PfSMAD4 plays a role in biomineralization and can transduce bone morphogenetic protein-2 signals in the pearl oyster Pinctada fucata
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Mollusca is the second largest phylum in nature. The shell of molluscs is a remarkable example of a natural composite biomaterial. Biomineralization and how it affects mollusks is a popular research topic. The BMP-2 signaling pathway plays a canonical role in biomineralization. SMAD4 is an intracellular transmitter in the BMP signaling pathway in mammals, and some genomic data show SMAD4’s involvment in BMP signaling in invertbrates, but whether SMAD4 plays a conservative role in pearl oyster, Pinctada fucata, still need to be tested.
In this study, we identified a SMAD4 gene (hereafter designated PfSMAD4) in pearl oyster Pinctada fucata. Bioinformatics analysis of PfSMAD4 showed high identity with its orthologs. PfSMAD4 was located in the cytoplasm in immunofluorescence assays and analyses of PfSMAD4 mRNA in tissues and developmental stages showed high expression in ovaries and D-shaped larvae. An RNA interference experiment, performed by PfSMAD4 double-stranded RNA (dsRNA) injection, demonstrated inhibition not only of nacre growth but also organic sheet formation with a decrease in PfSMAD4 expression. A knockdown experiment using PfBMP2 dsRNA showed decreased PfBMP2 and PfSMAD4 mRNA and irregular crystallization of the nacreous layer using scanning electron microscopy. In co-transfection experiments, PfBMP2-transactivated reporter constructs contained PfSMAD4 promoter sequences.
Our results suggest that PfSMAD4 plays a role in biomineralization and can transduce BMP signals in P. fucata. Our data provides important clues about the molecular mechanisms that regulate biomineralization in pearl oyster.
KeywordsSMAD4 Biomineralization BMP signaling pathway Pinctada fucata
bone morphogenic proteins
dulbecco's modified eagle media
fetal bovine serum
green fluorescent protein
scanning electron microscope
mothers against DPP homologs
transforming growth factor
Pearl oyster, Pinctada fucata, is distributed over the southern coast of China and is the most popular farming shellfish for pearl production. The plain outer surface of pearl oyster shells conceal the lustrous beauty of the mother-of-pearl lining ‘nacre’. It combines a high mechanical strength similar to many ceramics, with elasticity, reducing the brittleness of the shell [1, 2]. The nacreous layer of molluskan shells, which consist of highly oriented aragonitic crystals and an organic matrix (including chitin and proteins), is a product of biomineralization [3, 4, 5].
Bone morphogenic proteins (BMP) are the largest subgroup in the transforming growth factor-beta (TGF-β) superfamily  and play a canonical role in biomineralization [7, 8]. In the BMP family, BMP-2 has one of the strongest signals for stimulating biomineralization. BMP-2 stimulates bone or tooth mineralization via the canonical BMP pathway [9, 10, 11]; SMAD 1, 5, and presumably 8, propagate BMP signals and are structurally related to Mad that acts downstream of Dpp, a BMP homolog in Drosophila . SMAD4 is the only Co-SMAD in mammals , and Medea acts as a common SMAD in flies . In the cytoplasm, receptor-regulated SMADs (R-SMADs) are directly phosphorylated by BMP-like ligands and then associate with common SMADs (Co-SMADs) that are essential to distinct signaling pathways. The heteromeric complexes are translocated to the nucleus, where they regulate transcription of target genes in concert with other transcription factors [15, 16].
SMADs have a domain structure consisting of highly conserved amino (NH2)- and (COOH)-terminal regions, referred to as Mad homology 1 (MH1) and MH2 domains [17, 18], respectively. The MH1 domain can bind to specific DNA sequences in the nucleus and the MH2 domain is responsible for interaction with other SMAD proteins .
Accumulating examples show that BMP orthologs play important roles in biomineralization in mollusca [20, 21, 22, 23, 24, 25]. In previous studies, the BMP-2 gene of P. fucata has been identified and defined as PfBMP2 . Further studies showed that a purified recombinant 10-kD mature fragment of PfBMP2 could induce osteogenic differentiation in C3H10T1/2 , demonstrating that PfBMP2 is conserved in terms of its function in the formation of hard tissuePreliminary studies of SMAD4 genes in Crassostrea gigas and Lingula anatina show their potential involvement in shell formation [28, 29], and Luo et al. showed SMAD4’s involvment in BMP-2 signaling based on Mollusca and brachiopod genomes . Although a SMAD4 homolog was found in P. fucata (designated PfSMAD4), whether the SMAD4 protein has the same function as their homologs still needs to be tested. In this study, we investigated if PfSMAD4 played a role in biomineralization. Additionally, we identified that PfBMP2 could activate the promoter of PfSMAD4, and PfSMAD4 expression decreased after interfering with the expression of PfBMP2.
Sequence analysis of PfSMAD4
PfSMAD4 expression in tissues and developmental stages
PfSMAD4 is localized to the cytoplasm
Knockdown of PfSMAD4 leads to disorder of the nacreous layer
Knockdown of PfBMP2 leads to reduced PfSMAD4 expression
PfBMP2 activates PfSMAD4-specific reporter genes
S278Luc is the basic promoter of the PfSMAD4 promoter. Deletions of the region from −778 to −653 resulted in 40-fold increases in promoter activity, suggesting that these regions function as silencers in controlling PfSMAD4 gene transactivation (Fig. 6, right graph). Over-expression of pCDNA3.1 vector had no obvious effect on the activities of S278Luc, S778Luc and S1065Luc, but when transfected with pCDNA3.1-BMP2, their activity significantly increased (Fig. 6, right graph). The results presented in this report show that PfBMP2, when expressed in transiently transfected mammalian cells, can activate transcription from the PfSMAD4 promoter and cis-regulatory DNA sequences may exist in the region from −202 to −278.
PfSMAD4 plays a role in biomineralization
The PfSMAD4 gene shows high expression in mantle and D-shaped larvae stages. The mantle tissue stage corresponds to shell formation and the D-shaped larval stage is a period in which mineral materials largely accumulate. These results may suggest that PfSMAD4 exerts a function in shell formation not only in the adult but also during the embryonic stage. High expression level of the SMAD4 gene reported in the shell fields of embryos at different stages in Crassostrea gigas  is consistent with our study. The high expression in the ovary may indicate that PfSMAD4 functions in reproduction and development.
It is well known that TGF-β/BMP signaling play important roles in osteoblast differentiation and bone formation . As a common mediator Smad of TGF-β and BMP signaling, SMAD4 is also required for maintaining normal bone homeostasis. Conditional deletion of Smad4 in osteoblasts leads to lower bone mineral density, decreased bone volume, decreased bone formation rate, and a reduced number of osteoblasts . Mutations at a single codon in Mad homology 2 domain of SMAD4 can cause Myhre syndrome, which is a developmental disorder characterized by a shortness in stature, hands, feet, and so on . Interference of PfSMAD4 caused nacre disorder showed that PfSMAD4 played a role in biomineralization in P. fucata.
Conserved BMP2/SMAD4 signaling pathway in P. fucata
In recent years, many alternatively spliced SMAD4 variants have been found in many species [36, 37, 38, 39]. Most isoforms lack one or more in-frame exons, compared with the full-length transcripts, and the activities of their encoded proteins depends on which region of the SMAD protein is missing or affected . Comparison of the deduced amino acid sequence of PfSMAD4 with SMAD4 from other organisms showed that PfSMAD4 has an overall 27.8–77.5 % identity with known sequences. The MH1 domain and MH2 domain showed higher identities, ranging from 62 to 93.7 % and 56.1–96.8 %, respectively. The high identities of the MH1 and MH2 domains of SMADs imply a highly conserved structure, further suggesting a conservation in function. The SMAD4 sequence is conserved in eukaryotes from sponges to mammals and the PfSMAD4 has a high similarity to vertebrate SMAD4, confirming the hypothesis by Westbroek et al.  that human and pearl oyster may have homogeneous signal transmitters in biomineralization.
Many developmental mechanisms have shown to be conserved throughout evolution . Gabrielle et al.  demonstrated that the BMP signaling pathway was in place prior to the divergence in the line of Cnidaria to the higher Metazoa, and that it has been substantially conservative during evolution. Based on Mollusca and brachiopod genomes, BMP-SMAD signaling pathway showed its conservation in verterbrates . The conserved SMAD4 was identified in many invertebrates like fly , ascidian  and amphioxus , demonstrating a conserved function in the BMP signalling pathway. RNAi technology has been applied in investigating the function of specific genes  and it has been used successfully in Mollusca [48, 49, 50, 51]. As a potential signal transducing molecule, SMAD4 protein is expected to be co-expressed with the BMP signaling molecule. The interference of PfBMP2 mRNA led to reduced PfSMAD4 expression, indicating that PfSMAD4 could transduce a BMP2 signaling pathway. Moreover, the nacre pattern after PfSMAD4 interference bore similar resemblance to that after PfBMP2 interference, highlighting an essential role of PfSMAD4 in mediating the BMP signaling pathway in P. fucata. These results are reinforced by our luciferase assays showing PfBMP2 could activate the PfSMAD4 promoter.
Our results suggest that PfSMAD4 plays a role in biomineralization and can transduce BMP signals in P. fucata. Our data provide important clues about the molecular mechanisms that regulate biomineralization in pearl oyster.
Bioinformatics analysis of PfSMAD4
PfSMAD4 sequence was obtained from GenBank, accession number AGY49100.1. Multiple sequence alignments of the deduced amino acids were performed using ClustalX2  and protein domains were predicted by ExPASy translate tool (http://web.expasy.org/translate/). A neighbor-joining phylogenetic tree was constructed using the MEGA5.0 package . Reliability of branching was tested using bootstrap re-sampling with 1000 pseudo-replicates.
Cloning the 5' flanking region of the PfSMAD4 gene
GenomeWalker libraries were constructed using a GenomeWalker Universal kit according to manufacturer’s instructions (Clontech, Mountain View, CA, USA). Pearl oyster genomic DNA (2.5–5 μg) in each reaction was digested at 37 °C overnight with a restriction enzyme. Four enzymes (DraI, EcoRV, PvuII and StuI) were used in four reactions, respectively. After purification with phenol and chloroform extraction and ethanol precipitation, the digested DNA was ligated to GenomeWalker adapters (5'-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3') at 16 °C overnight. Primers for PCR-based DNA walking in GenomeWalker libraries were gene-specific: PfSMAD4-specific primer 1 (5'-ACCTGCCATCCAGAGTTCTT-3') and nested primer 2 (5'-CCAGACTTTCTATGGCTCGT-3'). The longest fragment from the four genomic libraries was gel-purified, and subcloned for sequencing. According to the sequence, the nested primer 3 (5'-GGAGGTCAATTCTCGGAAAC-3') was designed. The second round PCR used nested primer 2 and nested primer 3. From two rounds of PCR, we got a 2524 bp 5' UTR-intron and a 1065 bp 5' flanking sequence [GenBank:KJ530991].
RNA isolation and quantitative PCR analysis
P. fucata samples were isolated using TRIzol (Invitrogen, Carlsbad, CA, USA). Total RNA (1 μg) was treated with DNase I (Fermentas, Shenzhen, China) to prevent DNA contamination and subsequently reverse transcribed with Toyobo RT-PCR kit (Toyobo, Osaka, Japan). Quantitative PCR (qPCR) primers for tissue and developmental stage distribution were as follows: PfSAMD4, 5'- ATGCACCCGGTAGCTCTA-3' and 5'-TCACCGACTCCGAAACAGG-3'; β-actin, 5'- TGGTATGGGACAGAAGGAC-3' and 5'- GACAATGCCGTGCTCAAT -3'.
qPCR was carried out using a LightCycler 480 Real-Time PCR System (Roche, Basel, Switzerland), with SYBR green fluorescent dye, according to the manufacturer’s protocol (Toyobo). qPCR conditions were as follows: denaturation at 94 °C for 1 min, followed by 40 cycles at 94 °C for 15 s, 55 °C for 15 s and 72 °C for 60 s. We analyzed the relative gene expression using the typical cycle threshold (Ct) method (2-ΔΔCt method).
The cDNA encoding the full-length PfBMP2 was amplified with sequence specific primers, 5'-CGGGGTACCATGATTTACGGATTTGGACAT-3' containing a KpnI restriction site, and 5' -CCGCTCGAGCCGACATCCGCATCCTTC-3' containing an XhoI restriction site. After double digestion with KpnI and XhoI, the cDNA was cloned in-frame into the KpnI/XhoI sites of pcDNA3.1/myc-His (A) vector (Invitrogen). The construct was verified by sequencing. The pCDNA3.1-PfSMAD4 was constructed using the same strategy as above. Specific primers for PfSMAD4: F, 5'- CGGGGTACCATGACGACACAAGCACCAACG-3' (KpnI restriction site is underscored) and R, 5'-CCGCTCGAGGCCTAGGAAGAATCCTCT-3’ (XhoI restriction site is underscored).
A 1065 bp PfSMAD4 promoter fragment was subcloned into the KpnI and BglII sites of the pGL3-basic luciferase reporter vector (Promega, Madison, WI, USA) to generate S1065Luc. The fragments of the PfSMAD4 gene between S778Luc, S563Luc, S278Luc, S202Luc and S118Luc were amplified by PCR using S1065Luc as a template (transcriptional initiation site was defined as +1).
Cell culture, transfection
The 293 T human kidney cell line (HEK293T) was cultured at 37 °C in a humidified atmosphere of 5 % CO2 using DMEM (Gibco, Grand Island, NY, USA) supplemented with 10 % FBS (Gibco), 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco). The cultures were split every 2 to 3 days. Lipofectamine 2000 (Invitrogen) was used for the DNA transfections according to the manufacturer’s protocol.
PfSMAD4 distribution in P. fucata
Adult pearl oysters (shell length 4.5–5.5 cm) were obtained from Daya Bay (China Marine Biology Research Station, South China Sea Institute of Oceanology, the Chinese Academy of Sciences) in Shenzhen, China. They were acclimated in indoor cement ponds, at ambient seawater temperature for 1 week, before the experiment. Tissue expression profiles of PfSMAD4 were analyzed in ovaries, testes, gills, adductor muscles, mantles, hearts, and digestive glands. Each tissue was dissected from three oysters. Developmental stage expression profiles of PfSMAD4 were analyzed in fertilized eggs, 2–4 cell stage, blastocysts, the trochophore, D-shaped larvae, umbo larvae, eye-spot larvae, spats and juveniles. β-actin was expressed stably in all tested tissues and developmental stages. Three repetitions of the reaction were performed.
Subcellular localization of PfSMAD4 was performed by immunofluorescence assays. The HEK293T cells were seeded onto cover slips (10 mm × 10 mm) in a 12-well plate. After transfection for 48 h, the HEK293T cells were fixed with 4 % paraformaldehyde and then the coverslips were blocked using 2 % bovine serum albumin at room temperature for 30 min. Cells were incubated either with anti-myc antibody (1:60) or preimmune mouse serum (1:60) for 1 h, rinsed with PBS three times for 10 min and then incubated with FITC-conjugated goat anti-mouse antibodies (Pierce, Rockford, IL, USA) for a further hour. Finally, cells were stained with DAPI (1 mg/ml) and observed under fluorescence microscopy.
RNA interference (RNAi) was performed as described in Suzuki et al. , with some modifications. The primers used for generating PfBMP2 and PfSMAD4 dsRNA were RBMP2F:GCGTAATACGACTCACTATAGGGAGACATCCCGCAGTATTAAAGTGG, RBMP2R:GCGTAATACGACTCACTATAGGGAGACCGACATCCGCATCCTTCAAC; RSMAD4F:GCGTAATACGACTCACTATAGGGAGATTATGCCAGGATTTGGAGAT; RSMAD4R:GCGTAATACGACTCACTATAGGGAGAGAGGCTTGAGACTGAGGAG. The T7 promoter sequence is bold. For GFP, pEGFP-C1 (Clontech) was used as the template. A RiboMAX Large Scale RNA Production System (T7) kit (Promega) was used to synthesize and purify the dsRNA. RNase-free DNase I (TaKaRa, Otsu, Japan) was used to digest the template DNA. The PfBMP2 dsRNA and PfSMAD4 dsRNA were diluted to 80 μg/100 μl using PBS, and 100 μl solutions were injected into pearl oyster adductors. PBS and dsRNA-GFP were used as controls. Total RNA from the mantle tissue of each oyster was extracted 7 days after injection and used to synthesize the first strand cDNA as described above. qPCR was used to quantify the expression levels of PfBMP2 and PfSMAD4, where β-actin was used as an internal reference. The qPCR primers that were designed for PfSMAD4 and β-actin were the same sequences as in the distribution experiments above. The shell of each oyster was thoroughly washed with Milli-Q water and air-dried. It was then cut into pieces and mounted on the scanner with the inner nacreous surface face-up, sputter-coated with 10 nm-thick gold, and analyzed using scanning electron microscopy (SEM, S-3400 N, Hitachi, Tokyo, Japan).
HEK293T cells (1.5 × 105 cells/well) were seeded onto 48-well plates. Cells were transfected with the pGL3 reporter gene in the absence or presence of PfBMP2 expression vectors. The total amount of DNA (1.0 μg) was kept constant with empty vectors. For normalization of transfection efficiencies, 0.1 μg of Renilla (sea pansy) luciferase expression plasmid (pRL-TK, Promega) was included in the transfection experiments. Transfected cells were lysed and subjected to luciferase assays using luciferin substrate (Promega) following the manufacturer's instructions. The assays were performed in triplicates.
Data were analyzed by one-way analysis of variance (ANOVA) with default parameters or the Student’s t-test to identify differences between groups. Differences were considered statistically significant when P values were lower than 0.05.
This work was supported by the National Natural Science Foundation of China (41376159), the National Science and technology program of China (2012AA10A410) and the Marine Fishery Science and Technology Promotion Program of Guangdong Province, China (A201201A05, A201301A03).
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