A retinoic acid-responsive gene, MK, found in the teratocarcinoma system. Heterogeneity of the transcript and the nature of the translation product

A newly identified gene, MK, is transiently expressed in the early stages of embryonal carcinoma cell differentiation and in the mid-gestation period of mouse embryogenesis (Kadomatsu, K., Tomomura, M., and Muramatsu, T. (1988) Biochem. Biophys. Res. Commun. 151, 1312-1318). Analysis of various MK cDNA clones revealed differences in the 5'-region. So far three classes of cDNA clones (MK1, MK2, and MK3) have been identified; they were different in the 5'-untranslated region but shared the rest of the sequence. Ribonuclease protection, RNA blotting, and primer extension revealed that MK2-type RNA was the major MK RNA in retinoic acid-treated embryonal carcinoma cells. In addition, the number of A residues in an oligo(A) stretch in the 5'-side of the common sequence differed from 9 to 29. The number was 9 in the most frequent cases, when the putative MK polypeptide had a molecular weight of about 15,500 and had a signal peptide-like sequence. Hybrid selected MK RNA yielded the predicted polypeptide upon in vitro translation. When pancreatic microsomal membranes were included in the translation system, the translation product of MK RNA was processed and entered into the lumen of the membranes. These results suggest that the product of the MK gene is an extracellular polypeptide.     

Effects of Different Conditions of Retinoic Acid Treatment on Expression of MK Gene,which is Transiently Activated during Differentiation of Embryonal Carcinoma Cells1

MK gene was intensely expressed, when aggregates of HM-1 embryonal carcinoma (EC) cells were treated with retinoic acid for 2 days to induce the differntiation to nerve cells, myoblasts and extraembryonic endoderm cells. The conditions inhibiting nerve cell diffrentiation or extraembryonic endoderm cell differentiation affected MK gene expression only slightly. The maximum level of MK RNA was detected 2 days after initiation of retionic acid treatment, when cells were morphologically indistinguishable from undifferentiated EC cells. Thus, MK gene appears to be expressed in differentiating EC cells irrespective of the direction of differentiation. The degree of MK gene expression in sparsely cultured HM-1 cells correlated with the concentration of retinoic acid, especially between 10^-8 and 10^-7 M. When retinoic acid treatment was terminated after 1 day, the amount of MK RNA started to decrease. These two results are consistent with the view that retionic acid complexed with the receptor is directly involved in expression of MK gene.     

Structure of a retinoic acid-responsive gene, MK, which is transiently activated during the differentiation of embryonal carcinoma cells and the mid-gestation period of mouse embryogenesis

MK is a gene that is expressed temporarily during the early stages of retinoic acid-induced differentiation of embryonal carcinoma (EC) cells and during the mid-gestation period of mouse embryogenesis. The 5'-regions of MK cDNAs and their mRNAs are heterogeneous; so far three kinds of MK cDNAs (MK1, MK2, and MK3) have been isolated. The MK gene was cloned from a genomic DNA library of a BALB/c mouse, and its structure was elucidated. 5'-Region sequences specific for MK1, MK2, and MK3 were arranged in the order of MK3, MK2, and MK1. Then, there was a sequence common to all MK cDNAs consisting of four exons. The results indicate that different species of MK mRNA are generated by the use of alternative promoters and different modes of splicing.     

Midkine (MK), a heparin-binding growth/differentiation factor, is regulated by retinoic acid and epithelial-mesenchymal interactions in the developing mouse tooth, and affects cell proliferation and morphogenesis

Midkine (MK) is the first cloned gene in a new family of heparin- binding growth/differentiation factors involved in the regulation of growth and differentiation. We have analyzed the expression of MK mRNA and protein during tooth development in mouse embryos and studied the regulation of MK expression and the biological effects of MK protein in organ cultures. MK expression was restricted and preferential in the tooth area as compared to the rest of the developing maxillary and mandibular processes suggesting specific functions for MK during tooth morphogenesis. MK mRNA and protein were expressed during all stages of tooth formation (initiation, morphogenesis, and cell differentiation), and shifts of expression were observed between the epithelial and mesenchymal tissue components. However, the expression of mRNA and protein showed marked differences at some stages suggesting paracrine functions for MK. Tissue recombination experiments showed that MK gene and protein expression are regulated by epithelial-mesenchymal interactions, and, moreover, that dental tissue induces the ectopic expression of MK protein in non-dental tissue. The expression of MK gene and protein in the mandibular arch mesenchyme from the tooth region were stimulated by local application of retinoic acid in beads. Cell proliferation was inhibited in dental mesenchyme around the beads releasing MK, but this effect was modulated by simultaneous application of FGF-2. Morphogenesis and cell differentiation were inhibited in tooth germs cultured in the presence of neutralizing antibodies for MK, whereas the development of other organs (e.g., salivary gland, kidney) was unaffected. These results suggest important roles for MK in the molecular cascade that regulates tooth development.     

MK表达载体的构建及在大肠杆菌中的表达

MK是属于肝素结合因子家族的一种多肽,仅在胚胎中期和成年期肾脏表达,在某些肿瘤细胞中也有异常表达.MK能够促进细胞特别是神经细胞的生长和分化,并抑制某些肿瘤细胞的生长.通过RT-PCR从胚胎肾脏中获得了MK成熟肽DNA编码序列,克隆入载体pBV221中,并转入大肠杆菌,建立了重组MK的高表达菌株.     

Biphasic Pattern of Gelsolin Expression and Variations in Gelsolin-Actin Interactions during Myogenesis

During myogenesis in vitro, the amount of gelsolin in myogenic cells increased by a factor of 3 from about 200 ng to a maximum of 750 ng per milligram of total protein. Gelsolin increased steadily from the myoblast state to terminally differentiated myotubes containing abundant cross-striated myofibrils. At the same time, the amount of total actin varied by only about 30%, the molar ratio of gelsolin:actin increased from 1:500 to approximately 1:150. This modulation of gelsolin expression was observed both in avian and mammalian myocultures. Once the state of terminal differentiation in myocultures was attained, the amount of gelsolin decreased again. On the other hand, gelsolin decreased continuously in the postnatal mouse muscle by a factor of 5 between Day 1 and Day 12 after birth. When myogenic cells from various stages of differentiation were extracted with Triton X-100, the majority of gelsolin was soluble, whereas a minor fraction was tightly associated with the cytoskeleton. The actual amount of insoluble gelsolin depended on both the Ca²⁺ concentration during extraction and the degree of differentiation. Whereas at [Ca²¹] > 10⁵M about one-third of the total gelsolin was associated with the cytoskeleton at all stages of differentiation, the amount of insoluble gelsolin after Triton extraction in the presence of EGTA increased from 3 to 17% during differentiation. The amount of soluble actin decreased from 40 to 25% during the same period, independent of the Ca²⁺ concentration. We calculated that the amount of gelsolin associated with the cytoskeletal or myofibrillar system is approximately 20-fold higher in differentiated myotubes than in early myotubes, indicating a functional role of gelsolin for myofibrillar assembly.     

NADPH oxidase and eNOS control cardiomyogenesis in mouse embryonic stem cells on ascorbic acid treatment

Ascorbic acid (AA) increases cardiomyogenesis of embryonic stem (ES) cells. Herein we show that treatment of mouse ES cells with AA enhanced cardiac differentiation accompanied by an upregulation of the NADPH oxidase isoforms NOX2 and NOX4, phosphorylation of endothelial nitric oxide synthase (eNOS), and cyclic GMP (cGMP) formation, indicating that reactive oxygen species (ROS) as well as nitric oxide (NO) may be involved in cardiomyogenesis. In whole mount embryoid bodies as well as isolated Flk-1-positive (Flk-1⁺) cardiovascular progenitor cells ROS elevation by AA was observed in early stages of differentiation (Days 4-7), and absent at Day 10. In contrast NO generation following incubation with AA was absent at Day 4 and increased at Days 7 and 10. AA-mediated cardiomyogenesis was blunted by the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin, the free radical scavengers N-(2-mercaptopropionyl)-glycine (NMPG) and ebselen, and the NOS inhibitor L-NAME. Downregulation of NOX4 by short hairpin RNA (shRNA) resulted in significant inhibition of cardiomyogenesis and abolished the stimulation of MHC-ß and MLC2v gene expression observed on AA treatment. Our data demonstrate that AA stimulates cardiomyocyte differentiation from ES cells by signaling pathways that involve ROS generated at early stages and NO at late stages of cardiomyogenesis.     

In situ hybridization analysis of the temporospatial expression of the midkine/pleiotrophin family in rat embryonic pituitary gland

Pituitary gland development is controlled by numerous signaling molecules, which are produced in the oral ectoderm and diencephalon. A newly described family of heparin-binding growth factors, namely midkine (MK)/pleiotrophin (PTN), is involved in regulating the growth and differentiation of many tissues and organs. Using in situ hybridization with digoxigenin-labeled cRNA probes, we detected cells expressing MK and PTN in the developing rat pituitary gland. At embryonic day 12.5 (E12.5), MK expression was localized in Rathke’s pouch (derived from the oral ectoderm) and in the neurohypophyseal bud (derived from the diencephalon). From E12.5 to E19.5, MK mRNA was expressed in the developing neurohypophysis, and expression gradually decreased in the developing adenohypophysis. To characterize MK-expressing cells, we performed double-staining of MK mRNA and anterior pituitary hormones. At E19.5, no MK-expressing cells were stained with any hormone. In contrast, PTN was expressed only in the neurohypophysis primordium during all embryonic stages. In situ hybridization clearly showed that MK was expressed in primitive (immature/undifferentiated) adenohypophyseal cells and neurohypophyseal cells, whereas PTN was expressed only in neurohypophyseal cells. Thus, MK and PTN might play roles as signaling molecules during pituitary development.     

玉米花粉胚状体发育过程中DNA,RNA蛋白质含量及合成动态变化

继代培养的玉米花粉胚状体的发育过程可划分为6个时期:胚性细胞团时期、球形胚时期、心形胚时期、梨形胚时期、子叶形胚时期以及分化期。我们应用微量生化分析技术以及放射性同位素液体闪烁计数技术研究了玉米花粉胚状体发育过程的DNA、RNA、蛋白质含量及合成动态,发现DNA、RNA和蛋白质含量在胚性细胞团期较高,然后下降,但到了分化期时又有所升高。DNA合成速度在胚性细胞团时期较高,在以后的各时期降低并保持平稳。RNA和蛋白质的合成动态呈相似的变化规律。这个结果说明DNA、RNA和蛋白质在胚状体发育早期的活跃代谢,可能与胚性细胞的快速分裂以及胚性结构的形成有关,而后期的活跃代谢可能与胚状体的分化有关。     

Modulation of activities and RNA level of the components of the plasminogen activation system during fusion of human myogenic satellite cells in vitro.

Primary cultures of human myogenic stem cells (satellite cells) mimic myogenic differentiation. During this process, the expression of the components of the plasminogen activation system underwent modulation. Activities and mRNA levels of tissue-type and urokinase-type plasminogen activator were increased in a reproducible pattern during differentiation. A modulation of the mRNA level of PAI-2 was also observed. Human satellite cells expressed a urokinase receptor and also the mRNA level of this component underwent modulation. With the exception of PAI-1 mRNA, the level of all mRNAs increased from Day 4 to Day 8, i.e., just before myoblasts fusion, and then remained high at later stages. The modulation of the plasminogen activating activity indicates that this system is directly involved in the fusion process of myogenic differentiation.     

Immunohistochemical localization of truncated midkine in developing human bile ducts

Midkine (MK) is a heparin-binding growth factor whose gene has been identified in embryonal carcinoma cells in early stages of retinoic acid-induced differentiation. In the present study, we investigated the developmental localization of truncated MK protein in human bile ducts. Thirty specimens of the livers from 25 fetuses (from 9 to 40 gestational weeks) and from five neonates less than 4 weeks old were examined. Immunohistochemical analysis was performed using a mouse IgG2b monoclonal antibody against recombinant-truncated MK. Truncated MK was expressed moderately in the fetal liver from 9 to 15 gestational weeks. The immunoreactivities were found in the primitive hepatocytes, ductal plates, migrating biliary cells and immature bile ducts. The reaction products were localized in the cytoplasm heterogeneously. The intensity of immunostaining was weak from 15 gestational weeks to 26 gestational weeks. After 27 gestational weeks, truncated MK was not detected in the fetal livers. It was suggested that primitive hepatocytes, ductal plates and immature bile ducts produced truncated MK transiently during human bile ducts development.     

Changes in concentrations of follicular inhibin alpha and beta A subunit messenger ribonucleic acids and inhibin immunoactivity during preovulatory maturation in the pig.

Ovarian tissues were collected from 5 pigs on each of days 1, 3, 5, and 7 after withdrawal of an orally active progestin to determine changes in follicular inhibin subunit mRNAs during preovulatory maturation. Follicles (N = 146) were aspirated for fluid and homogenized in guanidinium isothiocyanate for RNA isolation. Follicular RNA and inhibin alpha and beta A subunit mRNA standards were dot-blotted, hybridized with [32P]-cDNA probes, and quantified by densitometry. Mean concentrations of alpha mRNA (pg/micrograms of RNA) increased (p < or = 0.05) by 140% as healthy follicles grew from medium (3-5 mm) to large (> 5 mm). Inhibin immunoactivity was greater (p < or = 0.05) in large than medium follicles. In contrast, mean concentrations of inhibin beta A subunit mRNA did not differ between healthy medium and large follicles. However, both alpha mRNA and beta A mRNA increased (p < or = 0.05) linearly as follicular diameter increased from 3 to 5 mm on Day 1 and from 3 to 9 mm on Day 3. On Day 5, alpha mRNA remained elevated, but was not significantly correlated with diameter. In contrast, beta A mRNA decreased linearly (p < 0.05) as diameter increased from 6 to 11 mm on Day 5. The molar ratio alpha mRNA to beta A mRNA was 20:1 in healthy, large follicles on Days 3 and 5. Mean concentration of alpha mRNA in large follicles decreased (p < 0.05) by 72% between Days 5 and 7, while beta A mRNA decreased to non-detectable levels on Day 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequential metabolic events and morphological changes during in vivo large granular lymphocyte activation and proliferation

Interferon (IFN) and IFN inducers are known to boost natural killer (NK) activity in vivo and in vitro. In vivo enhancement of NK activity results from activation of preexisting NK cells as well as from an increased number of large granular lymphocytes (LGL), with a portion of them undergoing cell division. Our study was addressed to analyze the sequence of metabolic events occurring within the LGL population of Fischer rats treated with poly(I:C), as an IFN inducer. The increase in cytotoxic activity and LGL number in the peripheral blood already reached maximal levels by 12 hr after poly(I:C) injection, remained on a plateau 24 to 48 hr later, then slightly decreased on Day 4, and returned to control levels by Day 6. A similar kinetics was observed for RNA synthesis. In contrast DNA synthesis first increased at 24 hr, peaked at 48 hr, then decreased on Day 4, and was not detectable on Day 6. Percoll fractionation resulted in 92-97% of LGL in fraction 1, and cells in this fraction accounted for the increase of cytotoxicity as well as for newly synthesized RNA and DNA. However, LGL recovered on Day 1 or 2 after poly(I:C) stimulation displayed quite heterogeneous morphology, and a number of mitotic configurations were seen on Day 2 within the LGL population. Our results indicate that the boosting of NK activity by poly(I:C) is always associated with an increase in LGL numbers, the enhanced lytic capacity is associated in vivo with new RNA synthesis by the NK cells, and only in a later phase NK cell proliferation may account for the increase in LGL numbers.     

Differential expression of keratin genes during mouse development

Suprabasal layers of the newborn mouse epidermis contain two mRNAs of 2.0 and 2.4 kb which are translated into keratins of 59 and 67 kDa, respectively. To study their expression during development, cDNA sequences corresponding to the 2.0- and the 2.4-kb mRNAs were cloned, characterized by hybridization selection assay, and used as probes to detect keratin sequences in polyadenylated RNA from Day 11, 13, 15, and 17 embryos. In RNA from Day 11 of gestation, two RNAs of 2.8 and 1.8 kb were identified. They were found to have homologies with both epidermal RNAs, suggesting that they are coding for proteins of the keratin family. These two sequences were not detected in sample of later stages. RNAs comigrating with the two epidermal keratin RNAs were identified only in Day 15 and 17 embryos indicating that their expression was induced between Day 13 and 15. Finally, the localization of the 59-kDa keratin mRNA was examined by in situ hybridization. The spinous and granulous cell layers were found to be heavily covered with grains while other regions of the tissue sections were unlabeled. All these results support the hypothesis of a sequential expression of keratins during differentiation of epidermal cells and suggest that proteins related to the keratins expressed specifically in keratinizing cells are expressed earlier during development.     

ROS-mediated platelet generation: a microenvironment-dependent manner for megakaryocyte proliferation,differentiation, and maturation

Platelets have an important role in the body because of their manifold functions in haemostasis, thrombosis, and inflammation. Platelets are produced by megakaryocytes (MKs) that are differentiated from haematopoietic stem cells via several consecutive stages, including MK lineage commitment, MK progenitor proliferation, MK differentiation and maturation, cell apoptosis, and platelet release. During differentiation, the cells migrate from the osteoblastic niche to the vascular niche in the bone marrow, which is accompanied by reactive oxygen species (ROS)-dependent oxidation state changes in the microenvironment, suggesting that ROS can distinctly influence platelet generation and function in a microenvironment-dependent manner. The objective of this review is to reveal the role of ROS in regulating MK proliferation, differentiation, maturation, and platelet activation, thereby providing new insight into the mechanism of platelet generation, which may lead to the development of new therapeutic agents for thrombocytopenia and/or thrombosis.