Response to metals treatment of Fra1, a member of the AP-1 transcription factor family, in P. lividus sea urchin embryos
Introduction
Heavy metals are natural components of the earth, variously distributed in the atmosphere, the waters and the soil (Järup, 2003). They are introduced in the environment by diverse natural processes, such as soil erosion or atmospheric agents, and by various human activities, such as mining, industries, agriculture and others (Jaishankar et al., 2014). When they are present at very low concentrations, heavy metals act as elements important for various cellular metabolic processes in living organisms. They become chronic and toxic when they go beyond threshold concentrations, cannot be eliminated and accumulate in the animals body. Thus, heavy metals are significant environmental pollutants for both marine and terrestrial ecosystems (Jaishankar et al., 2014). Metal toxicity is dose-dependent, relies on the duration of exposure and finally can lead to various disorders and diseases, such as degenerative processes and in some cases cancer (Järup, 2003).
Lithium (Li) is the lightest of the metals, with a very low density. It is present in trace quantities and shows many biochemical and molecular effects on signal transduction cascades, hormonal and neural regulation, ion transport, and gene expression (Pandey and Davis, 1980). Moreover, Li is one of the most effective drugs used in medicine to cure mental diseases (Manji and Lenox, 2000). In the sea urchin embryo, Li has a vegetalizing effect, inducing presumptive ectoderm to differentiate as endo-mesoderm, sometimes producing exogastrulae (Ghiglione et al., 1993; Herbst, 1893). Among heavy metals, Nickel (Ni) and Zinc (Zn) are normally present in the seawater at low concentrations, thus it seems that they are not naturally dangerous. However, at high concentrations, they can have adverse effects on the marine ecosystem and may be toxic for the embryonic development of many organisms (Blewett and Leonard, 2000). Indeed, it has been reported that Ni is teratogen, carcinogen, immunotoxic and neurotoxic for living organisms (Adjroud, 2013). In the sea urchin Lythechinus variegatus, Ni disrupts the dorso-ventral axis forming radialized embryos, mainly affecting ectodermal differentiation and pattern formation of mesenchyme cells, as described by Hardin (Hardin et al., 1992). Moreover, Ni-treated embryos overexpress ventral genes at the expenses of the dorsal genes (Hardin et al., 1992).
Zn is an essential element for living organisms and plays a physiological role in many biological processes. It is functional for a great number of enzymes, for the stabilization of DNA and for gene expression; it shows antioxidant properties and has a role in cancer prevention (Frassinetti et al., 2006). In the sea urchin embryo, sub-lethal concentrations of Zn induce “animalization”, i.e. increase the specification of the ectoderm cells at the animal pole and reduce the specification of endo-mesoderm territories (Poustka et al., 2007; Timourian, 1968).
A central question in environmental studies is how organisms face adverse external conditions. The need to deal with environmental pollutants has led to the evolution of a range of genes and proteins to be collectively considered a “defensome”. The comparison of genomes from distantly related organisms suggests the conservation of a chemical defensome in marine invertebrates (Goldstone et al., 2006; Goldstone, 2008; Yadetie et al., 2012). Numerous genes involved in the defense against toxic chemicals may also have developmental and differentiation roles in embryos and adults, and thus it seems interesting to understand how they can fulfill the dual functions in harmony.
In all organisms, transcription factors (TFs) are very important key molecules in the complex networks that regulate embryogenesis. They have many targets, among which other TFs, able to trigger cascades of events which finally serve to determine and specify the embryonic territories. They are grouped in large families related to the structures of their DNA binding domains, such as the basic Leucine zipper (bZIP), the Zinc Finger, the homeodomain Leucine zipper (HD-Zip) and the Forkhead box (FOX) (Katoh and Katoh, 2004; Landschulz et al., 1988).
The bZIP proteins function as dimers using the basic residues of α-helices to bind the phosphate groups in two different major grooves of DNA, in addition to interactions with other specific bases. The heterodimeric association confers DNA specificity (Vinson et al., 1989).
One of the most important component of the bZIP family is the AP-1 transcription factor complex, first isolated from HeLa cells (Lee et al., 1987), and involved in the regulation of cell proliferation, osteoclast differentiation, immune and stress response, cancer (Angel and Karin, 1991; Shaulian and Karin, 2002; Uchil et al., 2013). It consists of two principal protein families: the JUN family (c-Jun, JunB and JunD), and the FOS family (c-Fos, FosB, Fra-1 and Fra-2) (Milde-Langosch, 2005). The human FOS proteins can exclusively heterodimerize with members of the JUN family, whereas the JUN proteins can both homo- and heterodimerize with FOS members to form transcriptionally active AP-1 factors (Vinson et al., 2002).
These TFs have been described in many invertebrates, such as Drosophila melanogaster (Kockel et al., 2001), Caenorhabditis elegans (Sherwood et al., 2005), and in vertebrates (Herdegen and Waetzig, 2001; Shaulian and Karin, 2002). The availability of the Strongylocentrotus purpuratus annotated genome (Sodergren et al., 2006) has allowed the systematic identification of all the TFs encoded in the sea urchin (Howard-Ashby et al., 2006; Materna et al., 2006; Rizzo et al., 2006). In particular three genes have been annotated as component of AP-1 family, named Sp-fos (SPU_021173), Sp-jun (SPU_003102) and Sp-fra2 (SPU_021172) (Howard-Ashby et al., 2006). Recently, we have isolated and characterized Pl-jun from the P. lividus species, and based on our findings we suggested its involvement in skeleton patterning (Russo et al., 2014b).
To date, very little is known about the relationships among metals effects and the expression of the genes included in the AP-1 family. Among the few examples, c-Fos gene was found to be induced by Cadmium in mammalian kidney cells (Matsuoka and Call, 1995), as well as by Ni, as reported in a very recent study on the fish rainbow trout brain (Topal et al., 2015). Moreover, Murata et al. reported that human c-Fos was induced by a variety of heavy metals in dose-dependent manners, similarly to other two metal-inducible genes, MT-IIA and hsp70 (Murata et al., 1999).
In the present study, we investigated the temporal and spatial expression patterns of TFs of the AP-1 family in the P. lividus sea urchin embryo in response to treatments with Li, Ni and Zn. In particular, we focused on Pl-Fra TF, a member of the FOS related protein family, which we isolated for the first time in P. lividus sea urchin, and compared its response to the three metals to that of Pl-jun (Russo et al., 2014b), a potential partner for AP-1 complex formation, and Pl-MT, known to have a protective role against heavy metals (Russo et al., 2013). Besides, here we report the characterization of the cDNA coding for Pl-Fra, and its phylogenetic relationship with FRA proteins from the cognate sea urchin S. purpuratus and from other invertebrate and vertebrate organisms. We then determined the temporal and spatial expression of Pl-Fra mRNA, as well as the protein localization, during P. lividus embryo development.
Our findings associate the activity of genes of the AP-1 family to the mechanisms of response of the sea urchin embryo to metal injuries, thus providing a good model for forthcoming studies for the evaluation of environmental hazards.
Section snippets
Sampling of animals
Gametes were collected from adult sea urchin P. lividus, fished in the North-Western coast of Sicily of Mediterranean Sea. Eggs were fertilized and embryos were grown in Millipore (Billerica, MA, USA, 0.22 μm) filtered sea water (MFSW) containing antibiotics (50 mg/L streptomycin sulfate and 30 mg/L penicillin), at the dilution of 4.000 embryos/mL and at the moderate temperature of 18 ± 1 °C.
Metals exposure and morphological analysis
Caution: LiCl, NiCl2 and ZnSO4 are hazardous chemical and should be handled carefully.
Experiments were
Morphological analysis of embryos exposed to Li, Ni and Zn
To establish the sublethal or lethal doses for each selected metal in our sea urchin species, we carried out preliminary experiments with different concentrations of Li, Ni and Zn (ranging from 10−4 to 10−1 M) and analysed the morphologies obtained (data not shown). We selected the sublethal doses of 30 mM LiCl, 0.5 mM NiCl2 and 0.1 mM ZnSO4, and continuously treated P. lividus embryos with them starting from 30 min after fertilization by the time of each sampling.
All these metals had no
Discussion
In this study, we report the toxic effects of some metals (Li, Ni, Zn) on the development of P. lividus sea urchin embryos and the potential correlation between the stress response and the TFs belonging to the AP-1 superfamily, as they are known to be involved in many physiological cellular events, i.e. cell proliferation and differentiation, immune responses, as well as in cancer growth and stress responses. We show that Pl-Fra and Pl-jun mRNA expression is significantly induced by treatments
Acknowledgments
This research was partially supported by the Joint Research Project 2015–2017, as part of the Bilateral Agreement of Scientific and Technological Cooperation between the Consiglio Nazionale delle Ricerche of Italy (CNR) and the Russian Foundation for Basic Research (RFBR). We thank Mr Mauro Biondo for his technical assistance. We are also indebted to Professor David McClay for the kind gift of monoclonal antibody 1D5.
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This work is devoted to Valeria Matranga who is no longer with us.