Pinctada fucata mantle gene 3 (PFMG3) promotes differentiation in mouse osteoblasts (MC3T3-E1)

Nacre is secreted from the mantle of pearl oysters. In vivo and in vitro experiments have demonstrated that water-soluble extracts of nacre stimulate osteoblast differentiation and matrix mineralization, but the component responsible for this activity is unclear. It was reported that Pinctada fucata mantle gene 3 (PFMG3) with an N-terminal signal peptide could be secreted into the nacre of P. fucata. Here we report that PFMG3 is specifically expressed at the outer fold of the mantle and could promote calcium carbonate crystal formation in vitro. Consistent with this observation, we found that matrix mineralization of MC3T3-E1 cells, a murine osteoblast cell line, is accelerated upon treatment with PFMG3. Intriguingly, we observed that alkaline phosphatase activity and cell viability are increased after treating MC3T3-E1 cell with PFMG3. mRNA levels of osteoblast-specific marker genes osteocalcin and osteopontin are also increased. We conclude that PFMG3 from the mantle of P. fucata promotes MC3T3-E1 osteoblast cell differentiation, matrix mineralization, and calcium carbonate deposition in vitro. Our findings provide new evidence that PFMG3 may be used as a potential therapeutic molecule for the treatment of osteoporosis.     

Identification and characterization of a biomineralization related gene PFMG1 highly expressed in the mantle of Pinctada fucata

To elucidate the mechanism of nacre biomineralization, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca volvulus calcium-binding protein CBP-1 and had two EF-hand calcium-binding domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallization showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc-HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiation, and mineralization, and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralization, and the SSH method can pave the way for the bulk cloning and characterization of new genes involved in biomineralization in P. fucata and may accelerate research on the mechanism of pearl formation.     

Wound healing after excision of mantle tissue from the Akoya pearl oyster, Pinctada fucata

Pearl oysters are usually sacrificed to donate mantle tissue for pearl production. However, if oysters are anaesthetized, they are able to survive mantle excision and regenerate this tissue. Mantle excision causes a large wound and severs the pallial artery that necessitates rapid wound repair to avoid death by bleeding. This study was undertaken to assess the wound healing process in the mantle of the Akoya pearl oyster, Pinctada fucata, following mantle excision. Forty-seven P. fucata were relaxed with 2.5 mL L(-1) propylene phenoxetol before mantle tissue was excised. Oysters were relaxed and sacrificed 1, 3, 6, 12, 25, 36, 48, 66, 80 and 105 h after excision to assess mantle healing using histological techniques. Muscular contraction that effectively reduced the size of the wound was observed within 1 h after mantle excision. Accumulation of haemocytes and connective tissue occurred 3-6 h after excision and wound plugging was achieved within 6 h of excision. Proliferation of epithelial cells to cover the wound site was observed within the first 25 h after mantle excision and growth of connective tissue and formation of the pallial artery were observed within 105 h after mantle excision.     

马氏珠母贝Sox11基因的克隆及时序表达模式分析

为探究Sox(SRY-related HMG-box genes)基因家族在马氏珠母贝个体发育及性别分化中的作用, 研究首先利用兼并引物从马氏珠母贝基因组中克隆到一个HMG框(high mobility group box), 利用RACE-PCR技术从SMART cDNA文库中克隆到一个Sox基因的cDNA全长, 通过Clustal X和MEGA 4软件对该序列进行比对分析并构建系统进化树; 通过荧光定量PCR技术对该基因在不同组织及发育不同时期性腺中的表达情况进行分析。结果显示, 马氏珠母贝该Sox基因的cDNA全长为1579 bp, 其中开放阅读框(ORF)为1008 bp, 编码336个氨基酸, 5'非编码区为126 bp, 3'非编码区为445 bp。同源性分析表明, 马氏珠母贝Sox基因与太平洋牡蛎Sox11基因的同源性(Identity)最高, 为80%, 故命名为pmSox11; 系统进化树分析也显示pmSox11与太平洋牡蛎Sox11基因的亲缘关系最近。荧光定量PCR分析组织表达特异性显示, pmSox11在马氏珠母贝神经节分布较多的组织如外套膜、鳃、足、消化盲囊等大量表达, 在神经节相对较少的闭壳肌和卵巢中表达量较少; 时序表达图谱显示, pmSox11在3月龄幼贝性腺和1年龄发育早期精巢中表达量最高, 在2年龄成熟精巢和2年龄性转换性腺中表达量降低, 而在2年龄卵巢中表达量最低。研究表明, pmSox11基因可能在马氏珠母贝早期神经系统发育和性别发育的调控方面起到重要作用。     

A Novel Matrix Protein p10 from the Nacre of Pearl Oyster (Pinctada fucata) and Its Effects on Both CaCO3 Crystal Formation and Mineralogenic Cells

A novel matrix protein, designated as p10 because of its apparent molecular mass of 10 kDa, was isolated from the nacreous layer of pearl oyster (Pinctada fucata) by reverse-phase high-performance liquid chromatography. In vitro crystallization experiments showed that p10 could accelerate the nucleation of calcium carbonate crystals and induce aragonite formation, suggesting that it might play a key role in nacre biomineralization. As nacre is known to contain osteogenic factors, two mineralogenic cell lines, MRC-5 fibroblasts and MC3T3-E1 preosteoblasts, were used to investigate the biological activity of p10. The results showed that p10 could increase alkaline phosphatase activity, an early marker of osteoblast differentiation, while the viability of MRC-5 and MC3T3-E1 remained unchanged after treatment of p10. Taken together, the findings led to identification of a novel matrix protein from the nacre of P. fucata that plays a role in both the mineral phase and in the differentiation of the cells involved in biomineralization.     

Peptide-based activation of alpha5 integrin for promoting osteogenesis

Promoting osteoblastogenesis remains a major challenge in disorders characterized by defective bone formation. We recently showed that the alpha 5 integrin subunit (ITGA5) is critically involved in human mesenchymal cell osteoblast differentiation. In this study, we determined the potential of pharmacological ITGA5 activation by a synthetic cyclic peptide (GA-CRRETAWAC-GA) on murine osteoblast differentiation and function in vitro and bone formation in vivo. Peptide-mediated activation of ITGA5 in murine C3H10T1/2 mesenchymal cells resulted in the generation of the integrin-mediated cell signals FAK and ERK1/2-MAPKs. In vitro, peptide-based activation of ITGA5 protected from cell apoptosis but did not affect cell adhesion or replication, while it enhanced the expression of the osteoblast marker genes Runx2 and type I collagen and increased extracellular matrix (ECM) mineralization as also found with bone morphogenetic protein-2 (BMP2), a standard bone anabolic factor. When injected on adult mouse cranial bone for 3 weeks, the peptide-mediated activation of ITGA5 increased bone thickness by twofold, an effect also induced by BMP2. Histomorphometric analysis showed that this anabolic effect resulted from decreased cell apoptosis and increased bone forming surfaces and bone formation rate (BFR). We conclude that pharmacological activation of ITGA5 in mesenchymal cells is effective in promoting de novo bone formation as a result of increased osteoprogenitor cell differentiation into osteoblasts and increased cell protection from apoptosis. This peptide-based approach could be used therapeutically to promote the osteogenic capacity of osteoblast progenitor cells and to induce de novo bone formation in conditions where osteoblastogenesis is compromised.     

A novel carbonic anhydrase from the mantle of the pearl oyster (Pinctada fucata)

A novel carbonic anhydrase (CA) has been purified from the mantle of the pearl oyster, Pinctada fucata, by ammonium sulfate precipitation and affinity chromatography. Its molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be approximately 38 kDa. Native-PAGE shows that the novel CA can bind a fluorescent probe, 5-dimethylamino-1-naphthalenesulfonamide (DNSA), known to specifically bind carbonic anhydrase. Compared to carbonic anhydrase I (CAI) from human erythrocytes, the novel CA migrates faster indicating that it is more acidic. The effect of an inhibitor on the enzyme activity was also examined. The CA from the mantle showed a weak resistance to acetazolamide (AZ), a specific inhibitor of CA. When DNSA was bound to CA, it caused the wavelength of emission maximum intensity to blue shift to 454 nm upon excitation at 326 nm. Histochemical data indicates that the enzyme is distributed widely throughout the mantle tissue, being concentrated at the edge of the mantle. The evidence presented indicates a function for CA in the process of pearl formation and biomineralization.     

Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling

The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass. In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293T cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by overexpression of Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transduction of canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the high bone mass phenotype associated with the loss of sclerostin may be attributed, at least in part, to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.     

cDNA Microarray Analysis Revealing Candidate Biomineralization Genes of the Pearl Oyster, <Emphasis Type="Italic">Pinctada fucata martensii</Emphasis>

Biomineralization is a common biological phenomenon resulting in strong tissue, such as bone, tooth, and shell. Pinctada fucata martensii is an ideal animal for the study of biomineralization. Here, microarray technique was used to identify biomineralization gene in mantle edge (ME), mantle center (MC), and both ME and MC (ME-MC) for this pearl oyster. Results revealed that 804, 306, and 1127 contigs expressed at least three times higher in ME, MC, and ME-MC as those in other tissues. Blast against non-redundant database showed that 130 contigs (16.17 %), 53 contigs (17.32 %), and 248 contigs (22.01 %) hit reference genes (E?≤??10), among which 91 contigs, 48 contigs, and 168 contigs could be assigned to 32, 26, and 63 biomineralization genes in tissue of ME, MC, and ME-MC at a threshold of 3 times upregulated expression level. The ratios of biomineralization contigs to homologous contigs were similar at 3 times, 10 times, and 100 times of upregulated expression level in either ME, MC, or ME-MC. Moreover, the ratio of biomineralization contigs was highest in MC. Although mRNA distribution characters were similar to those in other studies for eight biomineralization genes of PFMG3, Pif, nacrein, MSI7, mantle gene 6, Pfty1, prismin, and the shematrin, most biomineralization genes presented different expression profiles from existing reports. These results provided massive fundamental information for further study of biomineralization gene function, and it may be helpful for revealing gene nets of biomineralization and the molecular mechanisms underlining formation of shell and pearl for the oyster.     

Regulation of osteoblast differentiation by Nurr1 in MC3T3-E1 cell line and mouse calvarial osteoblasts

The orphan nuclear receptor Nurr1 is primarily expressed in the central nervous system. It has been shown that Nurr1 is necessary for terminal differentiation of dopaminergic (DA) neurons in ventral midbrain. The receptor, however, is also expressed in other organs including bone, even though the role of Nurr1 is not yet understood. Therefore, we investigated the role of Nurr1 in osteoblast differentiation in MC3T3-E1 cells and calvarial osteoblasts derived from Nurr1 null newborn pups. Our results revealed that reduced Nurr1 expression, using Nurr1 siRNA in MC3T3-E1 cells, affected the expression of osteoblast differentiation marker genes, osteocalcin (OCN) and collagen type I alpha 1 (COL1A1), as measured by quantitative real-time PCR. The activity of alkaline phosphatase (ALP), another osteoblast differentiation marker gene, was also decreased in Nurr1 siRNA-treated MC3T3-E1 cells. In addition, Nurr1 overexpression increased OCN and COL1A1 expression. Furthermore, consistent with these results, during osteoblast differentiation, the expression of osteoblast marker genes was decreased in primary cultured mouse calvarial osteoblasts derived from Nurr1 null mice. Collectively, our results suggest that Nurr1 is important for osteoblast differentiation.     

Identification of calconectin, a calcium-binding protein specifically expressed by the mantle of Pinctada margaritifera

Nacre or mother-of-pearl in the shell of Pinctada margaritifera is composed of 95-99% calcium carbonate and 1-5% organic matrix. In this study, we developed an original technique to characterize the genes differentially expressed in nacre-forming cells (NFC) by combining suppression subtractive hybridization (SSH), to establish a cDNA subtractive library, with rapid amplification of cDNA ends (RACE)-PCR. Seventy-two specific cDNA sequences have been obtained so far. These include a protein containing two EF-hand Ca2+-binding domains which was completely sequenced after amplification by RACE-PCR. Its specific expression as well as the specificity of the SSH method was confirmed by semi-quantitative RT-PCR on NFC and mantle cells.     

Ingestion of particulate matter by the outer surface cells of the mollusc mantle

A study of the ingestion of particulate matter from the pallial space located between the shell and the outer surface of the mantle of Isognomon alatus and Pinctada radiata was undertaken with the aid of the electron microscope. For this purpose colloidal thorium dioxide (thorotrast) was introduced into the pallial space for periods of 1–5 days after which time the mantle was excised and prepared for examination with the electron microscope. After 24 hours thorotrast micelles were observed in the pallial space, on the surface of the microvilli, in small pinocytotic vesicles between the bases of the microvilli, in vacuoles undergoing coalescence and finally in large dense bodies (lysosomes). Amoebocytes in the pallial space also participate in the removal of particulate matter in a manner similar to that described for the surface epithelium. During active ingestion the Golgi apparatus changes from a vesicular to a lamellar form. The method of ingestion observed in the surface epithelia and the amoebocytes is similar to the ingestion of protein and other particulate material reported for a variety of vertebrate tissues.     

The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts

Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone.     

Monolayer formation and DNA synthesis of the outer epithelial cells from pearl oyster mantle in coculture with amebocytes

Summary In vitro experiments were conducted to clarify the involvement of the epithelium-amebocyte interaction in epithelial regeneration of bivalves. The outer epithelia of the pallial mantle of the pearl oyster, Pinctada fucata martensii, were separated in cell sheets from the inner connective tissue layers by digestion with Dispase. Clumps of the separated mantle epithelia were inoculated onto the amebocyte layers prepared on the bottom of culture dishes and maintained at 20° C in 5% CO₂:95% air for 1 wk. Balanced salt solution with 0.03% (wt/vol) glucose was used as a culture medium. The epithelial cells adhered to the amebocyte layers within 24 h, changed their shape from cuboidal to squamous, and migrated and formed monolayer sheets within 3 d. Electron microscopy confirmed maintenance of epithelial polarity and cell to cell junction in the sheets; 6 d after the inoculation, 5-bromo-2′-deoxyuridine was added to the culture at 30 μM. After labeling for 24 h, the cultures were fixed and stained with anti 5-bromo-2′-deoxyuridine antibody. Cells with immunoreactive nuclei were clearly observed in the epithelial cell sheets, indicating active DNA synthesis in the epithelial sheets. Thus, cocultured with amebocytes, the outer epithelial cells from pallial mantle tissue formed a monolayer sheet and started DNA synthesis. The morphological features of the mantle outer epithelial cells are analogous to those described for the in vivo cutaneous wound healing process, suggesting that the epithelium-amebocyte interaction is important in the regeneration of epithelium in bivalves.     

MODULATION OF OSTEOGENIC DIFFERENTIATION IN HUMAN SKELETAL CELLS IN VITRO BY 5-AZACYTIDINE

Cellular differentiation is controlled by a variety of factors including gene methylation, which represses particular genes as cell fate is determined. The incorporation of 5-azacytidine (5azaC) into DNA in vitro prevents methylation and thus can alter cellular differentiation pathways. Human bone marrow fibroblasts and MG63 cells treated with 5azaC were used as models of osteogenic progenitors and of a more mature osteoblast phenotype, respectively. The capacity for differentiation of these cells following treatment with glucocorticoids was investigated. 5azaC treatment led to significant expression of the osteoblastic marker alkaline phosphatase in MG63 osteosarcoma cells, which was further augmented by glucocorticoids; however, in human marrow fibroblasts alkaline phosphatase activity was only observed in glucocorticoid-treated cultures. MG63 cells represent a phenotype late in the osteogenic lineage in which demethylation is sufficient to induce alkaline phosphatase activity. Marrow fibroblasts are at an earlier stage of differentiation and require stimulation with glucocorticoids. In contrast, the expression of osteocalcin, an osteoblastic marker, was unaffected by 5azaC treatment, suggesting that regulation of expression of the osteocalcin gene does not involve methylation. These models provide novel approaches to the study of the control of differentiation in the marrow fibroblastic system.