PDGF-C controls proliferation and is down-regulated by retinoic acid in mouse embryonic palatal mesenchymal cells

BACKGROUND: Platelet-derived growth factor C (PDGF-C) was recently identified as a member of the PDGF ligand family. Some observation suggests that PDGF-C could play an important role in palatogenesis highlighted by the Pdgfc(-/-) mouse with cleft palate, which led us to examine the mechanism of PDGF-C signaling in palatogenesis. It is well known that retinoic acid (RA) is a teratogen that can effectively induce cleft palate in the mouse. Due to the critical roles of PDGF-C and RA in cleft palate, the link between cleft palate induced by RA and loss of PDGF-C was investigated. METHODS: Retarded mesenchymal proliferation is an important cause for cleft palate. To clarify the mechanism of PDGF-C in palatogenesis, we evaluated the effects of PDGF-C and anti-PDGF-C neutralizing antibody on proliferation activity in mouse embryonic palatal mesenchymal (MEPM) cells. RESULTS: Briefly, our results show PDGF-C promotes proliferation, anti-PDGF-C antibody inhibits it in MEPM cells, and RA downregulates the PDGF-C expression both at the mRNA and protein levels. CONCLUSIONS: These demonstrate that PDGF-C is a potent mitogen for MEPM cells, implying that inactivated PDGF-C by gene-targeting or reduced PDGF-C by RA may both cause inhibition of proliferation in palatal shelves, which might account for the pathogenesis of cleft palate in Pdgfc(-/-) mouse or RA-treated mouse. In conclusion, our results suggest that PDGF-C signaling is a new mechanism of cleft palate induced by RA.     

Effect of all-trans-retinoic acid on the expression of primordial germ cell differentiation-associated genes in mESC-derived EBs

atRA (all-trans-retinoic acid) is known to induce the differentiation of mESCs (mouse embryonic stem cells) into PGCs (primordial germ cells) in vitro. However, it is not clear as to what changes occur in PGC differentiation-associated genes or what mechanisms are involved when EBs (embryoid bodies) derived from mESCs are induced by atRA. EBs derived from mESCs were treated with 1, 2 or 5 μM atRA for 16 h, 2 days or 5 days. Real-time PCR and Western blot analysis were performed to detect the relative levels of PGC differentiation-associated genes (Lin28, Blimp1, Stra8 and Mvh) and the corresponding proteins respectively. Immunofluorescence was used to detect the protein location and distribution in EBs. The expression characteristics of genes could be divided into three categories: rapidly reached the peak value in 16 h and then decreased (Stra8, Lin28), initially low and then increased to reach the peak value in 5 days (Mvh) and relatively unchanged (Blimp1). A low level of Lin28 was expressed in EBs treated with atRA for 2 days or 5 days. The variation in the level of Lin28 mRNA did not influence the change in the level of Blimp1 mRNA. The changes in Stra8/Lin28 were consistent with the corresponding changes in the levels of their respective mRNAs, but the changes for Mvh/Blimp1 were not consistent with the corresponding changes in the levels of their respective mRNAs. Blimp1 expression may be independent of the effect of atRA on PGC differentiation. atRA may promote the start of a period in which there is a low level of Lin28 expression during PGC differentiation.     

Pre‐activation of retinoid signaling facilitates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into neurons in an appropriate cellular environment. Retinoid signaling pathway is required in neural development. However, the effect and mechanism through retinoid signaling regulates neuronal differentiation of MSCs are still poorly understood. Here, we report that all‐trans‐retinoic acid (ATRA) pre‐induction improved neuronal differentiation of rat MSCs. We found that, when MSCs were exposed to different concentrations of ATRA (0.01–100 μmol/L) for 24 h and then cultured with modified neuronal induction medium (MNM), 1 μmol/L ATRA pre‐induction significantly improved neuronal differentiation efficiency and neural‐cell survival. Compared with MNM alone induced neural‐like cells, ATRA/MNM induced cells expressed higher levels of Nestin, neuron specific enolase (NSE), microtubule‐associated protein‐2 (MAP‐2), but lower levels of CD68, glial fibrillary acidic protein (GFAP), and glial cell line‐derived neurotrophic factor(GDNF), also exhibited higher resting membrane potential and intracellular calcium concentration, supporting that ATRA pre‐induction promotes maturation and function of derived neurons but not neuroglia cells from MSCs. Endogenous retinoid X receptors (RXR) RXRα and RXRγ (and to a lesser extent, RXRβ) were weakly expressed in MSCs. But the expression of RARα and RARγ was readily detectable, whereas RARβ was undetectable. However, at 24 h after ATRA treatment, the expression of RARβ, not RARα or RARγ, increased significantly. We further found the subnuclear redistribution of RARβ in differentiated neurons, suggesting that RARβ may function as a major mediator of retinoid signaling during neuronal differentiation from MSCs. ATRA treatment upregulated the expression of Vimentin and Stra13, while it downregulated the expression of Brachyury in MSCs. Thus, our results demonstrate that pre‐activation of retinoid signaling by ATRA facilitates neuronal differentiation of MSCs.     

Differential regulation of protein expression, growth and apoptosis by natural and synthetic retinoids

All-trans retinoic acid (ATRA) can down regulate the anti-apoptotic protein Bcl-2 and the cell cycle proteins cyclin D1 and cdk2 in estrogen receptor-positive breast cancer cells. We show here that retinoids can also reduce expression of the inhibitor of apoptosis protein, survivin. Here we have compared the regulation of these proteins in MCF-7 and ZR-75 breast cancer cells by natural and synthetic retinoids selective for the RA receptors (RARs) alpha, beta, and gamma then correlated these with growth inhibition, induction of apoptosis and chemosensitization to Taxol. In both cell lines ATRA and 9-cis RA induced the most profound decreases in cyclin D1 and cdk2 expression and also mediated the largest growth inhibition. The RARalpha agonist, Ro 40-6055 also strongly downregulated these proteins although did not produce an equivalent decrease in S-phase cells. Only ATRA induced RARbeta expression. ATRA, 9-cis RA and 4-HPR initiated the highest level of apoptosis as determined by mitochondrial Bax translocation, while only ATRA and 9-cis RA strongly reduced Bcl-2 and survivin protein expression. Enumeration of dead cells over 96 h correlated well with downregulation of both survivin and Bcl-2. Simultaneous retinoid-mediated reduction of both these proteins also predicted optimal Taxol sensitization. 4-HPR was much weaker than the natural retinoids with respect to Taxol sensitization, consistent with the proposed requirement for reduced Bcl-2 in this synergy. Neither the extent of cell cycle protein regulation nor AP-1 inhibition fully predicted the antiproliferative effect of the synthetic retinoids suggesting that growth inhibition requires regulation of a spectrum of RAR-regulated gene products in addition even to pivotal cell cycle proteins.     

PAK4 is required for regulation of the cell-cycle regulatory protein p21, and for control of cell-cycle progression

The serine/threonine kinase PAK4 regulates cytoskeletal architecture, and controls cell proliferation and survival. In most adult tissues PAK4 is expressed at low levels, but overexpression of PAK4 is associated with uncontrolled proliferation, inappropriate cell survival, and oncogenic transformation. Here we have studied for the first time, the role for PAK4 in the cell cycle. We found that PAK4 levels peak dramatically but transiently in the early part of G1 phase. Deletion of Pak4 was also associated with an increase in p21 levels, and PAK4 was required for normal p21 degradation. In serum-starved cells, the absence of PAK4 led to a reduction in the amount of cells in G1, and an increase in the amount of cells in G2/M phase. We propose that the transient increase in PAK4 levels at early G1 reduces p21 levels, thereby abrogating the activity of CDK4/CDK6 kinases, and allowing cells to proceed with the cell cycle in a precisely coordinated way.     

Expression of the Wnt inhibitor Dickkopf-1 is required for the induction of neural markers in mouse embryonic stem cells differentiating in response to retinoic acid

Cultured mouse D3 embryonic stem (ES) cells differentiating into embryoid bodies (EBs) expressed several Wnt isoforms, nearly all isotypes of the Wnt receptor Frizzled and the Wnt/Dickkopf (Dkk) co-receptor low-density lipoprotein receptor-related protein (LRP) type 5. A 4-day treatment with retinoic acid (RA), which promoted neural differentiation of EBs, substantially increased the expression of the Wnt antagonist Dkk-1, and induced the synthesis of the Wnt/Dkk-1 co-receptor LRP6. Recombinant Dkk-1 applied to EBs behaved like RA in inducing the expression of the neural markers nestin and distal-less homeobox gene (Dlx-2). Recombinant Dkk-1 was able to inhibit the Wnt pathway, as shown by a reduction in nuclear beta-catenin levels. Remarkably, the antisense- or small interfering RNA-induced knockdown of Dkk-1 largely reduced the expression of Dlx-2, and the neuronal marker beta-III tubulin in EBs exposed to RA. These data suggest that induction of Dkk-1 and the ensuing inhibition of the canonical Wnt pathway is required for neural differentiation of ES cells.     

雷氏大疣蛛毒液对人肝癌HepG2细胞p21基因表达的影响

本文研究了雷氏大疣蛛毒液对人肝癌细胞株HepG2增殖抑制作用及其分子机制。采用XTT法观察到雷氏大疣蛛毒液剂量依赖抑制HepG2细胞增殖;流式细胞仪检测发现,经过雷氏大疣蛛毒液作用的HepG2细胞周期发生明显的选择性改变;RT-PCR方法检测到p21基因表达增强;Western blot检测发现,p21蛋白表达增加。结果提示,雷氏大疣蛛毒液抑制人肝癌细胞HepG2增殖的可能机制之一是使p21基因和蛋白表达增加,G2IM细胞周期被阻滞,从而诱导细胞凋亡。     

全反式维甲酸抑制猪脂肪间充质干细胞的成脂分化

分离培养猪脂肪间充质干细胞（adipose mesenchymal stem cells, AMSCs）,流式细胞仪鉴定其表面标记.利用MTT比色检测不同浓度的全反式维甲酸（all trans retinoic acid, ATRA）对猪AMSCs增殖的影响;光学显微镜下观察猪AMSCs向脂肪细胞分化的形态学变化;油红O染色提取法分析不同浓度ATRA对猪AMSCs成脂分化的影响;RT PCR检测脂肪细胞分化标志基因LPL和aP2 mRNA的变化.MTT比色结果显示,生理浓度（1×10^－9～1×10^－8 mol/L）和药理浓度（1×10^－7～1×10^－5 mol/L）ATRA对猪AMSCs增殖均没有影响.油红O染色提取法结果表明,除1×10^－7　mol/L ATRA对猪AMSCs成脂分化没有影响外,生理浓度（1×10^－9～1×10^－8 mol/L）和其它药理浓度（1×10^－6～1×10^－5 mol/L）ATRA均显著抑制猪AMSCs成脂分化（P<0.05）.RT-PCR检测显示,ATRA显著抑制脂肪细胞分化标志基因LPL和aP2 mRNA表达（P<0.05）.     

p21 and retinoblastoma protein control the absence of DNA replication in terminally differentiated muscle cells

During differentiation, skeletal muscle cells withdraw from the cell cycle and fuse into multinucleated myotubes. Unlike quiescent cells, however, these cells cannot be induced to reenter S phase by means of growth factor stimulation. The studies reported here document that both the retinoblastoma protein (Rb) and the cyclin-dependent kinase (cdk) inhibitor p21 contribute to this unresponsiveness. We show that the inactivation of Rb and p21 through the binding of the adenovirus E1A protein leads to the induction of DNA replication in differentiated muscle cells. Moreover, inactivation of p21 by E1A results in the restoration of cyclin E-cdk2 activity, a kinase made nonfunctional by the binding of p21 and whose protein levels in differentiated muscle cells is relatively low in amount. We also show that restoration of kinase activity leads to the phosphorylation of Rb but that this in itself is not sufficient for allowing differentiated muscle cells to reenter the cell cycle. All the results obtained are consistent with the fact that Rb is functioning downstream of p21 and that the activities of these two proteins may be linked in sustaining the postmitotic state.     

Self-renewal and differentiation of mouse embryonic stem cells as measured by Oct4 gene expression: Effects of lif,serum-free medium,retinoic acid,and dbcAMP

Summary In this study we examined the interplay between serum, leukemia inhibitory factor (LIF), retinoic acid, and dibutyrl cyclic adenosine monophosphate (dbcAMP) in affecting IOUD2 embryonic stem cell self-renewal and differentiation as assessed by Oct4 expression, and cell proliferation as measured by total cell protein. Removal of LIF, reduced levels of fetal calf serum (FCS), and addition of retinoic acid all induced embryonic stem cell differentiation as measured by reduced Oct4 expression. Lower levels of retinoic acid (0.1–10 nM) promoted the formation of epithelial-like cells, whereas higher levels (100–10,000 nM) favored differentiation into fibroblastic-like cells. The effects of dbcAMP varied with the presence or absence of FCS and LIF and the concentration of dbcAMP. In FCS-containing media, a low level of dbcAMP (100 μM) increased self-renewal in the absence of LIF, but it had no effect in its presence. In contrast, at higher concentrations (1000 μM dbcAMP), regardless of LIF, differentiation was promoted. A similar effect of dbcAMP was seen in the presence of retinoic acid. In media without FCS but with serum replacement supplements, there was no effect of dbcAMP. This study shows that the Oct4 expression system of IOUD2 cells provides a novel, simple method for quantifying cellular differentiation.     

Retinoic acid induces CDK inhibitors and growth arrest specific (Gas) genes in neural crest cells

Retinoic acid (RA), the active metabolite of vitamin A, regulates cellular growth and differentiation during embryonic development. In excess, this vitamin is also highly teratogenic to animals and humans. The neural crest is particularly sensitive to RA, and high levels adversely affect migration, proliferation and cell death. We investigated potential gene targets of RA associated with neural crest proliferation by determining RA-mediated changes in gene expression over time, using microarrays. Statistical analysis of the top ranked RA-regulated genes identified modest changes in multiple genes previously associated with cell cycle control and proliferation including the cyclin-dependent kinase inhibitors Cdkn1a (p21), Cdkn2b (p15(INK4b)), and Gas3/PMP22. The expression of p21 and p15(INK4b) contribute to decreased proliferation by blocking cell cycle progression at G1-S. This checkpoint is pivotal to decisions regulating proliferation, apoptosis, or differentiation. We have also confirmed the overexpression of Gas3/PMP22 in RA-treated neural crests, which is associated with cytoskeletal changes and increased apoptosis. Our results suggest that increases in multiple components of diverse regulatory pathways have an overall cumulative effect on cellular decisions. This heterogeneity contributes to the pleiotropic effects of RA, specifically those affecting proliferation and cell death.     

Polypyrimidine tract‐binding protein is required for the repression of gene expression by all‐trans retinoic acid

All‐trans retinoic acid is a key regulator of early development. High concentrations of retinoic acid interfere with differentiation and migration of neural crest cells. Here we report that a dinucleotide repeat in the cis‐element of Snail2 (previously known as Slug) gene plays a role in repression by all‐trans retinoic acid. We analyzed the cis‐acting regulatory regions of the Xenopus Snail2 gene, whose expression is repressed by all‐trans retinoic acid. The analysis identified a TG/CA repeat as a necessary element for the repression. By performing a yeast one‐hybrid screen, we found that a polypyrimidine tract‐binding protein (PTB), which is known to be a regulator of the alternative splicing of pre‐messenger RNA, binds to the TG/CA repeat. Overexpression and knockdown experiments for PTB in HEK293 cells and Xenopus embryos indicated that PTB is required for repression by retinoic acid. The green fluorescent protein‐PTB fusion protein was localized in the nucleus of 293T cells. In situ hybridization for PTB in Xenopus embryos showed that PTB is expressed at the regions including neural crest at the early stages. Our results indicate that PTB plays a role in the repression of gene expression by retinoic acid through binding to the TG/CA repeats.     

Susceptibility to killer T cells of gastric cancer cells enhanced by Mitomycin-C involves induction of ATBF1 and activation of p21 (Waf1/Cip1) promoter

Alpha-fetoprotein (AFP) expression is observed in embryonic tissues and, the expression of this protein is absent in normal adult tissues. The re-elevation of serum AFP strongly suggests generation of a malignant tumor in an adult. We demonstrated here that AFP-producing gastric cancer (AFP-gastric cancer) could be treated by a combination therapy with a low dose of Mitomycin-C (MMC) and lymphokineactivated killer T (LAK-T) cells. Treatment with MMC of AFP-gastric cancer cells enhanced their susceptibility to LAK-T cells and induced ATBF1 gene expression. We revealed here a novel signal pathway for regulation of the cell cycle of AFP-gastric cancer cells through ATBF1, which enhances the promoter activity of the p21 (Waf1/Cip1) gene. Immunoprecipitation revealed the direct interaction between ATBF1 and p53. Overexpressed ATBF1 stimulated p21 (Waf1/Cip1) promoter activity up to 4-fold compared with basal activity. The expression level of ATBF1 mRNA was doubled by MMC (0.05 microg/ml) treatment. The MMC treatment and ATBF1 overexpression synergistically activated the p21 (Waf1/Cip1) promoter activity in a dose-dependent manner up to 7-fold compared with basal activity.