Molecular cloning and expression of gelatinases (MMP-2 and MMP-9) in the pufferfish Takifugu rubripes

To determine the metabolism location of the extra-cellular matrix proteins in fugu (Takifugu rubripes), we cloned the cDNAs of the fugu gelatinases, matrix metalloproteinase-2 (MMP-2) and MMP-9, and examined their expressions in various adult tissues using a quantitative real-time PCR. The expression profiles of fugu gelatinases were different among tissues. FgMMP-9 mRNA was abundant in tissues that contain blood cells abundantly where fgMMP-2 mRNA was little expressed. We also examined the expression of these genes in fugu embryos during development using a whole mount in situ hybridization. Fugu MMP-2 mRNA was expressed in the pharyngeal area and mesenchyme in embryos at 80 hours post fertilization (hpf). While fugu MMP-9 mRNA was expressed in the vent at 140 hpf and the caudal end of the fin fold at 172 hpf. Although fugu MMP-2 mRNA was expressed in the pectoral fin bud at 120 hpf, fugu MMP-9 mRNA did not appear in this tissue until 10 days post fertilization (dpf). These data show expression profiles differ between the fugu gelatinases and suggest expressions of these genes are controlled at the matrix protein degradation site in fugu embryos during development.     

Expression of fugu TIMP-3 and -4 genes in adult tissues and embryos

The tissue inhibitors of metalloproteinases (TIMPs) are involved in various processes of extra-cellular matrix (ECM) metabolism by inhibiting matrix metalloproteinases (MMPs). However, the fundamental information for these genes is little known in fish. Previously, we report cDNA cloning and gene expressions of two fugu (Takifugu rubripes) TIMP-2s. Here, we cloned cDNA of fugu TIMP-3 and performed an expression analysis of TIMP-3 and -4 mRNA in fugu adult tissues using a quantitative real-time PCR. The expression level of TIMP-3 mRNA was constitutive in all tissues, while TIMP-4 was significantly higher in the brain (P=0.05). Further, we performed a whole mount in situ hybridization in fugu embryos at different stages. In early stages, TIMP-3 mRNA was abundant in the somites and the caudal end of the notochord. At hatching larvae, the TIMP-3 mRNA was abundant in the pectoral fin, dorsal and ventral fin fold along the entire antero-posterior axis. TIMP-3 may be involved in axis elongation and somitogenesis. TIMP-4 mRNA was expressed in the tail bud, at the midbrain-hindbrain boundary and in the diencephalon from 72 to 104 hpf. This indicates TIMP-4 is highly expressed in the brain matrix in vivo.     

Tissue inhibitor of metalloproteinases-2 is expressed in the interstitial matrix in adult mouse organs and during embryonic development.

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a member of a family of inhibitors of matrix-degrading metalloproteinases. A better insight into the role of this inhibitor during development and in organ function was obtained by examining the temporospatial expression of TIMP-2 in mice. Northern blot analysis indicated high levels of TIMP-2 mRNA in the lung, skin, reproductive organs, and brain. Lower levels of expression were found in all other organs with the exception of the liver and gastrointestinal tissue, which were negative of these tissues with complete absence of TIMP-2 mRNA in the epithelium. In the testis, TIMP-2 was present in the Leydig cells, and in the brain, it was expressed in pia matter and in neuronal tissues. TIMP-2 expression in the placenta increased during late gestation and was particularly abundant in spongiotrophoblasts In mouse embryo (day 10.5-18.5), TIMP-2 mRNA was abundant in mesenchymal tissues that surrounded developing epithelia and maturing skeleton. The pattern of expression significantly differs from that observed with TIMP-1 and TIMP-3, therefore, suggesting specific roles for each inhibitor during tissue remodeling and development.     

High expression of the mRNA of cytochrome P450 and phase II enzymes in the lung and kidney tissues of cattle

The objective of this study was to investigate the tissue-specific mRNA expression of different cytochrome P450 (CYP) isoforms, UDP glucuronsyl transferase 1A1 (UGT1A1) and glutathione-S-transferase (GSTA1) in the different tissues (liver, mammary gland, lungs, spleen, kidney cortex, heart, masseter muscle and tongue) of cattle, using quantitative real-time polymerase chain reaction (qPCR). CYP1A1-like mRNA was expressed in all of the tissues examined, including the liver, with the highest expression level in the kidney. CYP1A2-, 2E1- and 3A4-like mRNAs were only expressed hepatically. Interestingly, significant expression of CYP2B6-like mRNA was recorded in the lung tissue, while CYP2C9-like mRNA was expressed in the liver and kidney tissues of the cattle examined. UGT1A1- and GSTA1-like mRNAs were expressed in all of the examined tissues, except the mammary glands, and the highest expression levels were recorded in the kidney. The high expression of UGT1A1 in the lung tissue and GSTA1 in the liver tissue was unique to cattle; this has not been reported for rats or mice. The findings of this study strongly suggest that the liver, kidneys and lungs of cattle are the major organs contributing to xenobiotics metabolism.     

Quantitative analysis of messenger RNA expression of matrix metalloproteinases (MMP-2 and MMP-9), tissue inhibitor-2 of matrix metalloproteinases (TIMP-2), and steroidogenic enzymes in bovine placentomes during gestation and postpartum

The relationship between the mRNA expression of proteolytic and steroidogenic enzymes in bovine placentomes was examined. Caruncle and cotyledon tissues were collected every 6 hr after spontaneous parturition until the fetal membranes were released. Based on the time of fetal membrane release after parturition, the specimens were classified as follows: (1) the early group, in which the fetal membranes were released within 6 hr after parturition; and (2) the late group, in which the fetal membranes were released 6-12 hr after parturition. The placentomes from a slaughterhouse were additionally collected as samples for the examination of enzymes during the gestation period. The mRNA expression of steroidogenic enzymes in the cotyledon was observed to be higher than that in caruncle tissues; however, the mRNA expression patterns of P450scc and StAR tended to be similar in both placental tissues. On the other hand, although the expression levels of TIMP-2 mRNA in both caruncle and cotyledon tissues were similar, during gestation and postpartum the expression levels of MMP-2 and MMP-9 mRNA were approximately 10 times higher in caruncle than in cotyledon tissue. Marked contrasting changes in mRNA expression patterns between pre- and postpartum periods were observed for MMP-2 and MMP-9 in caruncle tissues and for MMP-9 and TIMP-2 in cotyledon tissues. The present study provides the first evidence that MMP-2, MMP-9, and TIMP-2 mRNAs are expressed in bovine placentomes during the gestational and postpartum periods and suggests that these enzymes, in conjunction with steroidogenic enzymes, mediate fetal membrane detachment after parturition.     

A lineage specific enhancer drives Otx2 expression in teleost organizer tissues

In mouse Otx2 plays essential roles in anterior-posterior axis formation and head development in anterior visceral endoderm and anterior mesendoderm. The Otx2 expression in these sites is regulated by VE and CM enhancers at the 5' proximal to the translation start site, and we proposed that these enhancers would have been established in ancestral sarcoptergians after divergence from actinopterigians for the use of Otx2 as the head organizer gene (Kurokawa et al., 2010). This would make doubtful an earlier proposal of ours that a 1.1 kb fragment located at +14.4 to +15.5 kb 3' (3'En) of fugu Otx2a gene harbors enhancers phylogenetically and functionally homologous to mouse VE and CM enhancers (Kimura-Yoshida et al., 2007). In the present study, we demonstrate that fugu Otx2a is not expressed in the dorsal margin of blastoderm, shield and early anterior mesendoderm, and that the fugu Otx2a 3'En do not exhibit activities at these sites of fugu embryos. We conclude that the fugu Otx2a 3'En does not harbor an organizer enhancer, but encodes an enhancer for the expression in later anterior mesendodermal tissues. Instead, in fugu embryos Otx2b is expressed in the dorsal margin of blastoderm at blastula stage and shield at 50% epiboly, and this expression is directed by an enhancer, 5'En, located at -1000 to -800 bp, which is uniquely conserved among teleost Otx2b orthologues.     

Expression of gelatinases and their tissue inhibitors in rat corpus luteum during pregnancy and postpartum

Extensive tissue remodeling occurs in the corpus luteum (CL) during both formation and luteolysis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play pivotal roles in these processes. In the present study, to evaluate the potential roles of matrix degrading proteases in luteal development and regression, we examined gelatinases and TIMP-1, -2, -3 mRNA expressions, as well as gelatinase activity in rat CL during pregnancy and postpartum using Northern blot, in situ hybridization, and gelatin zymography, respectively. The results showed that MMP-2 mRNA was only expressed at the early stages of pregnancy; TIMP-2 mRNA was highly expressed at the early and late pregnancy and day 1 postpartum, but could not be detected during the mid-phase of pregnancy; TIMP-3 mRNA expression was abundant during early pregnancy and peaked at day 7, but was absent from other time points examined. MMP-9 and TIMP-1 mRNAs in rat CL were below detectable level in the current study. Furthermore, the active MMP-2 was only present during the early stages of pregnancy, and no MMP-9 activity was observed in the zymogram. Taken together, our results suggest that MMP-2 and TIMP-3 may have functional roles in rat luteal formation, while TIMP-2 may be implicated in both formation and regression of the pregnant CL.     

PP2A-A/在金鱼及斑马鱼发育中的分化表达模式

以金鱼和斑马鱼为研究对象,运用RT-PCR和Western Blot技术分析蛋白磷酸酶2A(PP2A)结构亚基A(PP2A-A/)在金鱼、斑马鱼成体9种组织和12个发育时期胚胎中mRNA和蛋白水平的表达情况,得到其分化表达模式为:(1)在mRNA水平上,PP2A-A/在金鱼、斑马鱼9种组织中具有较强表达;种属差异性和组织差异性均较大;结构亚基A的两亚型A和A的表达存在差异。(2)在蛋白水平上,PP2A-A/在金鱼、斑马鱼9种组织中均有表达;种属差异性不大但出现明显的组织差异性。(3)PP2A-A/mRNA在金鱼和斑马鱼卵裂期到囊胚期胚胎中大量存在,PP2A-AmRNA在金鱼眼色素期量剧增推测其对金鱼眼色素的形成至关重要。(4)PP2A-A/基因在金鱼、斑马鱼12个发育时期胚胎中均有较高水平的蛋白存在,提示其为维持胚胎的正常发育发挥重要作用。       

Occurrence of two distinct types of tissue inhibitor of metalloproteinases-2 in teleost fish

We have cloned for the first time two cDNAs encoding distinct types of tissue inhibitor of metalloproteinases-2 (TIMP-2) from teleost fish, Japanese flounder, and designated these types as jfTIMP-2a and jfTIMP-2b. The open reading frames of the jfTIMP-2a and jfTIMP-2b cDNAs are composed of 663 and 657 nucleotides and 221 and 219 amino acids, respectively. Both jfTIMP-2s contain 12 cysteine residues, which might form six disulfide bonds as in other animals' TIMP-2s. The predicted full-length amino acid sequence of jfTIMP-2a has lower identity to jfTIMP-2b (63%) than to those of human (74%) and chicken (73%) TIMP-2s, but higher than to those of other human TIMPs (TIMP-1: 39%, TIMP-3: 43%, TIMP-4: 45%), indicating that jfTIMP-2a is a common TIMP-2, while jfTIMP-2b is unique to Japanese flounder. However, the C-terminal region including the last three disulfide bonds of jfTIMP-2b has higher amino acid identity to those of other animal TIMP-2s than to that of jfTIMP-2a. Reverse-transcribed polymerase chain reaction (RT-PCR) analysis showed the mRNAs of jfTIMP-2a and jfTIMP-2b to be ubiquitously expressed in all tissues examined, but with different expression patterns. These findings suggest that the two distinct jfTIMP-2s might perform different functions in teleost tissues.     

Cloning and expression of an alpha-2,8-polysialyltransferase (STX) from Xenopus laevis

Polysialic acid (polySia) is a carbohydrate structure found on neural cell adhesion molecules (N-CAM). Two polysialyltransferases (polySiaTs) that catalyze synthesis of polySia have been described, and designated PST-1/PST/ST8SiaIV and STX/ST8SiaII. We cloned a polySiaT (xSTX) from a nonmammalian vertebrate, Xenopus laevis . xSTX had 80% amino acid similarity to the rat STX. This clone induced polySia expression when transfected into polySia-negative COS-1 cells. Northern blot analysis of whole embryos at different stages of development revealed that xSTX mRNA was most abundantly expressed in premetamorphic stages. The relative level of xSTX and N-CAM mRNAs was also examined and found to change in parallel to the extent of polysialylation on N-CAM. In adult tissues, the expression of xSTX mRNA was restricted to brain, eye and heart, which also expressed polySia. These results suggest that xSTX is the major enzyme responsible for the synthesis of polysialylated N-CAM in embryos at certain stages of development and also in adult tissues.      

Kindlin-2 expression in adult tissues correlates with their embryonic origins

Kindlin-2 functions in the maintenance of homeostasis and in human diseases. This study investigated the interrelationship between Kindlin-2 expression in tissues and the corresponding germ layers from which these tissues originated. Kindlin-2 expression was examined in normal adult human organs and human cancer tissues by immunohistochemical analyses. Analysis of Kindlin-2 mRNA levels in adult human organs in the Oncomine dataset revealed Kindlin-2 is highly expressed in mesoderm-derived organs. However, Kindlin-2 was negative or weakly expressed in endoderm/ectoderm-derived organs. Interestingly, the abnormal expression of Kindlin-2 was observed in a variety of human cancers. In agreement with its expression profile in humans, Kindlin-2 was also highly expressed in mesoderm-derived organs in mouse embryos with the exception of strong Kindlin-2 expression in ectoderm-derived spinal cord and ganglia, tissues that are highly mobile during embryonic development. Importantly, we demonstrated the expression level of Kindlin-2 in adult organs correlated with their embryonic dermal origins and deregulation of Kindlin-2 in tissues is associated with tumor progression. This finding will help us understand the dual role of Kindlin-2 in the regulation of tumor progression and embryonic development.     

cdx4/lacZ and cdx2/lacZ protein gradients formed by decay during gastrulation in the mouse

Expression of the mouse caudal genes cdx4 and cdx2 is examined by use of lacZ reporter constructs expressed in transgenic mouse embryos. During early gastrulation, up to at least 8.5 days of development, reporter mRNA distributions are apparently similar to those of endogenous cdx mRNAs. By 8.25 to 8.8 days, cdx/lacZ protein activities have become distributed as posterior-to-anterior gradients along the neural and mesoderm tissues. The gradients form by decay of activity as cells become distanced from the regressing tailbud. In situ hybridization studies indicate that the decay is primarily in cdx/lacZ protein activities rather than mRNAs. As gastrulation proceeds, the locations of the gradients regress progressively posteriorly along the growing axis. Our results indicate how cdx4 and cdx2 protein gradients might be generated by decay during normal development. The smoothness of the gradients that we detect shows that there cannot be extensive mixing of cells once they leave the tailbud to contribute to the growing axis. An enhancer element located in the first intron of the cdx4 gene is essential for correct transgene expression.     

Expression of matrix metalloproteinase -2, -9 and tissue inhibitors of metalloproteinase -1, -2, -3 mRNAs in rat uterus during early pregnancy

Zymography and in situ hybridizition were used to investigate matrix metalloproteinase-2, -9 (MMP-2, -9) activities, and expression of mRNAs for MMP-2, -9 and tissue inhibitors of matrix metalloproteinases (TIMP-1, -2, -3) in the rat uterus during early pregnancy (day 1-7). The zymography results showed two forms of MMP-2 (64 and 67 kDa) in the rat uteri during early pregnancy. The 64-kDa MMP-2 activity was the highest on day 2 (P < 0.01) and higher on day 5 and 6 (P < 0.05). The 67-kDa MMP-2 activity reached the highest on day 5 and 6 (P < 0.01). The 64-kDa MMP-2 activity at the implantation sites was higher than those at interimplantation sites (P < 0.05). Furthermore, the 67 kDa MMP-2 can be converted to 64 kDa forms by incubation with p-aminophenylmercuric acetate (APMA) and trypsin in vitro. The 92-kDa MMP-9 activity was only detected on day 5 and 6 of pregnancy (P < 0.01). In situ hybridization showed that on day 1-4 of pregnancy, both MMP-2 and TIMP-2 mRNAs were evidently localized in the basal stromal cells. On day 5, MMP-2 mRNA signals were decreased in the basal stromal cells and mRNA for TIMP-2 was expressed in the epithelial cells and subepithelial stromal cells. The mRNAs for MMP-9, TIMP-1, and -3 were mainly expressed in epithelial cells on day 1-5. At the implantation site on day 6, the mRNAs for MMP-2, -9, TIMP-1, -2, and -3 were highly expressed in the primary decidual zone surrounding the implanting embryo, and in the whole decidualized stromal cells (the primary and secondary decidual zones) at the implantation site on day 7. The intensities of mRNAs for the TIMPs in decidualized stromal cells at the implantation site on day 6 and 7 were stronger than those for the MMPs. The weak mRNAs for MMP-2, -9, TIMP-1, and -3 but not TIMP-2 were also observed in the ectoplacental cone/trophoblastic cells of the implanting embryos. However, at the interimplantation sites on day 6 and 7, MMP-2, -9, TIMP-1, -2, and -3 mRNAs were weakly expressed in the epithelial cells, subepithelial stromal cells, and myometrium. The results suggested that the implanting rat embryo strongly induced MMP-2 and -9 proteins and gene expression for decidulization and embryo invasion, which were strictly controlled and balanced by the simultaneous expression of TIMP-1, -2 and -3.     

Molecular cloning and expression of a second zebrafish aldehyde dehydrogenase 2 gene (aldh2b).

Aldehyde dehydrogenase 2 (ALDH2) is primarily responsible for detoxification of short-chain aldehydes in vivo. Previously it was reported that zebrafish has an aldh2 gene. Here we report the presence of a second aldh2 gene (aldh2b) in zebrafish. Zebrafish aldh2b locates adjacently to aldh2 on Chromosome 5 and the two genes share the same genomic organizations. aldh2b was predicted to encode a protein comprising 516 amino acids. The protein exhibits 95% amino acid identity with zebrafish ALDH2 and more than 76% identity with other vertebrate ALDH2s, respectively. Employing RT-PCR analysis, we demonstrated that both aldh2 and aldh2b mRNAs were present in embryos at cleavage stage (2 hpf: hour post fertilization) throughout protruding-mouth stage (72 hpf) and in different adult tissues of zebrafish. Taken together, our results reveal that zebrafish has two orthologues of aldh2 gene and the two genes share similar expression patterns during early development and in adult tissues.