio of multinucleated osteoclasts/mononucleated osteoclasts in PTH-treated scales was significantly higher
Acknowledgments
This study was supported in part by grants to N.S. [Kurita Water and Environment Foundation; Grant-in-Aid for Scientific Research (C) No. 21500404 by JSPS; Grant-in-Aid for Space Utilization by Japan Aerospace Exploration Agency], to A.H. [Grant-in-Aid for Scientific Research (C) No. 21570062 by JSPS], to H.M. [Grant-in-Aid for Scientific Research (C) No.20592168 by JSPS], and to K.H. [Grant-in-Aid for Scientific Research of ExTEND2005 and the Environment Research and Technology Development
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