Enhancement of prostaglandin D2-D prostanoid 1 signaling reduces intestinal permeability by stimulating mucus secretion.

Introduction: The intestinal barrier plays a crucial role in distinguishing foods from toxins. Prostaglandin D2 (PGD2) is one of the lipid-derived autacoids synthesized from cell membrane-derived arachidonic acid. We previously reported that pharmacological stimulation of PGD2 receptor, D prostanoid 1 (DP1) attenuated the symptoms of azoxymethane/dextran sodium sulfate-induced colitis and ovalbumin-induced food allergy in mouse models. These observations suggested that DP1 stimulation protects the intestinal barrier. The present study aimed to uncover the effects of DP1 stimulation on intestinal barrier function and elucidate the underlying mechanisms. Materials and methods: Intestinal permeability was assessed in mice by measuring the transfer of orally administered fluorescein isothiocyanate-dextran (40 kDa) into the blood. The DP1 agonist BW245C (1 mg/kg) was administered 10 min prior to dextran administration. The intestinal permeability was confirmed using the ex vivo everted sac method. Tight junction integrity was evaluated in vitro by measuring the transepithelial electrical resistance (TER) in the human intestinal epithelial cell line Caco-2. Mucus secretion was assessed by observing Alcian Blue-stained intestinal sections. Results: Pharmacological DP1 stimulation reduced intestinal permeability both in vivo and ex vivo. Immunohistochemical staining showed that DP1 was strongly expressed on the apical side of the epithelial cells. DP1 stimulation did not affect TER in vitro but induced mucus secretion from goblet cells. Mucus removal by a mucolytic agent N-acetyl-l-cysteine canceled the inhibition of intestinal permeability by DP1 stimulation. Conclusion: These observations suggest that pharmacological DP1 stimulation decreases intestinal permeability by stimulating mucus secretion.

Possibilities of skin coat color-dependent risks and risk factors of squamous cell carcinoma and deafness of domestic cats inferred via RNA-seq data.

The transcriptome data of skin cells from domestic cats with brown, orange, and white coats were analyzed using a public database to investigate the possible relationship between coat color-related gene expression and squamous cell carcinoma risk, as well as the mechanism of deafness in white cats. We found that the ratio of the expression level of genes suppressing squamous cell carcinoma to that of genes promoting squamous cell carcinoma might be considerably lower than the theoretical estimation in skin cells with orange and white coats in white-spotted cat. We also found the possibility of the frequent production of KIT lacking the first exon (d1KIT) in skin cells with white coats, and d1KIT production exhibited a substantial negative correlation with the expression of SOX10, which is essential for melanocyte formation and adjustment of hearing function. Additionally, the production of d1KIT was expected to be due to the insulating activity of the feline endogenous retrovirus 1 (FERV1) LTR in the first intron of KIT by its CTCF binding sequence repeat. These results contribute to basic veterinary research to understand the relationship between cat skin coat and disease risk, as well as the underlying mechanism.

Implication of HpEts in gene regulatory networks responsible for specification of sea urchin skeletogenic primary mesenchyme cells.

The large micromeres of the 32-cell stage of sea urchin embryos are autonomously specified and differentiate into primary mesenchyme cells (PMCs), giving rise to the skeletogenic cells. We previously demonstrated that HpEts, an ets-related transcription factor, plays an essential role in the specification of PMCs in sea urchin embryos. In order to clarify the function of HpEts in the gene regulatory network involved in PMC specification, we analyzed the zygotic expression pattern and the cis-regulatory region of HpEts, and examined the activity of the HpEts protein as a transcription factor. Intron-based PCR reveals that zygotic expression of HpEts starts at the cleavage stage, and that the rate of transcription reaches maximum at the unhatched blastula stage. A series of progressive deletions of the fragments from -4.2 kbp to +1206 bp of the HpEts, which directs PMC-specific expression, caused a gradual decrease in the specificity, implying that coordination of several cis-regulatory elements regulates the expression in PMCs. A minimum cis-element required for the temporal expression is located within a 10 bp from -243 bp to -234 bp. The HpEts protein remains in the cytoplasm of entire embryonic cells in the cleavage stage. At the unhatched blastula stage, the HpEts protein translocates into the nucleus in presumptive PMCs. Transactivation assays demonstrate that the HpEts protein activates a promoter of Spicule Matrix Protein 50 (SM50), which is a target of HpEts, which binds to the regulatory region of SM50.

HpSulf, a heparan sulfate 6-O-endosulfatase, is involved in the regulation of VEGF signaling during sea urchin development.

Cell surface heparan sulfate proteoglycans (HSPGs) play significant roles in the regulation of developmental signaling, including vascular endothelial growth factor (VEGF), fibroblast growth factor, Wnt and bone morphogenetic protein signaling, through modification of their sulfation patterns. Recent studies have revealed that one of the functions of heparan sulfate 6-O-endosulfatase (Sulf) is to remove the sulfate from the 6-O position of HSPGs at the cell surface, thereby regulating the binding activities of heparan sulfate (HS) chains to numerous ligands and receptors in animal species. In this study, we focused on the sea urchin Hemicentrotus pulcherrimus homolog of Sulf (HpSulf), and analyzed its expression pattern and functions during development. HpSulf protein was present throughout development and localized at cell surface of all blastomeres. In addition, the HS-specific epitope 10E4 was detected at the cell surface and partially colocalized with HpSulf. Knockdown of HpSulf using morpholino antisense oligonucleotides (MO) caused abnormal morphogenesis, and the development of MO-injected embryos was arrested before the hatched blastula stage, indicating that HpSulf is necessary for the early developmental process of sea urchin embryos. Furthermore, we found that injection of HpSulf mRNA suppressed the abnormal skeleton induced by overexpression of HpVEGF mRNA, whereas injection of an inactive form of HpSulf mRNA, containing mutated cysteines in the sulfatase domain, did not have this effect. Taken together, these results suggest that HpSulf is involved in the regulation of various signal transductions, including VEGF signaling, during sea urchin development.

Role of the nanos homolog during sea urchin development.

The nanos genes play important roles in the development of primordial germ cells in animal species. In the sea urchin, Hemicentrotus pulcherrimus, small micromere descendants specifically express HpNanos mRNA and this expression continues in the left coelomic pouch, which produces the major component of the adult rudiment. In this study, we showed that morpholino knockdown of HpNanos resulted in a delay of primary mesenchyme cell ingression and a decrease in the number of cells comprising the left coelomic pouch. Knockdown analysis in chimeras and whole embryos revealed the disappearance of small micromere descendants from the archenteron tip. Furthermore, the expression of HpNanos mRNA was induced in other cell lineages in the HpNanos-knockdown and micromere-deleted embryos. Taken together, our results suggest that HpNanos is involved in the inductive interaction of small micromere descendants with other cell lineages, and that HpNanos is required for the survival of small micromere descendants.

Decreased expression of hypothalamic neuropeptides in Huntington disease transgenic mice with expanded polyglutamine-EGFP fluorescent aggregates.

Huntington disease is caused by polyglutamine (polyQ) expansion in huntingtin. Selective and progressive neuronal loss is observed in the striatum and cerebral cortex in Huntington disease. We have addressed whether expanded polyQ aggregates appear in regions of the brain apart from the striatum and cortex and whether there is a correlation between expanded polyQ aggregate formation and dysregulated transcription. We generated transgenic mouse lines expressing mutant truncated N-terminal huntingtin (expanded polyQ) fused with enhanced green fluorescent protein (EGFP) and carried out a high-density oligonucleotide array analysis using mRNA extracted from the cerebrum, followed by TaqMan RT-PCR and in situ hybridization. The transgenic mice formed expanded polyQ-EGFP fluorescent aggregates and this system allowed us to directly visualize expanded polyQ aggregates in various regions of the brain without performing immunohistochemical studies. We show here that polyQ-EGFP aggregates were intense in the hypothalamus, where the expression of six hypothalamic neuropeptide mRNAs, such as oxytocin, vasopressin and cocaine-amphetamine-regulated transcript, was down-regulated in the transgenic mouse brain without observing a significant loss of hypothalamic neurons. These results indicate that the hypothalamus is susceptible to aggregate formation in these mice and this may result in the down-regulation of specific genes in this region of the brain.

Pro-apoptotic protein kinase C delta is associated with intranuclear inclusions in a transgenic model of Huntington's disease.

In order to investigate any effect of truncated mutant huntingtin (tNhtt) aggregation on protein kinase C (PKC) signaling in Huntington's disease (HD), we studied a possible association of PKC isoforms with the aggregates using cellular and transgenic models of HD. In this report we describe an association of mutant tNhtt with at least three PKC isoforms (alpha, delta, zeta), as revealed by co-immunoprecipitation assays and immunocytochemistry in a cellular model of HD (Neuro2a cells expressing tNhtt-150Q-EGFP), as well as a specific association of PKC delta with intranuclear aggregates in a transgenic model (R6/2 mice). Immunoblot analysis of isolated nuclear fractions shows an elevation of nuclear PKC delta in transgenic brain tissue. The observed elevation has a strong similarity with the apoptotic translocation of PKC delta detected in experiments with the mouse neuroblastoma Neuro2a cells. Using a Neuro2a cell line expressing tNhtt with the nuclear localization signal, we demonstrate the association of PKC delta with intranuclear aggregates and present evidence that accumulation of PKC delta in cell nuclei does not depend on mutant htt nuclear translocation. Our results suggest that the association of PKC delta with intranuclear htt-aggregates may affect its apoptotic function in a transgenic model of HD.