Modulation of allergic responses by mitochondrial STAT3 inhibitors.

BACKGROUND: Recently, we have shown that mast cell mitochondrial STAT3 could serve as a new target for the regulation of the allergic response as it plays an essential role in immunologically mediated degranulation of mast cells. In the present work, we explored how two recently developed mitochondrial STAT3 inhibitors (Mitocur-1 and Mitocur-3) modulate the allergic response. METHODS: Experiments were performed both in vitro in cultured human/mouse mast cells and with rat basophilic leukemia (RBL) cells and also in vivo in mice. The effect of mitochondrial STAT3 inhibition on mast cell function was determined via checking degranulation and several cytokines secretion levels. RESULTS: Here, we show that treatment of rodent and human cultured mast cells with low concentrations of mitochondrial STAT3 inhibitors had no effect on STAT3 target gene expression. However, these inhibitors caused a significant reduction in mast cell exocytosis and cytokine release, due to a decrease in OXPHOS activity and STAT3 serine 727 phosphorylation. It was also observed in an OVA mouse model of allergic asthma that one of the inhibitors used significantly reduced eosinophilia and neutrophilia compared to the control mice group. Furthermore, it was observed that treatment with this inhibitor resulted in a significant reduction in blood histamine levels in mice after IgE-Ag challenge. CONCLUSION: The present data strongly suggest that the development of mitochondrial STAT3 inhibitors could serve as a potential treatment for allergy-associated diseases.

Transgenic expression of syndecan-1 uncovers a physiological control of feeding behavior by syndecan-3.

Transgenic expression in the hypothalamus of syndecan-1, a cell surface heparan sulfate proteoglycan (HSPG) and modulator of ligand-receptor encounters, produces mice with hyperphagia and maturity-onset obesity resembling mice with reduced action of alpha melanocyte stimulating hormone (alphaMSH). Via their HS chains, syndecans potentiate the action of agouti-related protein and agouti signaling protein, endogenous inhibitors of alphaMSH. In wild-type mice, syndecan-3, the predominantly neural syndecan, is expressed in hypothalamic regions that control energy balance. Food deprivation increases hypothalamic syndecan-3 levels several-fold. Syndecan-3 null mice, otherwise apparently normal, respond to food deprivation with markedly reduced reflex hyperphagia. We propose that oscillation of hypothalamic syndecan-3 levels physiologically modulates feeding behavior.

Syndecan-1 shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas aeruginosa.

Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.

Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns.

The syndecans are a gene family of four transmembrane heparan sulfate proteoglycans that bind, via their HS chains, diverse components of the cellular microenvironment. To evaluate the expression of the individual syndecans, we prepared cDNA probes to compare mRNA levels in various adult mouse tissues and cultured mouse cells representing various epithelial, fibroblastic, endothelial, and neural cell types and B cells at various stages of differentiation. We also prepared antibody probes to assess whether the extracellular domains of the individual syndecans are shed into the conditioned media of cultured cells. Our results show that all cells and tissues studied, except B-stem cells, express at least one syndecan family member; most cells and tissues express multiple syndecans. However, each syndecan family member is expressed selectively in cell-, tissue-, and development-specific patterns. The extracellular domain of all syndecan family members is shed as an intact proteoglycan. Thus, most, if not all, cells acquire a distinctive repertoire of the four syndecan family members as they differentiate, resulting in selective patterns of expression that likely reflect distinct functions.

Mapping of the syndecan genes in the mouse: linkage with members of the myc gene family.

The syndecans are a family of four cell surface heparan sulfate proteoglycans in vertebrates that mediate a variety of cell behaviors, including cell adhesion and the action of growth factors. Their core proteins contain conserved transmembrane and cytoplasmic domains but divergent extracellular regions in which only the glycosaminoglycan attachment sites are conserved. By extensive PCR analyses based on the conserved sequences, we find only four syndecan-related sequences in the mouse. These correspond to the previously described core proteins of syndecan proteoglycans from other vertebrates. We have mapped the genes for syndecan-2 to chromosome 15, syndecan-3 to chromosome 4, and syndecan-4 to chromosome 2 in the mouse. Together with the previous localization of the gene for syndecan-1 to chromosome 12, these data establish that the four syndecan genes are dispersed on different chromosomes and that each syndecan gene is located near a member of the myc gene family. Synd1 is next to Nmyc, Synd2 close to myc, Synd3 near Lmyc, and Synd4 on the same chromosome as Bmyc. The physical relationship between the members of these two gene families appears to be ancient and conserved after the two genome duplications thought to have occurred during vertebrate evolution.

Organization and promoter activity of the mouse syndecan-1 gene.

Syndecan-1, the prototype of a family of heparan sulfate-containing integral membrane proteoglycans, associates extracellularly with a variety of matrix molecules and growth factors and intracellularly with the actin cytoskeleton. Expressed constitutively on epithelia in mature tissues and in a developmentally regulated manner on epithelial and induced mesenchymal cells during embryogenesis, syndecan-1 appears to be involved in controlling the shape and organization of cells and tissues. To better understand the function and regulation of syndecan-1, we determined the structure of the mouse syndecan-1 gene (Synd-1). Synd-1 is approximately 19.5 kilobases in size and is organized into five exons that appear conserved in other family members. Exon 1 encodes the signal peptide; exon 2, the N-terminal glycosaminoglycan attachment region; exon 3, the bulk of the extracellular domain; exon 4, the protease-susceptible site; and exon 5, the transmembrane and cytoplasmic domains which are highly homologous between syndecan family members. Synd-1 has three transcriptional start sites, two polyadenylation sites, and is not alternatively spliced to produce its 2.6- and 3.4-kilobase mRNA species. Upstream sequences have promoter activity and contain TATA and CAAT boxes as well as a variety of other potential binding sites for transcription factors, including Sp1 (GC box), NF-kappa B, MyoD (E box), and Antennapedia. The structure of the promoter region suggests that control of Synd-1 expression is both constitutive and developmentally regulated. Because Synd-1 exons encode discrete functional domains of the syndecan-1 protein that are conserved throughout the syndecan family, all syndecan genes are likely derived from a common ancestor.