Membrane palmitoylated proteins regulate trafficking and processing of nectins

Nectins are cell-cell adhesion molecules involved in the formation of various intercellular junctions and the establishment of apical-basal polarity at cell-cell adhesion sites. To have a better understanding of the roles of nectins in the formation of cell-cell junctions, we searched for new cytoplasmic binding partners for nectin. We report that nectin-1α associates with membrane palmitoylated protein 3 (MPP3), one of the human homologues of a Drosophila tumor suppressor gene, Disc large. Two major forms of MPP3 at 66 and 98 kDa were detected, in conjunction with nectin-1α, suggesting that an association between the two may occur in various cell types. Nectin-1α recruits MPP3 to cell-cell contact sites, mediated by a PDZ-binding motif at the carboxyl terminus of nectin-1α. Association with MPP3 increases cell surface expression of nectin-1α and enhances nectin-1α ectodomain shedding, indicating that MPP3 regulates trafficking and processing of nectin-1α. Further study showed that MPP3 interacts with nectin-3α, but not with nectin-2α, showing that the association of nectins with MPP3 is isoform-specific. MPP5, another MPP family member, interacts with nectins with varying affinity and facilitates surface expression of nectin-1α, nectin-2α, and nectin-3α. These data suggest that wide interactions between nectins and MPP family members may occur in various cell-cell junctions and that these associations may regulate trafficking and processing of nectins.     

Biphasic regulation of tissue plasminogen activator activity in ischemic rat brain and in cultured neural cells: essential role of astrocyte-derived plasminogen activator inhibitor-1

In brain, the serine protease tissue plasminogen activator (tPA) and its endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1) have been implicated in the regulation of various neurophysiological and pathological responses. In this study, we investigated the differential role of neurons and astrocytes in the regulation of tPA/PAI-1 activity in ischemic brain. The activity of tPA peaked transiently and then decreased in cortex and striatum along with delayed induction of PAI-1 in the inflammatory stage after MCAO/reperfusion injury. In cultured primary cells, glutamate stimulation increased tPA activity in neurons but not in other cells such as microglia and astrocytes. With LPS stimulation, a model of neuroinflammatory insults, robust PAI-1 induction was observed in astrocytes but not in neurons and microglia. The upregulation of PAI-1 by LPS in astrocytes was also verified by RT-PCR analysis as well as PAI-1 promoter reporter assay. Lastly, we checked the effects of hypoxia on tPA/PAI-1 activity. Hypoxia increased tPA release from neurons without effects on microglia, while the activity of tPA in astrocyte was decreased consistent with increased PAI-1 activity in astrocyte. Taken together, the results from the present study suggest that neurons are the major source of tPA and that the glutamate-induced stimulated release is mainly governed by neurons in the acute phase. In contrast, the massive up-regulation of PAI-1 in astrocytes during subchronic and chronic inflammatory conditions, leads to decreased tPA activity in the later stages of MCAO. Differential regulation of tPA and PAI-1 in neurons, astrocytes and microglia suggest more attention is required to understand the role of local tPA activity in the vicinity of individual cell types.     

SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells

Purpose

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.

Methods

3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.

Results

AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.

Conclusion

SRJ23 inhibited the growth of prostate cancer cells by inducing G₂/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.     

Lack of association between AQP4 polymorphisms and risk of inflammatory demyelinating disease in a Korean population

Multiple sclerosis (MS) and neuromyelitis optica (NMO) are demyelinating autoimmune inflammatory diseases that affect the central nervous system (CNS). Previous genome-wide or candidate gene studies have suggested that genetic variants might be associated with the risk of MS or NMO. Aquaporin 4 (AQP4) is a commonly distributed water channel in astrocytes of the CNS, and its expression is decreased in NMO lesions due to astrocyte cytotoxicity. Previous studies have suggested the associations of AQP4 single nucleotide polymorphisms (SNPs) with MS and/or NMO. However, there have been few replication studies in various ethnic populations. This study, as the first of its kind performed in an Asian population, investigated associations of AQP4 SNPs with the risk of inflammatory demyelinating disease (IDD), including MS and NMO, in a Korean population. A total of seven common AQP4 SNPs were selected based on status of linkage disequilibrium (LD), and then genotyped in 178 IDD cases (79 MS and 99 NMO patients) and 237 normal controls. Statistical analyses showed no significant associations between AQP4 SNPs/haplotypes and development of IDD, including MS and NMO (P > 0.05). Further replications in larger cohorts and other ethnic groups are needed.     

Association of CHRDL1 Mutations and Variants with X-linked Megalocornea,Neuh?user Syndrome and Central Corneal Thickness

We describe novel CHRDL1 mutations in ten families with X-linked megalocornea (MGC1). Our mutation-positive cohort enabled us to establish ultrasonography as a reliable clinical diagnostic tool to distinguish between MGC1 and primary congenital glaucoma (PCG). Megalocornea is also a feature of Neuhäuser or megalocornea-mental retardation (MMR) syndrome, a rare condition of unknown etiology. In a male patient diagnosed with MMR, we performed targeted and whole exome sequencing (WES) and identified a novel missense mutation in CHRDL1 that accounts for his MGC1 phenotype but not his non-ocular features. This finding suggests that MMR syndrome, in some cases, may be di- or multigenic. MGC1 patients have reduced central corneal thickness (CCT); however no X-linked loci have been associated with CCT, possibly because the majority of genome-wide association studies (GWAS) overlook the X-chromosome. We therefore explored whether variants on the X-chromosome are associated with CCT. We found rs149956316, in intron 6 of CHRDL1, to be the most significantly associated single nucleotide polymorphism (SNP) (p = 6.81×10⁻⁶) on the X-chromosome. However, this association was not replicated in a smaller subset of whole genome sequenced samples. This study highlights the importance of including X-chromosome SNP data in GWAS to identify potential loci associated with quantitative traits or disease risk.     

CD4+CD25hiCD127low Regulatory T Cells Are Increased in Oral Squamous Cell Carcinoma Patients

Regulatory T cells (Tregs), a subset of CD4⁺ T cells plays a pivotal role in regulating the immune system. An increase in Treg numbers enables cancer progression by dampening the immune system and allowing tumor cells to evade immune detection and destruction. An increase in Treg numbers and expression of inhibitory cytokines including TGF-β and IL-10 are mechanisms by which Tregs exert their immune suppressive function. However, the presence of Tregs and inhibitory cytokines in oral cancer patients is still unclear. In this study, the presence of circulating Tregs in 39 oral cancer patients and 24 healthy donors was examined by studying the presence of the CD4⁺CD25^hiCD127^low cell population in their peripheral blood mononuclear cells using flow cytometry. Serum levels of TGF-β and IL-10 were measured by ELISA. T cell subsets of OSCC patients were found to differ significantly from healthy donors where a decrease in CD8⁺ cytotoxic T cells and an increase in Tregs (CD4⁺CD25^hiCD127^low) were observed. Further, the ratio of CD8⁺ T cells/Tregs was also decreased in patients compared to healthy donors. The presence of Tregs was accompanied by a decrease in IL-10 but not TGF-β secretion in OSCC patients when compared to donors; in addition, the analysis also revealed that an increased presence of Tregs was accompanied by better patient survival. Amongst OSCC patients, smokers had significantly higher levels of TGF-β. It is apparent that the immune system is compromised in OSCC patients and the characterization of the Treg subpopulation could form a basis for improving our understanding of the perturbations in the immune system that occur during OSCC tumorigenesis.