Comparative proteomic analysis of proteins involved in cell aggregation during neural differentiation of P19 mouse embryonic carcinoma cells.

Cell-cell interactions play a crucial role during embryogenesis and are enhanced during cell aggregation. P19 mouse embryonic carcinoma cells can differentiate into neural cells by the addition of retinoic acid (RA) or by overexpression of the Wnt1 gene, with both processes dependent on cell aggregation. To identify molecules involved in the cell aggregation process, two-dimensional gel electrophoresis (2DE) was used to establish the cell aggregation-associated protein profiles. MALDI-TOF/TOF was used to identify 71 protein spots with differential expression patterns. Among these spots, 54 were differentially expressed in both P19 and Wnt1-overexpressing P19 (Wnt1/P19) cell aggregates, with 42 proteins up-regulated and 12 proteins down-regulated. The other 17 spots were differentially expressed only in Wnt1/P19 cells. The expression patterns of 5 cell aggregation-associated proteins, N-myc downstream-regulated gene 1 (NDRG1), 14-3-3 epsilon, 14-3-3 gamma, acid calponin and cell division control protein 2 homologue (Cdc2), were confirmed by immunoblot and RT-PCR. To further investigate the relationship between cell aggregation and neural differentiation, NDRG1 expression was inhibited by RNA interference during P19 cell aggregation. Silencing of NDRG1 reduced the size of cell aggregates and the expression of N-cadherin, and it also impaired the RA-induced P19 cell neural differentiation. In conclusion, this study provides new clues for the possible mechanism underlying cell aggregation during pluripotent stem cell neural differentiation.

Synergistic function of DNA methyltransferases Dnmt3a and Dnmt3b in the methylation of Oct4 and Nanog.

DNA methylation plays an important role in gene silencing in mammals. Two de novo methyltransferases, Dnmt3a and Dnmt3b, are required for the establishment of genomic methylation patterns in development. However, little is known about their coordinate function in the silencing of genes critical for embryonic development and how their activity is regulated. Here we show that Dnmt3a and Dnmt3b are the major components of a native complex purified from embryonic stem cells. The two enzymes directly interact and mutually stimulate each other both in vitro and in vivo. The stimulatory effect is independent of the catalytic activity of the enzyme. In differentiating embryonic carcinoma or embryonic stem cells and mouse postimplantation embryos, they function synergistically to methylate the promoters of the Oct4 and Nanog genes. Inadequate methylation caused by ablating Dnmt3a and Dnmt3b is associated with dysregulated expression of Oct4 and Nanog during the differentiation of pluripotent cells and mouse embryonic development. These results suggest that Dnmt3a and Dnmt3b form a complex through direct contact in living cells and cooperate in the methylation of the promoters of Oct4 and Nanog during cell differentiation. The physical and functional interaction between Dnmt3a and Dnmt3b represents a novel regulatory mechanism to ensure the proper establishment of genomic methylation patterns for gene silencing in development.

SNAP-25 in hippocampal CA3 region is required for long-term memory formation.

SNAP-25 is a synaptosomal protein of 25 kDa, a key component of synaptic vesicle-docking/fusion machinery, and plays a critical role in exocytosis and neurotransmitter release. We previously reported that SNAP-25 in the hippocampal CA1 region is involved in consolidation of contextual fear memory and water-maze spatial memory (Hou et al. European J Neuroscience, 20: 1593-1603, 2004). SNAP-25 is expressed not only in the CA1 region, but also in the CA3 region, and the SNAP-25 mRNA level in the CA3 region is higher than in the CA1 region. Here, we provide evidence that SNAP-25 in the CA3 region is also involved in learning/memory. Intra-CA3 infusion of SNAP-25 antisense oligonucleotide impaired both long-term contextual fear memory and water-maze spatial memory, with short-term memory intact. Furthermore, the SNAP-25 antisense oligonucleotide suppressed the long-term potentiation (LTP) of field excitatory post-synaptic potential (fEPSP) in the mossy-fiber pathway (DG-CA3 pathway), with no effect on paired-pulse facilitation of the fEPSP. These results are consistent with the notion that SNAP-25 in the hippocampal CA3 region is required for long-term memory formation.

Second intron of mouse nestin gene directs its expression in pluripotent embryonic carcinoma cells through POU factor binding site.

Nestin, an intermediate filament protein, is expressed in the neural stem cells of the developing central nervous system. This tissue-specific expression is driven by the neural stem cell-specific enhancer in the second intron of the nestin gene. In this study, we showed that the mouse nestin gene was expressed in pluripotent embryonic carcinoma (EC) P19 and F9 cells, not in the differentiated cell types. This cell type-specific expression was conferred by the enhancer in the second intron. Mutation of the conserved POU factor-binding site in the enhancer abolished the reporter gene expression in EC cells. Oct4, a Class V POU factor, was found to be coexpressed with nestin in EC cells. Electrophoretic mobility-shift assays and supershift assays showed that a unique protein-DNA complex was formed specifically with nuclear extracts of EC cells, and Oct4 protein was included. Together, these results suggest the functional relevance between the conserved POU factor-binding site and the expression of the nestin gene in pluripotent EC cells.

The negative cell cycle regulator, Tob (transducer of ErbB-2), is involved in motor skill learning.

Tob (transducer of ErbB-2) is a negative cell cycle regulator with anti-proliferative activity in peripheral tissues. Our previous study identified Tob as a protein involved in hippocampus-dependent memory consolidation (M.L. Jin, X.M. Wang, Y.Y. Tu, X.H. Zhang, X. Gao, N. Guo, Z.Q. Xie, G.P. Zhao, N.H. Jing, B.M. Li, Y.Yu, The negative cell cycle regulator, Tob (Transducer of ErbB-2), is a multifunctional protein involved in hippocampus-dependent learning and memory, Neuroscience 131 (2005) 647-659). Here, we provide evidence that Tob in the central nervous system is engaged in acquisition of motor skill. Tob has a relatively high expression in the cerebellum. Tob expression is up-regulated in the cerebellum after rats receive training on a rotarod-running task. Rats infused with Tob antisense oligonucleotides into the 4th ventricle exhibit a severe deficit in running on a rotating rod or walking across a horizontally elevated beam.

Moderate hypothermia prevents neural cell apoptosis following spinal cord ischemia in rabbits.

Paraplegia is a disastrous complication after operations of descending and thoracoabdominal aortic aneurysm. Regional hypothermia protects against spinal cord ischemia although the protective mechanism is not well know. The objective of this study is to examine whether hypothermia protects the spinal cord by preventing apoptosis of nerve cell and also investigate a possible mechanism involved in hypothermia neuroprotection. Cell apoptosis with necrosis was evident in the spinal cord 24 h after 30 min of ischemia. Moderate hypothermia decreased the incidence of apoptotic nerve cells. Both cell apoptosis and necrosis were attenuated by hypothermia. p53 expression increased and bcl-2 expression declined after ischemia, while hypothermia mitigated these changes. This study suggests that apoptosis contributes to cell death after spinal cord ischemia, and that moderate hypothermia can prevent nerve cell apoptosis by a mechanism associated with bcl-2 and p53 genes.

Differential display of proteins involved in the neural differentiation of mouse embryonic carcinoma P19 cells by comparative proteomic analysis.

Mouse embryonic carcinoma P19 cell has been used extensively as a model to study molecular mechanisms of neural differentiation in vitro. After retinoic acid (RA) treatment and aggregation, P19 cells can differentiate into neural cells including neurons and glial cells. In this study, comparative proteomic analysis is utilized to approach the protein profiles associated with the RA-induced neural differentiation of P19 cells. Image analysis of silver stained two-dimensional gels indicated that 28 protein spots had significantly differential expression patterns in both quantity and quality. With mass spectrometry analysis and protein functional exploration, many proteins demonstrated an association with distinct aspects of neural differentiation. These proteins were gag polyprotein, rod cGMP-specific 3',5'-cyclic phosphodiesterase, 53 kDa BRG1-associated factor A, N-myc downstream regulated 1, Vitamin D receptor associated factor 1, stromal cell derived factor receptor 1, phosphoglycerate mutase, Ran-specific GTPase-activating protein, and retinoic acid (RA)-binding protein. While some cytoskeleton-related proteins such as beta cytoskeletal actin, gamma-actin, actin-related protein 1, tropomyosin 1, and cofilin 1 are related to cell migration and aggregation, other proteins have shown a relationship with distinct aspects of neural differentiation including energy production and utilization, protein synthesis and folding, cell signaling transduction, and self-protection. The differential expression patterns of these 28 proteins indicate their different roles during the neural differentiation of P19 cells. As an initial step toward unveiling the regulations involved in the commitment of pluripotent cells to a neural fate, information from this study may be helpful to uncover the molecular mechanisms of neural differentiation.

[Directed differentiation embryonic stem cells into the chondrocyte].

Cartilage is a tough, elastic, and fibrous connective tissue, composed of chondrocyte and extracellular matrix (ECM) structural entity. Because of the poor self-regeneration capabilities of chondrocytes, cell transplantation is necessanry once the cartilage is damaged. Traditionally autologous chondrocytes implantation is applied at the damage site, which has many problems such as the losing of its primary characteristics. A well-defined and efficient protocol to direct the differentiation of embryonic stem cells into the chondrocyte will provide a novel choice for cartilage repair. This article reviews some major progresses in this field.

Identification and characterization of a rat novel gene RSEP4 expressed specifically in central nervous system.

The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length of RSEP4 cDNA is 2006 bp, with a 501 nucleotide open reading frame (ORF) that encodes a 167 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1, CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

[Overexpression of STAT3 in COS7 cells causes prominent morphological changes].

Signal transducer and activator of transcription 3 (STAT3) plays a central role in mediating signals related to cell growth, differentiation, survival and movement. In this report, the results of immunocytochemistry and Western blot analysis showed that the active form of Stat3 protein existed in COS7 cells. The endogenous Stat3 protein could also induce the expression of m67-sequence-directed reporter genes. The transient transfection of Stat3 cDNA into COS7 cells increased Stat3 protein level and the expression of the reporter gene. The overexpression of Stat3 protein in COS7 cells caused prominent cell morphological changes. These cells had a much larger cell body, extended long processes with branches, lamellipodia and filopodia. These results suggest that Stat3 protein may play important roles in cell adhesion, migration and cytoskeleton reorganization.

Functional expression of guinea pig growth hormone receptor and its mutants in mammalian cells.

The cDNA of guinea pig (Cavia porcellus) growth hormone receptor (gpGHR) was cloned using RT-PCR in our laboratory. By sequence alignment, substitutions of amino acids conserved in other mammalian GHRs were found. For example, histidine-168 and tyrosine-332 equivalent to positions 170 and 333 in other mammalian GHRs, which were considered to be necessary for the dimerization of GHR and the specific GH-stimulated functions respectively, were replaced by tyrosine and serine in gpGHR. Here, we report the functional expression of gpGHR and its mutants, gpGHRY168H and gpGHRS332Y, in COS-7 cells and/or Chinese hamster ovary (CHO) cells. It was shown that the COS-7 cells transfected with pcDNA3-gpGHR possessed high affinity to bovine GH [K(a) = 1.3 x10(9) (mol/L)(-1)] and a protein band with molecular weight around 92 kD was detected by anti-mouse GHR monoclonal antibody (mAb263). When CHO cells were transfected with the expression vectors, pcDNA3-gpGHR, pcDNA3-gpGHRY168H and pcDNA3-gpGHRS332Y, the gpGHR and its mutants were expressed and the ligand binding, phosphorylation of JAK2, protein synthesis, and lipogenesis were studied. It was found that the mutation of serine to tyrosine at position 332 greatly increased the GH-stimulated protein synthesis and the phosphorylation of JAK2, while the mutation of tyrosine to histidine at position 168 increased the protein synthesis and decreased the phosphorylation of JAK2 only weakly. However, both mutations decreased the GH-stimulated lipogenesis. Thus, our study provides the experimental evidence that gpGHR may mediate the metabolic actions of GH and the substitutions of some conserved amino acids in gpGHR result in the changes of post-binding signaling.

Localization of CCTbeta in rat brain and overexpression in insect cells.

AIM: To study the localization of CTP: phosphocholine cytidylyltransferase beta isoform (CCTbeta) in rat brain, its expression in insect cells and enzymatic properties. METHODS: Using digoxigenin-labeled CCTbeta probes, in situ hybridization was carried out in rat brain wax sections. CCTbeta was overexpressed in Trichoplusia Ni (Tn) cells using baculovirus expression system. CTP:phosphocholine cytidylyltransferase assay (CT assay) and [3H] metabolic labeling experiment were used to study its activity, properties, and the effect on phosphatidylcholine (PC) synthesis. RESULTS: (1) CCbeta was abundant in CA1, CA2, CA4, and dentate gyrus (DG) region of hippocampus. (2) The content of CCTbeta in transfected Tn cells was over 1 104 times of that in rat brain, and CCTbeta increased the PC synthesis of Tn cells. (3) Hexadecylphosphocholine as well as some ions like Zn2+ and PO3-4 could inhibit the activity of CCTbeta, dCTP was another adaptive substrate of CCTbeta besides CTP. CONCLUSION: CCTbeta showed a similar localization in rat brain with the memory enhancing peptide argipressin (4-8).

Cloning and Preliminary Characterization of Regulatory Domain of Mouse Nestin Gene.

Mouse nestin genomic DNA(23.3 kb)was obtained after genomic library screening. Sequence comparison of the cDNA and genomic DNA revealed three introns in this gene. The positions and length of these introns are conserved among rat and human. The effect of the second intron of mouse nestin gene on the expression of luciferase reporter gene was investigated in vitro. Transient transfection of the second intron and its series deletions of mouse nestin gene showed that the second intron of mouse nestin gene could enhance reporter gene expression and the activity was contributed by a fragment about 800 bp long of its 3' end.

The Role of C-Terminus of Insulin B Chain and the Amino Group of B29Lys Side Chain in the Growth Promoting Activities of Insulin.

By growing the mouse mammary tumor-derived cell line GR2H6 in 96-well plates, we have developed an in vitro bioassay for the growth promoting activities of insulin. This bioassay system offers several advantages over currently used alternatives, such as higher sensitivity, better reproducibility and the processing of many samples simultaneously. Using this method, the mitogenic activities of insulin and its analogues were studied. The analogue with elongated C-tenminus of insulin B chain ( B31Lys Ins-NH(2)) had a higher mitogenic activity than insulin (130% of insulin). The mitogenic activities of analogues with B29Lys amino group blocked was one third of those with B29Lys amino group free, indicating that the C-terminal part of B chain and the amino group of B29Lys were important for the growth promoting activities of insulin.

N-cadherin Invovles in P19 Cell in vitro Neuronal Differentiation.

N-cadherin plays an important role in the early morphogenesis of neural tissue. For exploring the possible role of N-cadherin in neuronal differentiation in vitro, we chose RA induced P19 cell neuronal differentiation as a model system. Using RT-PCR, we examined the expression pattern of N-cadherin during P19 cell neuronal differentiation. The results showed that N-cadherin expression was increased rapidly during RA induction with its highest expression level at day 3 of neuronal differentiation, then down-regulated along with neuronal maturation. This expression pattern was similar with CNS development in vivo. Then, we constructed a N-cadherin expression-vector and transfected in P19 cells. The stable transfected cell lines, N-cadherin/P19 were selected, and we found that, after aggregated 4 days, the N-cadherin overexpression P19 cells could differentiate into neuron-like cells without the RA induction. The immunocytochemical staining showed that these neuron-like cells expressed neuron specific marker, NF160. These results suggest that N-cadherin gene is involved in P19 neuron differentiation.

Comparison of the Growth Promoting Effects of Serum Transferrins from Different Animals on Mouse Mammary Tumor Cell Line GR2H6.

The growth promoting effects of seven animal serum transferrins from mammalian, aves, reptilia, amphibian and osteichthyes on mouse mammary tumor cell line GR2H6 in serum-free medium were compared by MTT assays. The results indicated that the mitogenic activities of the transferrins from different species were different, and this discrepancy was approximately parallel to their binding capacities with transferrin receptors on human placenta and GR2H6 cells.

An Assay System for the Growth Promoting Activity of Insulin by (3)H-thymidine Incorporation.

In a chemically defined serum-free medium, insulin can stimulate DNA synthesis of the mouse mammary tumor-derived cell line GR2H6 up to 3 - 5 fold above the control group as measured by (3)H-thymidine incorporation. By growing GR2H6 cells in the 24-well and 96-well plates, a bioassay system for the growth promoting activity of insulin has been established. This method has the advantages such as easy operation, less time-consuming and suitable for measuring a large number of samples simultaneously. It may provide a novel model system for studies on the relationships between the growth promoting activity and the structure of insulin.

Growth Promoting Activity of Chimeric Molecules of "Insulin-Insulin-like Growth Factor-I"

The chimeric molecules of "Insulin-Insulin-like Growth Factor-I", Ins/IGFI(8) and Ins/IGF-I(11) were obtained by means of enzymatic semisynthesis. The growth promoting activity of these chimeric molecules were evaluated with a mouse mammary tumor derived cell line, GR2H6, and compared with that of IGF-I, insulin and desoctapeptide insulin (DOI). Both compounds have lower activities than IGF-I and effects similar to insulin, but are more potent mitogens than DOI. These results indicate that the C-terminus of insulin B-chain is importantly involved in its growth promoting activities.

Characterization of the Insulin and Transferrin Receptors of Two Cancer Cell Lines.

The insulin and transferrin receptors of the PA-1 and GR2H6 cell lines were characterized respectively. The optimum pH for the binding of PA-1 cell is 7.0 with insulin and with transferrin is 7.5 8.0. Scatchard plot and Hill plot analysis show respectively the receptor number of 2.97x10(6)/cell and 6.21x10(7)/cell for insulin receptors of PA-1 and GR2H6 with kD of 4.16x10(-7) M and 7.57x10(-7) M, Hill coefficient of 0.78 and 0.82, showing a negative cooperativity; transferring receptors of PA-1 and GR2H6 were found to have respectively receptor number of 1.25x10(5)/cell and 1.40x10(5)/cell, with kD of 1.38x10(-9) M and 1.66x10(-8) M. The Hill coefficient was 1.04 and 0.98, almost no different from 1, so transferrin receptors showed no cooperativity.