Yongchao Wang

The proprotein convertase furin is required for trophoblast syncytialization

The multinucleated syncytial trophoblast, which forms the outermost layer of the placenta and serves multiple functions, is differentiated from and maintained by cytotrophoblast cell fusion. Deficiencies in syncytial trophoblast differentiation or maintenance likely contribute to intrauterine growth restriction and pre-eclampsia, two common gestational diseases. The cellular and molecular mechanisms governing trophoblast syncytialization are poorly understood. We report here that the proprotein convertase furin is highly expressed in syncytial trophoblast in the first trimester human placentas, and expression of furin in the syncytiotrophoblast is significantly lower in the placentas from pre-eclamptic patients as compared with their gestational age-matched control placentas. Using multiple experimental models including induced fusion of choriocarcinoma BeWo cells and spontaneous fusion of primary cultured cytotrophoblast cells or placental explants, we demonstrate that cytotrophoblast cell fusion and syncytialization are accompanied by furin expression. Furin-specific siRNAs or inhibitors inhibit cell fusion in BeWo cells, as well as trophoblast syncytialization in human placental explants. Furthermore, type 1 IGF receptor (IGF1R) is indicated in this study as a substrate of furin, and processing of IGF1R by furin is an essential mechanism for syncytialization. Finally, using lentivirus-mediated RNAi targeting to mouse trophectoderm, we demonstrate that furin function is required for the development of syncytiotrophoblast structure in the labyrinth layer, as well as for normal embryonic development.

Phosphorylation of p90rsk during meiotic maturation and parthenogenetic activation of rat oocytes: correlation with MAP kinases.

This paper reports on the activation of p90rsk during meiotic maturation and the inactivation of p90rsk after electrical parthenogenetic activation of rat oocytes. In addition, the correlation between p90rsk and MAP kinases after different treatments was studied. We assessed p90rsk activity by examining its electrophoretic mobility shift on SDS-PAGE and evaluated ERK1+2 activity by both mobility shift and a specific antibody against phospho-MAP kinase. The phosphorylation of p90rsk during rat oocyte maturation was a sequential process that may be divided into two stages: the first stage was partial phosphorylation, which was irrelevant with MAP kinases because p90rsk phosphorylation took place prior to activation of MAP kinases. The second stage inferred full activation occurred at the time when MAP kinases began to be activated (3 h after germinal visicle breakdown). Evidence for the involvement of MAP kinases in the p90rsk phosphorylation was further obtained by the following approaches: (1) okadaic acid (OA) accelerated the phosphorylation of both MAP kinases and p90rsk; (2) OA induced phosphorylation of both MAP kinases and p90rsk in the presence of IBMX; (3) when activation of MAP kinases was inhibited by cycloheximide, p90rsk phosphorylation was also abolished; (4) dephosphorylation of p90rsk began to take place at 3 h post-activation, temporally correlated with the completion of MAP kinase inactivation; (5) phosphorylation of both kinases was maintained in oocytes that failed to form pronuclei after stimulation; (6) OA abolished the dephosphorylation of both kinases after parthenogenetic activation. Our data suggest that MAP kinases are not required for early partial activation of p90rsk but are required for full activation of p90rsk during rat oocyte maturation, and that p90rsk dephosphorylation occurs following MAP kinase inactivation after parthenogenetic activation of rat oocytes.

Participation of the ubiquitin-proteasome pathway in rat oocyte activation

The role of the ubiquitin-proteasome pathway (UPP) in mitosis is well known. However, its role in meiotic division is still poorly documented, especially in the activation of mammalian oocytes. In this study, the role of proteasome in the spontaneous and parthenogenetic activation of rat oocytes was investigated. We found that ALLN, an inhibitor of proteasome, when applied to metaphase II oocytes, inhibited spontaneous activation, blocked extrusion of the second polar body (PB) and caused the withdrawal of the partially extruded second PB. ALLN also inhibited the parthenogenetic activation induced by cycloheximide, but had no effect on the formation of pronuclei in activated eggs. In metaphase and anaphase, ubiquitin and proteasome localized to the meiotic spindle, concentrating on both sides of the oocyte-second PB boundary during PB extrusion. This pattern of cellular distribution suggests that UPP may have a role in regulating nuclear division and cytokinesis. Ubiquitin was seen to form a ring around the pronucleus, whereas proteasome was evenly distributed in the pronuclear region. Taken together, our results indicate that (1) UPP is required for the transitions of oocytes from metaphase II to anaphase II and from anaphase II to the end of meiosis; and (2) the UPP plays a role in cytokinesis of the second meiotic division.

Effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation

This paper investigates the effects of sense and antisense centromere/kinetochore complex protein-B (CENP-B) in cell cycle regulation. Full-length cenpb cDNA was subcloned into pBI-EGFP eukaryotic expression vector in both sense and antisense orientation. HeLa-Tet-Off cells were transfected with sense or antisense cenpb vectors. Sense transfection of HeLa-Tet-Off cells resulted in the formation of a large centromere/kinetochore complex, and apoptosis of cells following several times of cell division. A stable antisense cenpb transfected cell line, named HACPB, was obtained. The centromere/kinetochore complex of HACPB cells became smaller than control HeLa-Tet-Off cells and scattered, and the expression of CENP-B was down-regulated. In addition, delayed cell cycle progression, inhibited malignant phenotype, restrained ability of tumor formation in nude mice, and delayed entry from G2/M phase into next G1 phase were observed in HACPB cells. Furthermore, the expression of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors (CKIs) were modulated during different phases of the cell cycle. CENP-B is an essential protein for the maintenance of the structure and function of centromere/kinetochore complex, and plays important roles in cell cycle regulation.

Identification of a novel resident centrosomal protein

One human autoimmune serum was identified to react with centrosomes by immunofluorescence. We applied the affinity purification of membrane-bound antibody technique and demonstrated that the antibodies present in this antiserum reacted with a 31/29 ku centrosomal antigen. Immunofluorescence showed that this antigen is located at centrosome in a cell-cycle independent manner, and thereby it belongs to the family of centrosomal residents. We then utilized this autoimmune serum and antibodies against centrin and gamma-tubulin to investigate changes of centrosome cycle kinetics during premature chromosome condensation (PCC) artificially induced in V79-8 cells. We show here that centrosomal proteins continue to express when cells are synchronized at G1/S boundary and S phase by Hydroxyurea (HU). During this time, the addition of caffeine causes cells with unreplicated genome to go into mitosis, and induces the separation of the replicated centrosomes. These results suggest that the coordination of DNA synthesis and centrosome replication in the normal cell cycle can be uncoupled. Cells ensure that centrosome duplicates once, and only once during each DNA synthesis cycle through the tight and subtle coordination of cell cycle engine molecules, and thereby the assembly of bipolar spindle and the accurate transmission of genetic information.

Partial inhibition of CDK4 gene expression leads to the extension of G1 phase of V79-8 cells

V79-8 is an abnormal cell line which does not have detectable G1 and G2 phases in its cell cycle. This cell line is derived from V79 cell line which has G1 phase but lacks G2 phase. By using an anti-sense approach, CDK4 gene expression was partially inhibited to find whether CDK4 might contribute to the lack of G1 phase in V79-8 cells. Anti-CDK4 anti-sense plasmid was constructed and used to transfect V79-8 cells. Clones of transfected cells (V79-8-asCDK4) were examined, in comparison with V79-8 cells, to determine its growth curve, cell doubling-time (GT), the level of CDK4 gene expression and the levels of expression of some other growth related genes. V79-8-asCDK4 cells showed a slower growth rate with a doubling time 2.5-h longer than that of V79-8 cells. A flow cytometry (FCM) analysis demonstrated that the 2.5 h increase of the doubling time of V79-8-asCDK4 cells was mainly due to the appearance of G1 phase because its G2 + M phase was not significantly different from that of V79-8 cells. The decrease of CDK4 gene expression in V79-8-asCDK4 cells was shown by Northern-blot. Changes in the expression levels of the growth-related genes TGF-β, cyclin Dl and Rb were also detected in V79-8-asCDK4 cells. CDK4 functions mainly in G1 and at the transition between G1 and S phases. Expression of an anti-sense CDK4 gene fragment reduces the levels of endogenous CDK4, CDK4/cyclinD kinase activity and the phosphorylation of Rb. These events may postpone the inactivation of the check-point leading to the delay of entry into S phase and the reappearance of G1 phase in V79-8-asCDK4 cells.

Effects ofPKCα antisense RNA on human breast cancer cell proliferation and expression ofcyclinD1 andCDK4

The role of PKCα in human breast cancer cell proliferation and expression ofcyclinD1 andCDK4 has been investigated using inhibition ofPKCα expression by its antisense RNA. WhenPKCα expression was inhibited the rate of cell proliferation decreased apparently and the levels ofcyclinD1 andCDK4 mRNA were lower than the control. The results showed thatPKCα, a key member of signal transduction system, played an important role in human breast cancer cell proliferation and had a close relationship with expression ofcyclinD1 andCDK4 which control start of cell cycle.

Effects of CDK4 antisense RNA on Human breast cancer cell proliferation and expression of cyclinD1, cyclinE and CDK2

The role of CDK4 in human breast cancer cell proliferation and expression of cyclinD1, cyclinE and CDK2 has been investigated using inhibition of CDK4 expression by antisense RNA. When CDK4 expression was inhibited, the rate of cell proliferation and tumorigenecity decreased apparently. This indicates that CDK4 plays an important role in formation and development of breast tumor. The results of Northern blot analysis showed that the levels of cyclinDl and CDK2 mRNAs changed slightly whereas the level of cyclinE mRNA decreased obviously. It is suggested that the expression of CDK4 is necessary for imction of cyclinE expression. Thus, inhibition of CDK4 expression affects not only the role of CDK4 itself but also the role of other genes.

Effects of all-trans retinoic acid on proliferation of human aorta smooth muscle cells

The primary cultured human aorta smooth muscle cells (SMCs) were characterized and the effects of all-trans retinoic acid (atRA) on these cells were analysed. It is found that atRA could slow down the growth rate of SMCs, and decrease the expression levels of cyclinD1, CDK4, c-myc at 39%, 38% and 43.6% respectively, while atRA had no effects on the expression of α-actin which is specific for SMCs. Results demonstrated that atRA inhibited cell growth of SMCs but did not induce cell differentiation. These effects may be clue to the decreased translation levels of cyclinD1, CDK4 and transcription level of c-myc. The fact that atRA inhibited SMCs proliferation may become one of the theoretic basis for the clinical use of atRA in the treatment of SMCs proliferative diseases such as atherosclerosis.

Relationship between histone acetylation/deacetylation and gene transcription

In eukaryotes, nucleosome is the basic unit of chromatin. Nucleosorne is composed of an octamer of histone proteins (two molecules each of histones H2A, H2B, H3 and H4) and DNA strand wound around the octamer. Some data show that core histone octamer can affect gene transcription bothin vitro andin vivo. Recent results indicate that histone acetylation/deacetylation is a key step to regulate activity of genes. This article summarizes some coactivators, such as GCNSp, P300/CBP and TAFII 250, which are recently found to have histone acetyltransferase activity. The relationship between these coactivators and gene activation is also described. Besides, this article concerns some corepressors which have histone deacetylase activity, such as Rpd3p, HDAC2. These corepressors combine with other protein complex and then repress transcription. Finally, some problems to be solved and the future direction in this active field are discussed.