Putative midkine family protein up-regulation in Patella caerulea (Mollusca, Gastropoda) exposed to sublethal concentrations of cadmium

Aquatic Toxicology 75 (2005) 374–379

Putative midkine family protein up-regulation in Patella caerulea (Mollusca, Gastropoda) exposed to sublethal concentrations of cadmium Silvana Vanucci a,∗,1 , Daniela Minerdi b,1,2 , Kenji Kadomatsu c , Alessio Mengoni d , Marco Bazzicalupo d a

c

Department of Animal Biology and Marine Ecology, University of Messina, Salita Sperone 31, 98166 S Agata, Messina, Italy b Department of Animal Biology and Genetics, University of Florence, via Romana 19, 50125 Firenze, Italy Department of Biochemistry, University of Nagoya Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan d Department of Animal Biology and Genetics, University of Florence, via Romana 19, 50125 Firenze, Italy Received 9 February 2005; received in revised form 18 August 2005; accepted 30 August 2005

Abstract A cDNA sequence of a putative midkine (MK) family protein was identified and characterised in the mollusc Patella caerulea. The midkine family consists of two members, midkine and pleiotrophin (PTN), and it is one of the recently discovered cytokines. Our results show that this putative midkine protein is up-regulated in specimens of P. caerulea exposed to sublethal cadmium concentrations (i.e. 0.5 and 1 mg l−1 Cd) over a 10-day exposure period. Semiquantitative RT-PCR and quantitative Real time RT-PCR estimations indicate elevated expression of midkine mRNA in exposed specimens compared to controls. Moreover, RT-PCR Real time values were higher in the viscera (here defined as the part of the soft tissue including digestive gland plus gills) than in the foot (i.e. foot plus head plus heart) of the limpets. At present, information on the functional signalling significance of the midkine family proteins suggests that the up-regulation of P. caerulea putative midkine family protein is a distress signal likely with informative value on health status of the organism and with potential prognostic capability. © 2005 Elsevier B.V. All rights reserved. Keywords: Patella caerulea; Mollusc; Midkine; Pleiotrophin; Distress signal; Cadmium; RT-PCR Real time

∗ Corresponding author. Tel.: +39 347 5714315; fax: +39 090 393409. E-mail address: [email protected] (S. Vanucci). 1 These authors contributed equally to this work. 2 Present address: Di.Va.P.R.A.-Plant Pathology, University of Torino, Via L. da Vinci 44, 10095 Grugliasco, Italy.

0166-445X/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2005.08.014

In the last decade, coastal environments have been subjected to increasing monitoring and efforts have been spent to study distress signals in order to develop biomarkers with warning prognostic capability (Moore, 2002). Cadmium is one of the most toxic and widespread heavy metals found in the marine environment (Romeo et al., 1995); it promotes cellular

S. Vanucci et al. / Aquatic Toxicology 75 (2005) 374–379

oxidative stress and induction of anti-oxidant systems in many marine molluscs (e.g. Leung and Furness, 1999; Geret et al., 2002). Moreover, cadmium is recognised to be carcinogenic in mammals (IARC, 1999), exerting pronounced co-mutagenic effects. At present, these effects are explained not only by increasing the level of DNA strand breakage induced by oxidative stress, but also by impairing cell recovery (Hartwing and Schwerdtle, 2002; Fatur et al., 2003), and delaying the onset of apoptosis (Waalkes et al., 2000). Recently, it has been showed that in the mussel Mytilus edulis cadmium enhances genotoxicity by mechanisms similar to those reported for mammals (Pruski and Dixon, 2002). The midkine (MK) is a recently discovered family of cytokines (see reviews Deuel et al., 2002; Kadomatsu and Muramatsu, 2004) and consists of only two members, namely heparin-binding growth factors MK and pleiotrophin (PTN). Information on PTN and MK function is mostly from mammals; MK and PTN share receptors and show similar biological activities that include fibrinolytic, anti-apoptotic, mitogenic, transforming, angiogenic, chemotactic, and protooncogenic ones. In adults, a characteristic property of MK/PTN genes is a striking induction of gene expression in limited emergency conditions, such as wound repair following injuries. In stark contrast, MK and PTN are strongly expressed in neurodegenerative diseases and in malignant tumours. The blood MK level in human carcinomas is correlated with prognostic factors; thus, it is as a good marker for evaluating the progress of carcinomas (Ikematsu et al., 2003). In this work, we report on isolation and characterisation of a cDNA sequence of a putative MK family protein that is up-regulated in Patella caerulea exposed to sublethal concentrations of cadmium. To our knowledge, this is the first report regarding MK/PTN identification in molluscs and on the involvement of MK/PTN in response to heavy metal exposure. We also report data on the mRNA tissue/organ quantitative expression feature in terms of both RT-PCR and RT-PCR Real time estimations. We chose the gastropod limpet P. caerulea as it is widely distributed over Mediterranean coastal areas, accumulates cadmium and is considered as a sentinel organism for the Mediterranean sea (Campanella et al., 2001). Samples of P. caerulea were collected from the external side of a breakwater at the Marina di Cala Galera (42◦ 26 50 N, 11◦ 26 00 E, Tyrrhenian sea) on

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7 June 2003. To reduce size/age-related variability, limpets of similar wet flesh weight (1 ± 0.4 g without shell) were selected. Measurements taken in the surface water layer in front of the breakwater gave an average salinity of 35 g l−1 and a temperature of 20 ◦ C. Immediately after collection, limpets were transported to the laboratory (Florence) and acclimated for 2 days before being exposed to cadmium (Cd). Three holding tanks, containing 20 specimens each, with each specimen singly placed in an open Petridish (diameter 6 cm, height 1 cm), were prepared and supplied with continuously aerated artificial seawater (35 g l−1 Reef CrystalsTM salt, Aquarium Systems). The three tanks were located in a temperature-controlled room (20 ± 0.5 ◦ C) with a natural daylight cycle. Exposure concentrations of cadmium were chosen with reference to sublethal concentrations based on 96-h acute toxicity tests carried out on P. caerulea in the same laboratory (Parenti, 2003). The three groups of limpets were exposed for 10 days to 0 (control), 0.50 and 1.0 mg l−1 Cd, respectively. Cadmium concentrations were prepared using a 100 mg l−1 Cd stock solution obtained by adding an appropriate quantity of CdCl2 (Fluka Chemie® ). Water was changed every second day to ensure no build-up of toxic materials from animals, changing water quality. The animals were not fed for the entire exposure period. Mortalities were recorded daily and dead animals were discarded; at the end of the 10th day mortalities were 4, 20 and 39% for 0 (control), 0.5 and 1.0 mg l−1 Cd treated animals, respectively. Following exposure, the tissues of the control and cadmium treated limpets were separated from the shells, and for each specimen the whole soft body was dissected into two parts: the foot (i.e. the foot plus head plus heart) and the viscera (i.e. the remain soft tissue including digestive gland plus gills); foot (F) and viscera (V) were frozen in liquid nitrogen and stored at −70 ◦ C until analyses. Total RNA was extracted from 500 mg of tissues (viscera plus foot) of P. caerulea specimens exposed to 1 mg l−1 cadmium using Trizol (Invitrogen) solution according to the manufacturer’s protocol. The generation of cDNA was carried out by 3 RACE technique using a sense degenerated primer (Mtfw: 5 -GTGTGGNAGCVVGTGC-3 ) designed on cDNA consensus sequences from gastropod metallothioneins (the original objective was focused on MT, with unexpected isolation of MK). SMART PCR cDNA was

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S. Vanucci et al. / Aquatic Toxicology 75 (2005) 374–379

obtained from 2.5 ␮g of RNA following the manufacturer’s instructions (SMART cDNA Synthesis Kit, Clontech). First strand synthesis was performed using SuperScript III reverse transcriptase (Qiagen) according to manufacturer’s instructions. PCR was performed using buffer (1.5 mM MgCl2 ), Taq DNA polymerase (Invitrogen, 2.5 U), primers (350 ng each), reverse transcriptase products (500 ng), dNTPs (2.5 mM of each) and sterilised water. cDNA amplification was performed following these parameters: 94 ◦ C for 3 min (1 cycle), 94 ◦ C for 1.5 min, 50 ◦ C for 1.5 min, 72 ◦ C for 1.5 min (30 cycles), 72 ◦ C for 10 min (1 cycle). RACE–PCR product of about 500 bp was cloned in pGeM-T vector (Promega) and sequenced (GenBank accession number AY678118). Reverse transcription and PCR using specific primers for Patella vulgata ␣2-tubulin cDNA (␣tubf: 5 -ACGTCGACAAGACCGACTTC-3 ; ␣-tubr: 5 -TGAAACCAGTTGGACACCAG-3 ) and for P. caerulea putative MK cDNA (mdkf: 5 -AACATTAAGGGGAACCCAGG-3 ; mdkr: 5 -TTGTTTTGGTTTCGGTGTCA-3 ) sequences were performed on 2 ␮g total RNA extracted from the viscera and from the foot of P. caerulea specimens exposed to 0.5 and 1 mg l−1 Cd, and from control specimens. The housekeeping gene α2-tub was used as the internal reference gene. The analysis was carried out in a final volume of 20 ␮l containing 4 ␮l of 5× buffer, 400 ␮M dNTPs, 0.8 ␮M of ␣-tubr and mdkr primers, 10 U of RNase inhibitor (Amersham Biosciences), 100 U of enzyme and 2 ␮g of total RNA. Reverse transcription was allowed to proceed for 60 min at 50 ◦ C and was stopped by heating at 70 ◦ C for 15 min. Amplification reactions (45 s at 94 ◦ C, 45 s at 62 ◦ C, 45 s at 72 ◦ C), using primers ␣-tubf and mdkf, were allowed to proceed for 25 cycles. RT-PCR real time assay was carried out with a iCycler iQTM Real-Time PCR apparatus (Bio-Rad). Individual reactions were assembled with oligonucleotide primers (0.15 mM each), 12.5 ␮l of 2X iQTM SYBR Green Supermix (Bio-Rad Laboratories; containing 100 mM KCl, 40 mM Tris–HCl, pH 8.4, 0.4 mM dNTPs, 50 U ␮l−1 iTaq DNA polymerase, 6 mM MgCl2 , 20 nM SYBR Green I, 20 nM fluorescein) plus an appropriate volume of each cDNA preparation in a final volume of 25 ␮l. The following program was run: 95 ◦ C for 3 min (1 cycle), 94 ◦ C for 20 s, 62 ◦ C for 20 s (45 cycles). The primers utilised for Real-Time

PCR analysis were ␣-tubf, ␣-tubr, mdkf and mdkr, already described above. A melting curve (55–95 ◦ C with a heating rate of 0.5 ◦ C for 10 s and a continuous fluorescence measurement) was recorded at the end of every run to assess amplification product specificity. The ‘Comparative threshold cycle (Ct)’ method was used to calculate relative MK expression levels with the ␣2-tubulin RNA as a reference (Rasmussen, 2001). A 3 -end complete cDNA sequence of 538 bp was obtained and analysed for the presence of open reading frames (ORFs). The analysis revealed the presence of one not complete ORF of 417 bp, a 3 -untranslated region of 116 bp and a polyadenylation signal located 70 bp downstream of the stop codon (TGA). The deduced protein sequence contained 139 amino acids with a predicted molecular weight of 15 kDa and an estimated pI of 10.52 (the nucleotide sequence has the GenBank accession number AY678118.). BlastX analysis showed low E-values with MK family sequences belonging to vertebrates and invertebrates. The P. caerulea putative protein exhibited the highest degree of sequence similarity to the Anopheles gambiae MK/PTN protein (23.7% sequence identity, 47.5% sequence similarity). The P. caerulea putative MK Cterminal half domain is conserved: 6 out of the 10 conserved cysteine residues in vertebrates are present in the limpet sequence (Fig. 1). Moreover, the four residues that characterise the heparin binding cluster I of the C-terminal half domain are also conserved, although the third amino acid arginine is converted to lysine (Fig. 1). The domain corresponding to the Cterminal half domain of vertebrate MK and PTN is evolutionally conserved, and this stresses the importance of its structure in retaining biological activities (e.g. neurite outgrowth, fibrinolysis). Cluster I is especially important for the recognition of heparin sulphate as well as chondroitin sulphate proteoglycans, nerve cell migration, and interaction with protein tyrosin phosphatase (Kadomatsu and Muramatsu, 2004 and references therein). Semiquantitative RT-PCR analyses showed that the P. caerulea putative MK was overexpressed in exposed specimens with respect to controls, and expression was higher in the viscera than in the foot (Fig. 2), whereas no overexpression was observed in exposed animals when using ␣2-tubulin primers (data not shown). The RT-PCR Real time analysis (mdk correlation coefficient: 0.996, equation: Y = −3.347X + 15.325,

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Fig. 1. ClustalW alignment of the amino acid sequence deduced from the Patella caerulea putative midkine gene and from vertebrates and invertebrates orthologous genes. The amino acids are indicated by single-letter code. Gaps were introduced for optimal alignment. Identical residues between two sequences are indicated by an asterisk (*); similar residues are indicated by one (low similarity) or two dots (high similarity). Conserved cysteins are indicated in bold, conserved residues of the heparin binding cluster I are marked with I. A: Danio rerio PTN sequence B60042; B: Xenopus tropicalis PTN sequence AAH61275.1; C: Mus musculus PTN sequence AAH02064.1, D: Mouse PTN sequence I53001; E: Rattus norvegicus PTN sequence; F: Danio rerio midkine-related growth factor sequence NP 571145; G: Bos taurus PTN sequence NP 776380.1; H: Gallus gallus PTN sequence B60042; I: Patella caerulea putative MK/PTN sequence AY678118; L: Anopheles gambiae PTN/MK sequence XP315691.

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intrinsically high informative value on the health status of the organism and warning prognostic capability. At present, we speculate that MK may be involved in repairing cell injuries induced by cadmium oxidative stress and/or that MK may be activated by cadmium related genotoxicity. This study is the basis for future investigations that are needed to assess the expression pattern of the MK-like protein and its use as a biomarker of effect.

Acknowledgement Fig. 2. Agarose gel of RT-PCR product (size: 146 bp) amplified using specific primers (mdkf/mdkr) for Patella caerulea putative midkine gene. Lanes 1 and 2, cDNA from the viscera (V) and from the foot (F) of one unexposed specimen; lanes 3 and 4, cDNA from V and F of the first 0.5 mg l−1 Cd-exposed specimen; lanes 5 and 6, cDNA from V and F of the first 1 mg l−1 Cd-exposed specimen; lanes 7 and 8, cDNA from V and F of the second 0.5 mg l−1 Cd-exposed specimen; lanes 9 and 10, cDNA from V and F of the second 1 mg l−1 Cd-exposed; lane 11, RT negative control; DNA marker (1 kbp plus, Invitrogen) is shown on the left.

PCR efficiency: 98.9%; α2-tub correlation coefficient: 0.996, equation: Y = −3.276X + 16.665, PCR efficiency: 101.9%), performed on four P. caerulea specimens exposed to cadmium (i.e. two specimens at 0.5 and two specimens at 1 mg l−1 Cd, respectively), showed a dramatic MK over-expression in treated animals with respect to controls; mdk transcript levels, normalised to α2-tub transcript levels and relative to unexposed controls, were also higher in the viscera than in the foot (i.e. relative gene expressions in the first 0.5 mg l−1 Cd-exposed specimen were: 7900- and 2340-fold in V and F, respectively; in the second 0.5 mg l−1 Cd specimen: 9800- and 2730-fold, in V and F, respectively; in the first 1 mg l−1 Cd specimen: 25475- and 10603-fold, in V and F, respectively; in the second 1 mg l−1 Cd specimen: 18632- and 4534-fold, in V and F, respectively). Our results indicate that cadmium induces overexpression of a MK family protein; a tissue/organdependent up-regulation seems also to emerge. The higher MK expression found in the viscera than in the foot is consistent with observations that heavy metals accumulate mainly in the gills and digestive gland or in hepatopancreas in marine molluscs (Viarengo, 1989). The nature of the cytokine identified in P. caerulea has

The authors thank Dr. Maria Ceccherini, Faculty of Agriculture, Florence University, for facilities in using Cycler BIO-RAD instrument.

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