SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis

Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine as substrates to produce SM and diacylglycerol (DAG). To evaluate the role of SMS in apoptosis, we generated Chinese hamster ovary cells that stably express human SMS1 or SMS2. We found that SMS1 or SMS2 overexpression results in a significant increase in cellular levels of SM (24% or 20%) and DAG (35% or 31%), respectively, compared with controls. Cells overexpressing SMS1 or SMS2 were more likely to undergo lysis mediated by lysenin (a protein that causes lysis through its affinity with SM-rich microdomains in the plasma membrane) than were controls, indicating SM enrichment of the plasma membrane. SMS1 and SMS2 overexpression also led to higher retention of DiIC16 fluorescence compared with wild-type cells, indicating an increased number of detergent-insoluble microdomains and significantly increased tumor necrosis factor-alpha-mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 small interfering RNA (siRNA) to knock down their mRNA in THP-1-derived macrophages. We found that SMS1 or SMS2 siRNA significantly reduces intracellular SM (by 20% or 23%), plasma membrane SM (as indicated by the rate of lysenin-mediated cell lysis), and DAG levels (24% or 20%), respectively, while significantly reducing lipopolysaccharide-mediated apoptosis compared with controls. These results indicate that SMS1 and SMS2 are key factors in the control of SM and DAG levels within the cell and thus influence apoptosis.     

Quantitative Immunohistochemical Analysis Reveals Association between Sodium Iodide Symporter and Estrogen Receptor Expression in Breast Cancer

Background

Human sodium iodide symporter (hNIS) gene over-expression is under active consideration worldwide as an alternative target molecule for breast cancer (BC) diagnosis and targeted radio-iodine treatment. However, the field demands better stratified analysis of endogenous hNIS expression across major BC subtypes. Therefore, we have analyzed subtype-specific variation of hNIS overexpression in breast tumor tissue samples by immunohistochemistry (IHC) and also report the development of a homogeneous, quantitative analysis method of digital IHC images.

Methods

hNIS expression was analyzed from 108 BC tissue samples by IHC. Sub-cellular localization of hNIS protein was analyzed by dual immunofluorescence (IF) staining method using hNIS and HER2 antibodies. An ImageJ based two-step digital analysis method was developed and applied for the bias-free analysis of the images.

Results

Staining of the tumor samples show 70% cases are hNIS positive indicating high incidence of hNIS positive cases in BC. More importantly, a subtype specific analysis done for the first time shows that hNIS expression is overly dominated in estrogen receptor (ER) positive cases than the receptor negative cases. Further, 56% of the ER+ve, PgR+ve, HER2-ve and 36% of ER+ve, PgR+ve, HER2+ve cases show highest intensity staining equivalent to the thyroid tissue. A significant positive correlation is also observed between hNIS and estrogen receptor expression (p = 0.0033, CI = 95%) suggesting hNIS mediated targeted radio-iodine therapy procedures may benefit both ER+ve, PgR+ve, HER2–ve as well as HER2+ve cases. Further, in a few cases, hNIS and HER2 protein localization is demonstrated by overlapping membrane co-expression. ImageJ based image analysis method shows over 70% match with manual pathological scoring method.

Conclusion

The study indicates a positive link between hNIS and ER expression in BC. The quantitative IHC image analysis method reported here will further help in patient stratification and potentially benefit global clinical assessment where hNIS mediated targeted ¹³¹I radio-ablative therapy is aimed.     

Exploring the UDP pocket of LpxC through amino acid analogs

Lipopolysaccharide (LPS) biosynthesis is an attractive antibacterial target as it is both conserved and essential for the survival of key pathogenic bacteria. Lipid A is the hydrophobic anchor for LPS and a key structural component of the outer membrane of Gram-negative bacteria. Lipid A biosynthesis is performed in part by a unique zinc dependent metalloamidase, LpxC (UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase), which catalyzes the first non-reversible step in lipid A biosynthesis. The UDP portion of the LpxC substrate-binding pocket has been relatively unexplored. We have designed and evaluated a series of hydroxamate based inhibitors which explore the SAR of substitutions directed into the UDP pocket with a range of substituted α-amino acid based linkers. We also provide the first wild type structure of Pseudomonas aeruginosa LpxC which was utilized in the design of many of these analogs.     

Periostin Induces Intracellular Cross-talk between Kinases and Hyaluronan in Atrioventricular Valvulogenesis

Periostin (PN), a novel fasciclin-related matricellular protein, has been implicated in cardiac development and postnatal remodeling, but the mechanism remains unknown. We examined the role of PN in mediating intracellular kinase activation for atrioventricular valve morphogenesis using well defined explant cultures, gene transfection systems, and Western blotting. The results show that valve progenitor (cushion) cells secrete PN into the extracellular matrix, where it can bind to INTEGRINs and activate INTEGRIN/focal adhesion kinase signaling pathways and downstream kinases, PI3K/AKT and ERK. Functional assays with prevalvular progenitor cells showed that activating these signaling pathways promoted adhesion, migration, and anti-apoptosis. Through activation of PI3K/ERK, PN directly enhanced collagen expression. Comparing PN-null to WT mice also revealed that expression of hyaluronan (HA) and activation of hyaluronan synthase-2 (Has2) are also enhanced upon PN/INTEGRIN/focal adhesion kinase-mediated activation of PI3K and/or ERK, an effect confirmed by the reduction of HA synthase-2 in PN-null mice. We also identified in valve progenitor cells a potential autocrine signaling feedback loop between PN and HA through PI3K and/or ERK. Finally, in a three-dimensional assay to simulate normal valve maturation in vitro, PN promoted collagen compaction in a kinase-dependent fashion. In summary, this study provides the first direct evidence that PN can act to stimulate a valvulogenic signaling pathway.     

Spatial distribution of proteins and metabolites in developing wheat grain and their differential regulatory response during the grain filling process

Grain filling and grain development are essential biological processes in the plant’s life cycle, eventually contributing to the final seed yield and quality in all cereal crops. Studies of how the different wheat (Triticum aestivum L.) grain components contribute to the overall development of the seed are very scarce. We performed a proteomics and metabolomics analysis in four different developing components of the wheat grain (seed coat, embryo, endosperm, and cavity fluid) to characterize molecular processes during early and late grain development. In-gel shotgun proteomics analysis at 12, 15, 20, and 26 days after anthesis (DAA) revealed 15 484 identified and quantified proteins, out of which 410 differentially expressed proteins were identified in the seed coat, 815 in the embryo, 372 in the endosperm, and 492 in the cavity fluid. The abundance of selected protein candidates revealed spatially and temporally resolved protein functions associated with development and grain filling. Multiple wheat protein isoforms involved in starch synthesis such as sucrose synthases, starch phosphorylase, granule-bound and soluble starch synthase, pyruvate phosphate dikinase, 14-3-3 proteins as well as sugar precursors undergo a major tissue-dependent change in abundance during wheat grain development suggesting an intimate interplay of starch biosynthesis control. Different isoforms of the protein disulfide isomerase family as well as glutamine levels, both involved in the glutenin macropolymer pattern, showed distinct spatial and temporal abundance, revealing their specific role as indicators of wheat gluten quality. Proteins binned into the functional category of cell growth/division and protein synthesis/degradation were more abundant in the early stages (12 and 15 DAA). At the metabolome level all tissues and especially the cavity fluid showed highly distinct metabolite profiles. The tissue-specific data are integrated with biochemical networks to generate a comprehensive map of molecular processes during grain filling and developmental processes.