MK──一种新发现的细胞因子

MK是一种新发现的细胞因子,属于肝素结合因子家族,MK是一种小分子多肽,其基因表达仅见于胚胎中期及成年期肾脏,在许多肿瘤细胞中也可见MK基因不同程度的表达,MK能够促进正常细胞的生长和分化,特别是促进神经细胞的发育,它还可以抑制某些肿瘤细胞的生长．MK基因在成年肾脏中表达的原因尚未阐明．     

A new family of heparin binding growth/differentiation factors: differential expression of the midkine (MK) and HB-GAM genes during mouse development.

MK (midkine) and HB-GAM (heparin-binding growth-associated molecule) constitute a new family of heparin-binding growth differentiation factors. The modes of expression of MK and HB-GAM during mouse development were quantitatively examined by mRNA hybridization. The following three distinct patterns of expression were observed in the brain/head region. On the 11th-13th days of gestation, MK was intensely, but HB-GAM relatively weakly expressed; on the 15th-19th days, both MK and HB-GAM expression became weaker; and in the neonatal period, HB-GAM was intensely expressed and MK expression increased slightly. The level of HB-GAM expression was lower than that of MK in the whole embryo on the 11th to 13th days of gestation. HB-GAM mRNA was detected in the kidney of newborn and young mice, where MK was more highly expressed. The identity of the weakly expressed MK and HB-GAM signals was confirmed by means of the polymerase chain reaction in the neonatal brain (MK), the head of 13-day embryos (HB-GAM), and the kidney of 7-day-old mice (HB-GAM). In conclusion, MK and HB-GAM are frequently co-expressed in the same cells and anatomic regions of the fetus or new born mouse, while their modes of expression differ.     

A 178-kb BAC transgene imprints the mouse Gtl2 gene and localizes tissue-specific regulatory elements

The regulation of genomic imprinting, the allele-specific expression of an autosomal gene, is complex and poorly understood. Imprinted genes are organized in clusters, where cis-acting regulatory elements are believed to interact to control multiple genes. We have used BAC transgenesis in the mouse to begin to delineate the region of DNA required for proper expression and imprinting of the mouse Delta-like1 (Dlk1) and Gene-trap locus2 (Gtl2) imprinted genes. We demonstrate that the Gtl2 gene is expressed from a BAC transgene in mouse embryo and placenta only upon maternal inheritance, as is the endogenous Gtl2 gene. Gtl2 is therefore properly imprinted on the BAC in an ectopic chromosomal location and must carry with it all necessary imprinting regulatory elements. Furthermore, we show that the BAC Gtl2 gene is expressed at levels approaching those of the endogenous gene only in the brain of adult animals, not in other sites of endogenous expression such as the pituitary, adrenal, and skeletal muscle. These data localize the enhancer(s) for brain Gtl2 expression, but not those for other tissues, to the DNA contained within the BAC clone. As the Dlk1 gene is not expressed from the BAC in any tissues, it must require additional elements that are different from those necessary for Gtl2 expression. Our data refine the interval for future investigation of Gtl2 imprinting and provide evidence for distinct regulation of the linked Dlk1 and Gtl2 genes.     

N-myc proto-oncogene expression during organogenesis in the developing mouse as revealed by in situ hybridization

The N-myc proto-oncogene is expressed during embryogenesis, suggesting that it plays a role in normal development. Since the myc-family oncogenes have been implicated in the control of cell growth, the embryonic expression may reflect rapid proliferation known to occur in development. Alternatively, N-myc expression may be involved in specific differentiation stages. In many embryonic tissues, early and late differentiation events occur in different locations. By in situ hybridization of tissue sections, we now demonstrate a restricted expression of N-myc mRNA to a few tissues and to areas where the first differentiation stages occur. N-myc expression was most strongly expressed in the developing kidney, hair follicles, and in various parts of the central nervous system. In these tissues, expression was restricted to a few cell lineages. In all lineages, expression was confined to early differentiation stages, and, at onset of overt differentiation, the level of expression decreased dramatically. Several rapidly proliferating tissues showed very little, if any, N-myc expression. In the brain, post-mitotic but not yet differentiated cells expressed high levels of N-myc mRNA. Therefore, N-myc expression is not a simple marker for proliferation in the embryo. Rather, N-myc expression seems to be a feature of early differentiation stages of some cell lineages in kidney, brain, and hair follicles, regardless of the proliferative status of the cell. The results raise the possibility that N-myc may participate in the control of these early differentiation events.     

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.     

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.     

Transiently truncated and differentially regulated expression of midkine during mouse embryogenesis

Midkine (MK) is a retinoic acid response cytokine, mostly expressed in embryonic tissues. Aberrant expression of MK was found in numerous cancers. In human, a truncated MK was expressed specifically in tumor/cancer tissues. Here we report the discovery of a novel truncated form of MK transiently expressed during normal mouse embryonic development. In addition, MK is concentrated at the interface between developing epithelium and mesenchyme as well as highly proliferating cells. Its expression, which is closely coordinated with angiogenesis and vasculogenesis, is spatiotemporally regulated with peaks in extensive organogenesis period and undifferentiated cells tailing off in maturing cells, implying its role in nascent blood vessel (endothelial) signaling of tissue differentiation and stem cell renewal/differentiation. Cloning and sequencing analysis revealed that the embryonic truncated MK, in which the conserved domain is in-frame deleted, presumably producing a novel secreted small peptide, is different from the truncated form in human cancer tissues, whose deletion results in a frame-shift mutation. Our data suggest that MK may play a role in epithelium-mesenchyme interactions, blood vessel signaling, and the decision of proliferation vs differentiation. Detection of the transiently expressed truncated MK reveals its novel function in development and sheds light on its role in carcinogenesis.     

Dorsoventral patterning of the C. elegans postembryonic mesoderm requires both LIN-12/Notch and TGFbeta signaling

The heparin binding molecules MK and HB-GAM are involved in the regulation of growth and differentiation of many tissues and organs. Here we analyzed the expression of MK and HB-GAM in the developing mouse incisors, which are continuously growing organs with a stem cell compartment. Overlapping but distinct expression patterns for MK and HB-GAM were observed during all stages of incisor development (initiation, morphogenesis, cytodifferentiation). Both proteins were detected in the enamel knot, a transient epithelial signaling structure that is important for tooth morphogenesis, and the cervical loop where the stem cell niche is located. The functions of MK and HB-GAM were studied in dental explants and organotypic cultures in vitro. In mesenchymal explants, MK stimulated HB-GAM expression and, vice-versa, HB-GAM upregulated MK expression, thus indicating a regulatory loop between these proteins. BMP and FGF molecules also activated expression of both cytokines in mesenchyme. The proliferative effects of MK and HB-GAM varied according to the mesenchymal or epithelial origin of the tissue. Growth, cytodifferentiation and mineralization were inhibited in incisor germs cultured in the presence of MK neutralizing antibodies. These results demonstrate that MK and HB-GAM are involved in stem cells maintenance, cytodifferentiation and mineralization processes during mouse incisor development.     

Pleiotrophin gene expression is highly restricted and is regulated by platelet-derived growth factor.

Pleiotrophin (PTN) is a growth and neurite extension promoting polypeptide which is highly expressed in brain and in tissues derived from mesenchyme. The PTN gene is developmentally regulated and is closely related to the MK and RI-HB genes, both of which are developmentally regulated and induced by retinoic acid. We now have screened 17 cell lines and report that expression of the PTN gene in these cells is restricted to embryo fibroblasts and intestinal smooth muscle cells. However, NIH 3T3 cells stimulated by the platelet-derived growth factor (PDGF) express a marked increase in levels of PTN mRNA whereas retinoic acid failed to increase levels of PTN mRNA in NIH 3T3 cells or in F9 embryonal carcinoma cells within 72 hours of exposure. The results suggest that expression of the PTN gene is highly restricted and that the PTN gene is a new member of the PDGF-induced cytokine family.     

Identification of chicken embryo kinase 5, a developmentally regulated receptor-type tyrosine kinase of the Eph family.

Chicken embryo kinase 5 (Cek5) is a transmembrane tyrosine kinase of the Eph family that was identified by screening a 10-d chicken embryo cDNA expression library with anti-phosphotyrosine antibodies. The extracellular region of Cek5 contains a cysteine rich N-terminal subdomain and a C-terminal subdomain mostly devoid of cysteines and comprising two repeats similar to fibronectin type III repeats. Immunoblotting experiments with anti-Cek5 polyclonal antibodies indicated that Cek5 is a membrane-associated 120-kDa protein containing intramolecular (but not intermolecular) disulfide bonds. Cek5 is already expressed in 2-d-old chicken embryos and is also expressed, at higher levels, later in development. In 10-d-old chicken embryos, Cek5 is expressed at substantial levels in nearly all the tissues examined, whereas in adult it is expressed predominantly in the brain. The expression of Cek5 in the brain gradually diminishes during embryonic development, whereas in the skeletal muscle of the thigh a sharp decrease in Cek5 expression was detected at the time of terminal muscle differentiation. Its wide tissue distribution throughout development and its sustained expression in adult brain suggest that Cek5 is an important component of signal transduction pathways, likely to interact with a widely distributed and important ligand, which is as yet unknown.     

骨髓间充质干细胞和部分肿瘤细胞中Nucleostemin基因的表达

以分离的人胚胎和大鼠骨髓间充质干细胞 (MSCs) ,6种肿瘤细胞株 ,裸鼠肿瘤和转移瘤组织为实验材料 ,以大鼠心肌组织和人胎盘组织为对照 ,探讨nucleostemin基因的表达情况 .RT PCR结果显示 ,nucleostemin基因在MSCs、肿瘤细胞和肿瘤组织中均有不同程度的表达 ,而大鼠心肌和人胎盘组织中无表达 .DNA测序结果证明 ,扩增的PCR产物与GenBank提供的DNA序列完全同源 .SCID裸鼠肿瘤动物模型定量PCR结果证实 ,nucleostemin的mRNA在裸鼠肿瘤组织和转移瘤组织中表达较高 .研究结果表明 ,在细胞中nucleostemin基因不同水平的表达可能与MSCs、肿瘤细胞的增殖和肿瘤的发生、发展与转移有关 .     

The Loss of the Expression of α Catenin, the 102 kD Cadherin Associated Protein, in Central Nervous Tissues during Development

A monoclonal antibody specific for α catenin, the 102kD cadherin-associated protein, has been characterized and used to describe the expression and distribution pattern of α catenin in adult mice and mouse embryos. This monoclonal antibody recognized an epitope in the middle part of the α catenin molecule of various vertebrate species, and bound to neither vinculin nor αN catenin, which are cytoskeletal proteins with sequence similarity to α catenin. At the early mouse embryo stage (neurulae stage) α catenin was expressed and concentrated at cell-to-cell contact sites together with various types of cadherins in all tissues. In embryos at 12.5 days of gestation, the α catenin expression was gradually diminished selectively in central nervous tissues such as brain and spinal cord, and in most of the adult central nervous tissues the α catenin expression was hardly detected. In adult non-nervous tissues most of the cells examined expressed α catenin. Especially in well-polarized tissues such as epithelial cells, α catenin appeared to be highly concentrated at cell-to-cell adherens junctions where cadherins act as adhesion molecules.
This loss of α catenin expression in central nervous tissues was observed not only in mice but also in other vertebrate species such as fish and newt, suggesting that this phenomenon has important implications from the view point of nervous tissue development.