The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.     

Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo

Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.     

Developing with lethal RA levels: genetic ablation of Rarg can restore the viability of mice lacking Cyp26a1

We have previously reported that the retinoic acid (RA) catabolizing enzyme CYP26A1 plays an important role in protecting tail bud tissues from inappropriate exposure to RA generated in the adjacent trunk tissues by RALDH2, and that Cyp26a1-null animals exhibit spina bifida and caudal agenesis. We now show that, in the absence of Cyp26a1, retinoic acid receptor gamma (RARgamma) mediates ectopic RA-signaling in the tail bud. We also show that activated RARgamma results in downregulation of Wnt3a and Fgf8, which integrate highly conserved signaling pathways known for their role in specifying caudal morphogenesis. Ablation of the gene for RARgamma (Rarg) rescues Cyp26a1-null mutant animals from caudal regression and embryonic lethality, thus demonstrating that CYP26A1 suppresses the RA-mediated downregulation of WNT3A and FGF8 signaling pathways by eliminating ectopic RA in gastrulating tail bud mesoderm.     

Retinoic acid selectively activates the ERK2 but not JNK/SAPK or P38 map kinases when inducing myeloid differentiation

Summary Among the three major mitogen-activated protein kinase (MAPK) cascades—the extracellular signal regulated kinase (ERK) pathway, the c-JUN N-terminal/stress-activated protein kinase (JNK/SAPK) pathway, and the reactivating kinase (p38) pathway—retinoic acid selectively utilizes ERK but not JNK/SAPK or p38 when inducing myeloid differentiation of HL-60 human myeloblastic leukemia cells. Retinoic acid is known to active ERK2. The present data show that the activation is selective for this MAPK pathway. JNK/SAPK or p38 are not activated by retinoic acid. Presumably because it activates relevant signaling pathways including MAPK, the polyoma middle T antigen, as well as certain transformation defective mutants thereof, is known to promote retinoic acid-induced differentiation, although the mechanism of action is not well understood. The present results show that consistent with the selective involvement of ERK2, ectopic expression of either the polyoma middle T antigen or its dl23 mutant, which is defective for PLCγ and PI-3 kinase activation, or the Δ205 mutant, which in addition is also weakened for activation of src-like kinases, caused no enhanced JNK/SAPK or p38 kinase activity that promoted the effects of retinoic acid. However, all three of these polyoma antigens are known to enhance ERK2 activation and promote differentiation induced by retinoic acid. Polyoma-activated MAPK signaling relevant to retinoic acid-induced differentiation is thus restricted to ERK2 and does not involve JNK/SAPK or p38. Taken together, the data indicate that among the three parallel MAPK pathways, retinoic acid-induced HL-60 myeloid differentiation selectively depends on activating ERK but not the other two MAPK pathways, JNK/SAPK or p38, with no apparent cross talk between pathways. Furthermore, the striking ability of polyoma middle T antigens to promote retinoic acid-induced differentiation appears to utilize ERK, but not JNK/SPK or p38 signaling.     

Stimulation of premature retinoic acid synthesis in Xenopus embryos following premature expression of aldehyde dehydrogenase ALDH1.

In order for nuclear retinoic acid receptors to mediate retinoid signaling, the ligand retinoic acid must first be produced from its vitamin A precursor retinal. Biochemical studies have shown that retinal can be metabolized in vitro to retinoic acid by members of the aldehyde dehydrogenase enzyme family, including ALDH1. Here we describe the first direct evidence that ALDH1 plays a physiological role in retinoic acid synthesis by analysis of retinoid signaling in Xenopus embryos, which have plentiful stores of maternally derived retinal. The Xenopus ALDH1 gene was cloned and shown to be highly conserved with chick and mammalian homologs. Xenopus ALDH1 was not expressed at blastula and gastrula stages, but was expressed at the neurula stage. We used a retinoic acid bioassay to demonstrate that retinoic acid is normally undetectable in embryos from fertilization to the initial gastrula stage, but that a tremendous increase in retinoic acid occurs during neurulation when ALDH1 is first expressed. Overexpression of ALDH1 by injection of Xenopus embryos with mRNAs encoding the mouse, chick or Xenopus ALDH1 homologs induced high levels of retinoic acid detection during the blastula stage. Thus, premature expression of ALDH1 stimulates premature synthesis of retinoic acid. These findings reveal an important conserved role for ALDH1 in retinoic acid synthesis in vivo, and demonstrate that conversion of retinoids from the aldehyde form to the carboxylic acid form is a crucial regulatory step in retinoid signaling.     

Retinoic acid determines life span of leukemic cells by inducing antagonistic apoptosis-regulatory programs

As a single signal, retinoids induce terminal differentiation. This implies that they activate differentiation and apoptosis in a temporally defined order to allow expression of the differentiated phenotype well before death. We report that two apparently contradictory retinoid-induced programs have the capacity to define cellular life span. Anti-apoptotic factors are activated concomitantly with differentiation, while retinoids induce at the same time also pro-apoptotic signaling. We have assessed the roles of two key factors, Bcl2A1 and TRAIL, in the temporal programming of cell death and differentiation. We demonstrate that PLB985 are type II cells in which TRAIL induces apoptosis through the extrinsic and--via Bid activation--also the intrinsic death pathways. Bcl2A1, ectopically over-expressed, or endogenously induced by retinoic acid receptor agonists, protected cells from apoptosis triggered by TRAIL, whose induction required the activation of both the retinoic acid and retinoid X receptors. Bcl2A1 prevented loss of mitochondrial membrane potential and caspase-9, but not caspase-8, activation. The expression of anti-sense Bcl2A1 sensitized PLB985 cells to TRAIL. Co-culture experiments revealed protection from fraternicide if sister cells were pre-exposed to retinoic acid. Collectively, our data support a model in which retinoids orchestrate a life span-regulatory program comprising Bcl2A1 induction to temporally protect against concomitantly induced TRAIL death signaling. Termination of this life span in presence of Bcl2A1 is most likely a consequence of the Bid-independent TRAIL action. Thus, depending on the retinoic acid and retinoid X receptor activation potential of a ligand and the relative efficacies of the intrinsic and extrinsic death pathways in a given cell, a single retinoid triggers the life span of a differentiated phenotype.     

Augmentation of specific tumor immunity against a syngeneic SV40-induced sarcoma in mice by retinoic acid.

The effects of retinoic acid, a vitamin A derivative, which has been shown to have immune adjuvant properties, were studied in vivo in a syngeneic tumor system by the use of the tumor-cell neutralization assay. The effector activity of spleen cells of BALB/c mice immune against a syngeneic SV40-induced sarcoma, mKSA, was specifically augmented by low doses of retinoic acid, whereas high doses had a suppressive effect. In addition, the time required for generation of the effector activity was shortened and the immune activity lasted longer in the retinoic acid-treated mice. With the use of cell-depletion techniques it was demonstrated that thymus-derived lymphocytes were affected by retinoic acid.     

Interaction between the aryl hydrocarbon receptor and retinoic acid pathways increases matrix metalloproteinase-1 expression in keratinocytes

Exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) results in a variety of pathological lesions in humans via activation of the aryl hydrocarbon receptor (AhR) pathway. It has become apparent that this pathway interacts with a variety of signaling pathways that are believed to be involved in mediating TCDD/AhR biological effects. Our hypothesis is that TCDD mediates these pathological lesions by directly altering the expression of genes involved in matrix deposition and remodeling and that the retinoic acid signaling pathway is involved in modulating TCDD-induced effects. Therefore, we examined the effect of TCDD and all-trans retinoic acid (atRA) on the expression of matrix metalloproteinase-1 (MMP-1, interstitial collagenase), one of the proteolytic enzymes that degrade type I collagen, in normal human keratinocytes. The data show that TCDD exposure results in increased MMP-1 expression in keratinocytes that is further enhanced by co-treatment with all-trans retinoic acid. TCDD-induced expression of MMP-1 appears to be mediated through two AP-1 elements in the proximal promoter of the MMP-1 gene. However, retinoic acid-mediated induction of keratinocyte MMP-1 is a result of both promoter activation and increased mRNA stability. These findings are the first to demonstrate TCDD-induced expression of MMP-1 and to demonstrate interactions between the TCDD/AhR and retinoic acid pathways on MMP-1 expression.     

Sex specific retinoic acid signaling is required for the initiation of urogenital sinus bud development

The mammalian urogenital sinus (UGS) develops in a sex specific manner, giving rise to the prostate in the male and the sinus vagina in the embryonic female. Androgens, produced by the embryonic testis, have been shown to be crucial to this process. In this study we show that retinoic acid signaling is required for the initial stages of bud development from the male UGS. Enzymes involved in retinoic acid synthesis are expressed in the UGS mesenchyme in a sex specific manner and addition of ligand to female tissue is able to induce prostate-like bud formation in the absence of androgens, albeit at reduced potency. Functional studies in mouse organ cultures that faithfully reproduce the initiation of prostate development indicate that one of the roles of retinoic acid signaling in the male is to inhibit the expression of Inhba, which encodes the βA subunit of Activin, in the UGS mesenchyme. Through in vivo genetic analysis and culture studies we show that inhibition of Activin signaling in the female UGS leads to a similar phenotype to that of retinoic acid treatment, namely bud formation in the absence of androgens. Our data also reveals that both androgens and retinoic acid have extra independent roles to that of repressing Activin signaling in the development of the prostate during fetal stages. This study identifies a novel role for retinoic acid as a mesenchymal factor that acts together with androgens to determine the position and initiation of bud development in the male UGS epithelia.     

Retinoic acid and Wnt/β-catenin have complementary roles in anterior/posterior patterning embryos of the basal chordate amphioxus

A role for Wnt/β-catenin signaling in axial patterning has been demonstrated in animals as basal as cnidarians, while roles in axial patterning for retinoic acid (RA) probably evolved in the deuterostomes and may be chordate-specific. In vertebrates, these two pathways interact both directly and indirectly. To investigate the evolutionary origins of interactions between these two pathways, we manipulated Wnt/β-catenin and RA signaling in the basal chordate amphioxus during the gastrula stage, which is the RA-sensitive period for anterior/posterior (A/P) patterning. The results show that Wnt/β-catenin and RA signaling have distinctly different roles in patterning the A/P axis of the amphioxus gastrula. Wnt/β-catenin specifies the identity of the ends of the embryo (high Wnt = posterior; low Wnt = anterior) but not intervening positions. Thus, upregulation of Wnt/β-catenin signaling induces ectopic expression of posterior markers at the anterior tip of the embryo. In contrast, RA specifies position along the A/P axis, but not the identity of the ends of the embryo—increased RA signaling strongly affects the domains of Hox expression along the A/P axis but has little or no effect on the expression of either anterior or posterior markers. Although the two pathways may both influence such things as specification of neuronal identity, interactions between them in A/P patterning appear to be minimal.     

维甲酸与精子发生

维生素A的活性代谢物维甲酸在哺乳动物精子发生过程中发挥着重要的调节作用,但其具体调节机制并不十分清楚。该文拟对睾丸内维甲酸的运输、代谢、信号系统以及维甲酸调控精子发生的研究进展进行简单总结。     

Keeping an eye on retinoic acid signaling during eye development

Retinoic acid is a metabolic derivative of vitamin A that plays an essential function in cell-cell signaling by serving as a ligand for nuclear receptors that directly regulate gene expression. The final step in the conversion of retinol to retinoic acid is carried out by three retinaldehyde dehydrogenases encoded by Raldh1 (Aldh1a1), Raldh2 (Aldh1a2), and Raldh3 (Aldh1a3). Mouse Raldh gene knockout studies have been instrumental in understanding the mechanism of retinoic acid action during eye development. Retinoic acid signaling in the developing eye is particularly complex as all three Raldh genes contribute to retinoic acid synthesis in non-overlapping locations. During optic cup formation Raldh2 is first expressed transiently in perioptic mesenchyme, then later Raldh1 and Raldh3 expression begins in the dorsal and ventral retina, respectively, and these sources of retinoic acid are maintained in the fetus. Retinoic acid is not required for dorsoventral patterning of the retina as originally thought, but it is required for morphogenetic movements that form the optic cup, ventral retina, cornea, and eyelids. These findings will help guide future studies designed to identify retinoic acid target genes during eye organogenesis.     

Emerging role of NF-κB signaling in the induction of senescence-associated secretory phenotype (SASP)

The major hallmark of cellular senescence is an irreversible cell cycle arrest and thus it is a potent tumor suppressor mechanism. Genotoxic insults, e.g. oxidative stress, are important inducers of the senescent phenotype which is characterized by an accumulation of senescence-associated heterochromatic foci (SAHF) and DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS). Interestingly, senescent cells secrete pro-inflammatory factors and thus the condition has been called the senescence-associated secretory phenotype (SASP). Emerging data has revealed that NF-κB signaling is the major signaling pathway which stimulates the appearance of SASP. It is known that DNA damage provokes NF-κB signaling via a variety of signaling complexes containing NEMO protein, an NF-κB essential modifier, as well as via the activation of signaling pathways of p38MAPK and RIG-1, retinoic acid inducible gene-1. Genomic instability evoked by cellular stress triggers epigenetic changes, e.g. release of HMGB1 proteins which are also potent enhancers of inflammatory responses. Moreover, environmental stress and chronic inflammation can stimulate p38MAPK and ceramide signaling and induce cellular senescence with pro-inflammatory responses. On the other hand, two cyclin-dependent kinase inhibitors, p16INK4a and p14ARF, are effective inhibitors of NF-κB signaling. We will review in detail the signaling pathways which activate NF-κB signaling and trigger SASP in senescent cells.