合浦珠母贝外套膜细胞engrailed、BMP2和Smad3的mRNA表达水平的相关性

软体动物engrailed蛋白和骨形成相关蛋白对胚胎贝壳区域边界形成可能具有重要作用,engrailed还被推测为调节基质蛋白在外套膜组织区域化表达的重要调控因子．因此,弄清调控engrailed在软体动物中特征表达的分子机制有着重要的研究意义．但是,由于贝类基因组测序尚不完整,目前也没有建立获得贝类细胞系,以致于许多预测可能参与调控的基因需要通过克隆来鉴定,而且经典的研究细胞信号通路的方法也很难得到应用．目前,在中国南海广泛养殖的合浦珠母贝中,已获知其BMP2和Smad3的cDNA全长,以该贝的基因组为模板,PCR扩增获得了一段engrailed编码区片段．经软件分析,该片段含有EH4结构域,且与其他物种engrailed蛋白具有很高的同源性．研究的贝中,特别是外套膜组织中,engrailed、BMP2和Smad3三者表达之间的相关性,将有助于我们理解贝壳形成的分子机制．贝壳缺刻后半定量PCR试验结果表明,三者均参与贝壳修复,且在贝壳缺刻后的修复过程中,engrailed和Smad3的mRNA表达变化规律非常相似,提示它们之间可能存在相互影响的联系．用地塞米松(DXM)和过氧化氢(H₂O₂)分别处理原代培养的贝外套膜组织迁出细胞,实时相对定量PCR检测engrailed、BMP2和Smad3的mRNA表达水平,统计分析结果表明,三者具有显著的相关性．上述所有结果为进一步研究贝类生物矿化的发育和信号转导机制提供了新的思路和基础．     

cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca(2+)-binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca(2+)-dependent electrophoretic shift, Ca(2+)-binding activity and significant Ca(2+)-induced conformational changes. Ca(2+)-dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism.     

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata)

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.     

Smad proteins exist as monomers in vivo and undergo homo- and hetero-oligomerization upon activation by serine/threonine kinase receptors.

Smad proteins are signal transducers for the members of the transforming growth factor-beta (TGF-beta) superfamily. Here we show that, in the absence TGF-beta stimulation, Smads exist as monomers in vivo. Smad2 and Smad3 form homo-oligomers upon phosphorylation by the constitutively active TGF-beta type I receptor, and this oligomerization does not require Smad4. Major portions of Smad4, Smad6 and Smad7 are also present as monomers in vivo. Analysis using a cross-linking reagent suggested that the Smad2 oligomer induced by receptor activation is a trimer. Studies by gel chromatography demonstrated that the Smad2-Smad4 heteromer is not larger than the Smad2 homomer. Moreover, overexpression of Smad4 prevented Smad2 from forming a homo-oligomer. These findings suggest that Smad2 may form a homotrimer, or heterotrimers with Smad4, which are probably composed of two and one, or one and two molecules of Smad2 and Smad4, respectively, depending on the amount of each protein. Gel-mobility shift assay revealed that the Smad3 homomer and Smad3-Smad4 heteromer constitute DNA-binding complexes. Transition of the Smad proteins from monomers to oligomers is thus a critical event in the signal transduction of the TGF-beta superfamily members.     

Cloning and expression of a rat Smad1: Regulation by TGFß and modulation by the ras/MEK pathway

A new family of signaling intermediates for TGFß superfamily members and other growth factors has recently been identified and termed Smads. It has been suggested that the Smad1 subfamily is regulated primarily by the TGFß superfamily member bone morphogenetic protein (BMP). Here we demonstrate that TGFß induced phosphorylation of endogenous Smad1 in untransformed IECs and that the RI and RII TGFß receptors were detectable in Smad1 immunocomplexes. Expression of a dominant-negative mutant of Ras inhibited the ability of TGFß to phosphorylate endogenous Smad1. In a separate series of experiments, we have cloned a rat homologue of the drosophila mad gene (termed RSmad1) by screening an intestinal epithelial cell (IEC) cDNA library. By using an in vitro kinase assay with RSmad1 as the substrate, we demonstrate that the TGFß receptor complex can directly phosphorylate RSmad1. We show, further, that a dominant-negative mutant of MEK1 inhibited the ability of RSmad1 to induce the TGFß-responsive reporter p3TP-Lux in a human breast cancer cell line. Collectively, our data demonstrate that TGFß can regulate Smad1 and that the Ras and MEK signaling components are partially required for the ability of TGFß to regulate Smad1. J. Cell. Physiol. 178:387–396, 1999. © 1999 Wiley-Liss, Inc.     

Endoglin in liver fibrosis

Liver fibrosis occurs in most types of chronic liver diseases and is characterized by excessive accumulation of extracellular matrix proteins, leading to disruption of tissue function and eventually organ failure. Transforming growth factor (TGF)-β represents an important pro-fibrogenic factor and aberrant TGF-β action has been implicated in many disease processes of the liver. Endoglin is a TGF-β co-receptor expressed mainly in endothelial cells that has been shown to differentially regulates TGF-β signal transduction by inhibiting ALK5-Smad2/3 signalling and augmenting ALK1-Smad1/5 signalling. Recent reports demonstrating upregulation of endoglin expression in pro-fibrogenic cell types such as scleroderma fibroblasts and hepatic stellate cells have led to studies exploring the potential involvement of this TGF-β co-receptor in organ fibrosis. A recent article by Meurer and colleagues now shows that endoglin expression is increased in transdifferentiating hepatic stellate cells in vitro and in two different models (carbon tetrachloride intoxication and bile duct ligation) of liver fibrosis in vivo. Moreover, they show that endoglin overexpression in hepatic stellate cells is associated with enhanced TGF-β-driven Smad1/5 phosphorylation and α-smooth muscle actin production without altering Smad2/3 signaling. These findings suggest that endoglin may play an important role in hepatic fibrosis by altering the balance of TGF-β signaling via the ALK1-Smad1/5 and ALK-Smad2/3 pathways and raise the possibility that targeting endoglin expression in transdifferentiating hepatic stellate cells may represent a novel therapeutic strategy for the treatment of liver fibrosis.     

A novel ferritin subunit involved in shell formation from the pearl oyster (Pinctada fucata)

Iron is one of the most important minor elements in the shell of bivalves. This study was designed to investigate the involvement of ferritin, the principal protein for iron storage, in shell formation. A novel ferritin cDNA from the pearl oyster (Pinctada fucata) was isolated and characterized. The ferritin cDNA encodes a 206 amino acid polypeptide, which shares high similarity with snail soma ferritin and the H-chains of mammalian ferritins. Oyster ferritin mRNA shows the highest level of expression in the mantle, the organ for shell formation. In situ hybridization analysis revealed that oyster ferritin mRNA is expressed at the highest level at the mantle fold, a region essential for metal accumulation and contributes to metal incorporation into the shell. Taken together, these results suggest that ferritin is involved in shell formation by iron storage. The identification and characterization of oyster ferritin also helps to further understand the structural and functional properties of molluscan ferritins.     

In vitro regulation of CaCO(3) crystal polymorphism by the highly acidic molluscan shell protein Aspein

Biominerals, especially molluscan shells, generally contain unusually acidic proteins. These proteins are believed to function in crystal nucleation and inhibition. We previously identified an unusually acidic protein Aspein from the pearl oyster Pinctada fucata. Here we show that Aspein can control the CaCO(3) polymorph (calcite/aragonite) in vitro. While aragonite is preferentially formed in Mg(2+) -rich solutions imitating the extrapallial fluids of marine molluscs, Aspein exclusively induced calcite precipitation. Our results suggest that Aspein is involved in the specific calcite formation in the prismatic layer. Experiments using truncated Aspein demonstrated that the aspartic acid rich domain is crucial for the calcite precipitation.     

软体动物维甲酸X受体研究进展

维甲酸X受体(retinoid X receptor,RXR)作为配体依赖的转录因子,是核受体超家族重要的一员.脊椎动物RXR与配体及其辅调节因子相互作用,调控基因的协调表达,在胚胎发育、细胞分化、新陈代谢等许多生理过程中起着重要作用.软体动物RXR的研究因其与腹足类性畸变的关系越来越受到关注.本文综述了目前获得的软体动物RXR基因的结构,比较了软体动物RXR基因各功能结构域与人类和其他动物RXR的相似性.以RXR编码区的氨基酸序列为基础,构建了系统进化树,发现软体动物RXR与脊索动物而不是其他无脊椎动物的RXR聚成一支.软体动物和甲壳动物不同RXR亚型的氨基酸序列比较发现,两类动物可能存在不同的剪切酶或剪切位点.此外论文还针对软体动物RXR的配体、二聚体伙伴以及生理功能等方面的研究进行了综述.     

A Functional Study of Transforming Growth Factor-Beta from the Gonad of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The transforming growth factor (TGF)-β superfamily is a group of important growth factors involved in multiple processes such as differentiation, cell proliferation, apoptosis and cellular growth. In the Pacific oyster Crassostrea gigas, the oyster gonadal (og) TGF-β gene was recently characterized through genome-wide expression profiling of oyster lines selected to be resistant or susceptible to summer mortality. Og TGF-β appeared specifically expressed in the gonad to reach a maximum when gonads are fully mature, which singularly contrasts with the pleiotropic roles commonly ascribed to most TGF-β family members. The function of og TGF-β protein in oysters is unknown, and defining its role remains challenging. In this study, we develop a rapid bacterial production system to obtain recombinant og TGF-β protein, and we demonstrate that og TGF-β is processed by furin to a mature form of the protein. This mature form can be detected in vivo in the gonad. Functional inhibition of mature og TGF-β in the gonad was conducted by inactivation of the protein using injection of antibodies. We show that inhibition of og TGF-β function tends to reduce gonadic area. We conclude that mature og TGF-β probably functions as an activator of germ cells development in oyster.