Neonatal lactocrine deficiency affects the adult porcine endometrial transcriptome at pregnancy day 13.

Reproductive performance of female pigs that do not receive sufficient colostrum from birth is permanently impaired. Whether lactocrine deficiency, reflected by low serum immunoglobulin immunocrit (iCrit), affects patterns of endometrial gene expression during the periattachment period of early pregnancy is unknown. Here, objectives were to determine effects of low iCrit at birth on the adult endometrial transcriptome on pregnancy day (PxD) 13. On the first day of postnatal life, gilts were assigned to high or low iCrit groups. Adult high (n = 8) and low (n = 7) iCrit gilts were bred (PxD 0), and humanely slaughtered on PxD 13 when tissues and fluids were collected. The endometrial transcriptome was defined for each group using mRNAseq and microRNAseq. Reads were mapped to the Sus scrofa 11.1 genome build. Mature microRNAs were annotated using miRBase 21. Differential expression was defined based on fold change (≥ ±1.5). Lactocrine deficiency did not affect corpora lutea number, uterine horn length, uterine wet weight, conceptus recovery, or uterine luminal fluid estrogen content on PxD 13. However, mRNAseq revealed 1157 differentially expressed endometrial mRNAs in high versus low iCrit gilts. Differentially expressed genes had functions related to solute transport, endometrial receptivity, and immune response. Six differentially expressed endometrial microRNAs included five predicted to target 62 differentially expressed mRNAs, affecting similar biological processes. Thus, lactocrine deficiency on the first day of postnatal life can alter uterine developmental trajectory with lasting effects on endometrial responses to pregnancy as reflected at the level of the transcriptome on PxD 13.

Usp18 promotes conventional CD11b+ dendritic cell development.

Dendritic cells (DCs) represent the key cells linking innate and adaptive immune responses. It is critical to understand the molecular factors regulating DC differentiation. Usp18 is an IFN-inducible member of the ubiquitin-specific protease family, which deconjugates ubiquitin-like modifier ISG15 from target proteins and competitively inhibits IFN-α/ß-induced JAK/STAT activation. This study demonstrates that the frequency of conventional CD11b(+) DCs in the spleen of Usp18(-/-) mice was significantly reduced, whereas the frequencies of conventional CD8(+) DCs and plasmacytoid DCs remained normal. In addition, Usp18(-/-) bone marrow (BM) cells generate DCs less efficiently in GM-CSF-supplemented culture, demonstrating a fundamental defect throughout the DC differentiation pathway. Usp18(-/-) BM cells were rescued by exogenous expression of either wild-type or deconjugation-inactive Usp18, and superimposition of an IFN-α/ß receptor knockout returned in vivo DC populations to normal, clearly showing that the defect seen is due solely to Usp18's effect on IFN signaling. Finally, Usp18(-/-) BM-derived DCs expressed high levels of SOCS1/SOCS3, known inhibitors of GM-CSF signaling, providing a mechanistic explanation for the phenotype. In conclusion, we have identified a novel role of Usp18 in modulating conventional CD11b(+) DC development via its inhibitory effect on type I IFN signaling.

Maternal growth factor regulation of human placental development and fetal growth.

Normal development and function of the placenta is critical to achieving a successful pregnancy, as normal fetal growth depends directly on the transfer of nutrients from mother to fetus via this organ. Recently, it has become apparent from both animal and human studies that growth factors within the maternal circulation, for example the IGFs, are important regulators of placental development and function. Although these factors act via distinct receptors to exert their effects, the downstream molecules activated upon ligand/receptor interaction are common to many growth factors. The expression of numerous signaling molecules is altered in the placentas from pregnancies affected by the fetal growth complications, fetal growth restriction, and macrosomia. Thus, targeting these molecules may lead to more effective treatments for complications of pregnancy associated with altered placental development. Here, we review the maternal growth factors required for placental development and discuss their mechanism of action.

Colon carcinoma cells harboring PIK3CA mutations display resistance to growth factor deprivation induced apoptosis.

PIK3CA, encoding the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is mutated in a variety of human cancers. We screened the colon cancer cell lines previously established in our laboratory for PIK3CA mutations and found that four of them harbored gain of function mutations. We have now compared a panel of mutant and wild-type cell lines for cell proliferation and survival in response to stress. There was little difference in PI3K activity between mutant PIK3CA-bearing cells (mutant cells) and wild-type PIK3CA-bearing cells (wild-type cells) under optimal growth conditions. However, the mutant cells showed constitutive PI3K activity during growth factor deprivation stress (GFDS), whereas PI3K activity decayed rapidly in the wild-type cells. Importantly, constitutively active PI3K rendered the mutant cells resistant to GFDS-induced apoptosis relative to the wild-type cells, indicating a biological advantage under stress conditions that is imparted by the mutant enzymes. Compared with the wild-type cells, the mutant cells were hypersensitive to the apoptosis induced by the PI3K inhibitor LY294002. In addition, PIK3CA small interfering RNA significantly decreased DNA synthesis and/or induced apoptosis in the mutant cells but not in the wild-type cells. Furthermore, ecotopic expression of a mutant PIK3CA in a nontumorigenic PIK3CA wild-type cell line resulted in resistance to GFDS-induced apoptosis, whereas transfection of wild-type PIK3CA or empty vector had little effect. Taken together, our studies show that mutant PIK3CA increases the capacity for proliferation and survival under environmental stresses, such as GFDS while also imparting greater dependency on the PI3K pathway for proliferation and survival.

Arginase 1 regulation of nitric oxide production is key to survival of trophic factor-deprived motor neurons.

When deprived of trophic factors, the majority of cultured motor neurons undergo nitric oxide-dependent apoptosis. However, for reasons that have remained unclear, 30-50% of the motor neurons survive for several days without trophic factors. Here we hypothesize that the resistance of this motor neuron subpopulation to trophic factor deprivation can be attributed to diminished nitric oxide production resulting from the activity of the arginine-degrading enzyme arginase. When incubated with nor-N(G)-hydroxy-nor-L-arginine (NOHA), the normally resistant trophic factor-deprived motor neurons showed a drop in survival rates, whereas trophic factor-treated neurons did not. NOHA-induced motor neuron death was inhibited by blocking nitric oxide synthesis and the scavenging of superoxide and peroxynitrite, suggesting that peroxynitrite mediates NOHA toxicity. When we transfected arginase 1 into motor neurons to see whether it alone could abrogate trophic factor deprivation-induced death, we found that its forced expression did indeed do so. The protection afforded by arginase 1 expression is reversed when cells are incubated with NOHA or with low concentrations of nitric oxide. These results reveal that arginase acts as a central regulator of trophic factor-deprived motor neuron survival by suppressing nitric oxide production and the consequent peroxynitrite toxicity. They also suggest that the resistance of motor neuron subpopulations to trophic factor deprivation may result from increased arginase activity.

FOXO3a-dependent regulation of Puma in response to cytokine/growth factor withdrawal.

Puma is an essential mediator of p53-dependent and -independent apoptosis in vivo. In response to genotoxic stress, Puma is induced in a p53-dependent manner. However, the transcription factor driving Puma up-regulation in response to p53-independent apoptotic stimuli has yet to be identified. Here, we show that FOXO3a up-regulates Puma expression in response to cytokine or growth factor deprivation. Importantly, dysregulated Akt signaling in lymphoid cells attenuated Puma induction upon cytokine withdrawal. Our results suggest that Puma, together with another BH3 only member, Bim, function as FOXO3a downstream targets to mediate a stress response when PI3K/Akt signaling is down-regulated.

Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons.

A molecular basis of survival from neuronal injury is essential for the development of therapeutic strategy to remedy neurodegenerative disorders. In this study, we demonstrate that an EF-hand Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1), one of the key proteins for various neuronal functions, also acts as an important survival factor. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressors, whereas the dominant-negative mutant (E120Q) accelerated it. In addition, NCS-1 proteins increased upon treatment with glial cell line-derived neurotrophic factor (GDNF) and mediated GDNF survival signal in an Akt (but not MAPK)-dependent manner. Furthermore, NCS-1 is significantly up-regulated in response to axotomy-induced injury in the dorsal motor nucleus of the vagus neurons of adult rats in vivo, and adenoviral overexpression of E120Q resulted in a significant loss of surviving neurons, suggesting that NCS-1 is involved in an antiapoptotic mechanism in adult motor neurons. We propose that NCS-1 is a novel survival-promoting factor up-regulated in injured neurons that mediates the GDNF survival signal via the phosphatidylinositol 3-kinase-Akt pathway.

Enhanced cell survival of melanocyte spheroids in serum starvation condition.

Autologous melanocyte transplantation for vitiligo treatment by use of melanocyte suspension has drawbacks including cell damage in cell preparation and transportation, difficult manipulation and low engraftment rate in acral vitiligious lesions. We have proposed the concept of cellular patch as an alternative solution. In the development of melanocyte patches, we have shown that chitosan membrane supports the growth and phenotype expression of melanocytes. Surprisingly, melanocytes spontaneously grow into three-dimensional spheroids on chitosan-coated surface. In this work, we demonstrate that, compared with monolayered melanocytes, melanocyte spheroids show a better survival in growth factor and serum-deprived condition. Survival of melanocytes is further ameliorated when a greater portion of melanocytes is precultured into spheroidal morphology. Melanocyte spheroids disintegrate and the cells return to a physiological dendritic morphology after they are reinoculated on collagen I-coated surface. Our results show that melanocytes are morphologically transformable depending on the substratum used and spheroidal melanocytes have a superior survival to that of monolayered dendritic melanocytes in stringent conditions. Preculturing melanocytes into spheroids can provide melanocytes a survival advantage. Chitosan-based melanocyte patch can be a promising method to enhance the engraftment rate and facilitate the cell preparation and transplantation procedures in melanocyte transplantation for vitiligo treatment.

Regulation of mouse embryo development by autocrine throphic factors

Embryo development depends on maternal and embryonic factors. When occurs in vitro, embryos secrete factors that stimulate their development. The purpose of this study was to investigate the possible effects of embryos at morula stage on mouse embryo development in vitro. To obtain conditioned media (CM), morulas were cultured in groups of 5 (CM5) or 10 (CM10) in microdrops of Ham-Fl0 culture medium during 24h and later they were removed. Subsequently, 365 morulas were cultured in CM5 and CM10 or in Ham-F10 media (as control group). No differences in blastocyst formation could be found between embryos cultured for 24h in Ham-F1O, CM5 or CM10 (49.66, 53.04, 60.00% respectively). However, CM5 significantly increased differentiation in embryos cultured for 48h as compared to Ham-FlO medium (80.00% and 64.14 respectively). The CM5 caused a significant increase in the hatching rate compared to Ham-F10 evaluated at 78 and 96h of culture (66.96 vs. 52.41% and 70.43 vs. 55.17%, respectively). After 72, 78 and 96h of culture the hatching rate for embryos cultured in CM10 was significantly higher than that in Ham-F10 (64.76 vs. 47.59%, 67.62 vs. 52.41% and 73.33 vs. 55.17%, respectively). At 48h of culture, differences between CM5, CMl0 and Ham-F10 were not observed. These results suggest that preimplantational mouse embryos produce trophic factor/factors that enhance the differentiation and hatching process

Mutant PIK3CA promotes cell growth and invasion of human cancer cells.

PIK3CA is mutated in diverse human cancers, but the functional effects of these mutations have not been defined. To evaluate the consequences of PIK3CA alterations, the two most common mutations were inactivated by gene targeting in colorectal cancer (CRC) cells. Biochemical analyses of these cells showed that mutant PIK3CA selectively regulated the phosphorylation of AKT and the forkhead transcription factors FKHR and FKHRL1. PIK3CA mutations had little effect on growth under standard conditions, but reduced cellular dependence on growth factors. PIK3CA mutations resulted in attenuation of apoptosis and facilitated tumor invasion. Treatment with the PI3K inhibitor LY294002 abrogated PIK3CA signaling and preferentially inhibited growth of PIK3CA mutant cells. These data have important implications for therapy of cancers harboring PIK3CA alterations.

Autocrine regulation of human urothelial cell proliferation and migration during regenerative responses in vitro.

Regeneration of the urothelium is rapid and effective in order to maintain a barrier to urine following tissue injury. Whereas normal human urothelial (NHU) cells are mitotically quiescent and G0 arrested in situ, they rapidly enter the cell cycle upon seeding in primary culture and show reversible growth arrest at confluency. We have used this as a model to investigate the role of EGF receptor signaling in urothelial regeneration and wound-healing. Transcripts for HER-1, HER-2, and HER-3 were expressed by quiescent human urothelium in situ. Expression of HER-1 was upregulated in proliferating cultures, whereas HER-2 and HER-3 were more associated with a growth-arrested phenotype. NHU cells could be propagated in the absence of exogenous EGF, but autocrine signaling through HER-1 via the MAPK and PI3-kinase pathways was essential for proliferation and migration during urothelial wound repair. HB-EGF was expressed by urothelium in situ and HB-EGF, epiregulin, TGF-alpha, and amphiregulin were expressed by proliferating NHU cells. Urothelial wound repair in vitro was attenuated by neutralizing antibodies against HER-1 ligands, particularly amphiregulin. By contrast, the same ligands applied exogenously promoted migration, but inhibited proliferation, implying that HER-1 ligands provoke differential effects in NHU cells depending upon whether they are presented as soluble or juxtacrine ligands. We conclude that proliferation and migration during wound healing in NHU cells are mediated through an EGFR autocrine signalling loop and our results implicate amphiregulin as a key mediator.

Multiple death pathways in retina-derived 661W cells following growth factor deprivation: crosstalk between caspases and calpains.

During development of the mammalian retina, neurons that do not succeed in establishing functional synaptic connections are eliminated by apoptosis, allowing the formation of a finely tuned network. Growth factors play a crucial role in controlling the balance between apoptosis and survival signals not only at developmental stages but also in long-term preservation of retinal functions. In the present work, we explore the apoptotic mechanisms triggered by growth factor deprivation of retina-derived 661W cells. Under serum starvation conditions, these cone photoreceptors underwent cell death with participation of caspase-9, -3 and -12. Interestingly, inhibition of caspases did not prevent apoptosis but only resulted in a temporary delay. We show m-calpain activation in parallel with caspases, indicating that more than one execution pathway is available to cone photoreceptors. Moreover, crosstalk of the caspase and calpain pathways was detected, suggesting a loop that may act to amplify the apoptotic cascade.

Eating oneself and uninvited guests: autophagy-related pathways in cellular defense.

The eukaryotic cell uses an evolutionarily conserved lysosomal pathway of self-digestion (autophagy) for survival when extracellular nutrients are limited. In this issue of Cell, new evidence indicates that autophagy is used to for survival when intracellular nutrients are limited by growth factor deprivation (Lum et al., 2005). Other recent studies indicate that the autophagy machinery is also used to degrade foreign microbial invaders (xenophagy).

Growth factor regulation of autophagy and cell survival in the absence of apoptosis.

In animals, cells are dependent on extracellular signals to prevent apoptosis. However, using growth factor-dependent cells from Bax/Bak-deficient mice, we demonstrate that apoptosis is not essential to limit cell autonomous survival. Following growth factor withdrawal, Bax-/-Bak-/- cells activate autophagy, undergo progressive atrophy, and ultimately succumb to death. These effects result from loss of the ability to take up sufficient nutrients to maintain cellular bioenergetics. Despite abundant extracellular nutrients, growth factor-deprived cells maintain ATP production from catabolism of intracellular substrates through autophagy. Autophagy is essential for maintaining cell survival following growth factor withdrawal and can sustain viability for several weeks. During this time, cells respond to growth factor readdition by rapid restoration of the ability to take up and metabolize glucose and by subsequent recovery of their original size and proliferative potential. Thus, growth factor signal transduction is required to direct the utilization of sufficient exogenous nutrients to maintain cell viability.

HB-GAM inhibits proliferation and enhances differentiation of neural stem cells.

Proliferation of neural stem cells in the embryonic cerebral cortex is regulated by many growth factors and their receptors. Among the key molecules stimulating stem cell proliferation are FGF-2 and the FGF receptor-1. This ligand-receptor system is highly dependent on the surrounding heparan sulfates. We have found that heparin-binding growth-associated molecule (HB-GAM, also designated as pleiotrophin) regulates neural stem cell proliferation in vivo and in vitro. Deficiency of HB-GAM results in a pronounced, up to 50% increase in neuronal density in the adult mouse cerebral cortex. This phenotype arises during cortical neurogenesis, when HB-GAM knockout embryos display an enhanced proliferation rate as compared to wild-type embryos. Further, our in vitro studies show that exogenously added HB-GAM inhibits formation and growth of FGF-2, but not EGF, stimulated neurospheres, restricts the number of nestin-positive neural stem cells, and inhibits FGF receptor phosphorylation. We propose that HB-GAM functions as an endogenous inhibitor of FGF-2 in stem cell proliferation in the developing cortex.

Activation/division of lymphocytes results in increased levels of cytoplasmic activation/proliferation-associated protein-1: prototype of a new family of proteins.

We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

Alterations in the expression of MHC class I glycoproteins by B16BL6 melanoma cells modulate insulin receptor-regulated signal transduction and augment [correction of augments] resistance to apoptosis.

In a variety of malignancies, the immune-escape phenotype is associated, in part, with the inability of tumor cells to properly present their Ags to CTLs due to a deranged expression of MHC class I glycoproteins. However, these molecules were found to possess broader nonimmune functions, including participation in signal transduction and regulation of proliferation, differentiation, and sensitivity to apoptosis-inducing factors; processes, which are characteristically impaired during malignant transformation. We investigated whether the deranged expression of MHC class I expression by tumor cells could affect proper receptor-mediated signal transduction and accentuate their malignant phenotype. The malignant and H-2K murine MHC class I-deficient B16BL6 melanoma cells were characterized by an attenuated capacity to bind insulin due to the retention of corresponding receptor in intracellular stores. The restoration of H-2K expression in these cells, which abrogated their capacity to form tumors in mice, enhanced membrane translocation of the receptor, presumably, by modulating its glycosylation. The addition of insulin to H-2K-expressing melanoma cells cultured in serum-free conditions precluded apoptotic death by up-regulating the activity of protein kinase B (PKB)/Akt. In contrast, the deficiency for H-2K characteristic to the malignant clones was associated with a constitutive high activity of PKB/Akt, which rendered them resistant to apoptosis, induced by deprivation of serum-derived growth factors. The possibility to correct the regulation of PKB/Akt activity by restoration of H-2K expression in B16BL6 melanoma cells may be considered as an attractive approach for cancer therapy, since an aberrant activation of this enzyme is characteristic to resistant malignancies.

[Vasculogenesis and angiogenesis: molecular and cellular controls. part I: growth factors]. / Vasculogenèse et angiogenèse: contrôles moléculaires et cellulaires. lère Partie: les facteurs de croissance.

Angiogenesis characterizes embryonic development, but occurs also in adulthood, under physiological circumstances like adaptation to muscular exercise or in pathology like cancers. Knowledge of these mechanisms has step forward partly due to knock-out mice that have allowed to devoid an exact role to the different growth factors that are involved. Interestingly, the same growth factors and their receptors are equally involved during development and adulthood. We have detailed here their respective role and how interactions between them leads to a newly formed vessel.

Neural crest and cardiovascular development: a 20-year perspective.

BACKGROUND: Twenty years ago this year was the first publication describing a region of neural crest cells necessary for normal cardiovascular development. Ablation of this region in chick resulted in persistent truncus arteriosus, mispatterning of the great vessels, outflow malalignments, and hypoplasia or aplasia of the pharyngeal glands. METHODS: We begin with a historical perspective and then review the progress that has been made in the ensuing 20 years in determining the direct and indirect contributions of the neural crest cells, now termed cardiac neural crest cells, in cardiovascular and pharyngeal arch development. Many of the molecular pathways that are now known to influence the specification, migration, patterning and final targeting of the cardiac neural crest cells are also reviewed. RESULTS: Although much knowledge has been gained by using many genetic manipulations to understand the cardiac neural crest cells' role in cardiovascular development, most models fail to explain the phenotypes seen in syndromic and non-syndromic human congenital heart defects, such as the DiGeorge syndrome. CONCLUSIONS: We propose that the cardiac neural crest exists as part of a larger cardiocraniofacial morphogenetic field and describe several human syndromes that result from abnormal development of this field.

Caspase-2 redux.

It has been difficult to assign caspase-2 to the effector or initiator caspase groups. It bears sequence homology to initiators (caspase-9 and CED-3), but its cleavage specificity is closer to the effectors (caspase-3 and -7). Interest in caspase-2 was dampened by the lack of a dramatic phenotype in the caspase-2 null mouse. Studies have been inhibited by the lack of knowledge about its mechanism of activation and the lack of specific methods to assay its activity. Molecular studies have defined a unique role for caspase-2 in apoptosis initiated by beta-amyloid toxicity or by trophic factor deprivation. Recently, a role for caspase-2 as an upstream initiator of mitochondrial permeabilization has been proposed. Thus, while much remains to be deciphered about caspase-2, most critically the mode of activation, it is clear that caspase-2 plays critical and singular roles in the control of programmed cell death.