Serglycin secretion is part of the inflammatory response in activated primary human endothelial cells in vitro

Background

Endothelial cells have important functions in e.g. regulating blood pressure, coagulation and host defense reactions. Serglycin is highly expressed by endothelial cells, but there is limited data on the roles of this proteoglycan in immune reactions.

Methods

Cultured primary human endothelial cells were exposed to proinflammatory agents lipopolysaccharide (LPS) and interleukin 1β (IL-1β). The response in serglycin synthesis, secretion and intracellular localization and effect on the proteoglycan binding chemokines CXCL-1 and CXCL-8 were determined by qRT-PCR, Western blotting, immunocytochemistry, ELISA and serglycin knockdown experiments.

Results

Both LPS and IL-1β increased the synthesis and secretion of serglycin, while only IL-1β increased serglycin mRNA expression. Stimulation increased the number of serglycin containing vesicles, with a greater portion of large vesicles after LPS treatment. Also, increased intracellular and secreted levels of CXCL-1 and CXCL-8 were observed. The increase in CXCL-8 secretion was unchanged in serglycin knockdown cells. However, the increase in CXCL-1 secretion from IL-1β stimulation was reduced 27% in serglycin knockdown cells; while the LPS-induced secretion was not affected. In serglycin expressing cells CXCL-1 positive vesicles were evenly distributed throughout the cytoplasm, while confided to the Golgi region in serglycin knockdown cells. This was the case only for IL-1β stimulated cells. LPS-induced CXCL-1 distribution was unaffected by serglycin expression.

Conclusions

These results suggest that different signaling pathways are involved in regulating secretion of serglycin and partner molecules in activated endothelial cells.

General significance

This knowledge increases our understanding of the roles of serglycin in immune reactions. This article is part of a Special Issue entitled: Matrix-mediated cell behaviour and properties.     

Relatedness facilitates cooperation in the subsocial spider, Stegodyphus tentoriicola

Background  

Cooperative hunting and foraging in spiders is rare and prone to cheating such that the actions of selfish individuals negatively affect the whole group. The resulting social dilemma may be mitigated by kin selection since related individuals lose indirect fitness benefits by acting selfishly. Indeed, cooperation with genetic kin reduces the disadvantages of within-group competition in the subsocial spider Stegodyphus lineatus, supporting the hypothesis that high relatedness is an important pre-adaptation in the transition to sociality in spiders. In this study we examined the consequences of group size and relatedness on cooperative feeding in the subsocial spider S. tentoriicola, a species suggested to be at the transition to permanent sociality.     

Multi-site-specific 16S rRNA methyltransferase RsmF from Thermus thermophilus

Cells devote a significant effort toward the production of multiple modified nucleotides in rRNAs, which fine tune the ribosome function. Here, we report that two methyltransferases, RsmB and RsmF, are responsible for all four 5-methylcytidine (m⁵C) modifications in 16S rRNA of Thermus thermophilus. Like Escherichia coli RsmB, T. thermophilus RsmB produces m⁵C967. In contrast to E. coli RsmF, which introduces a single m⁵C1407 modification, T. thermophilus RsmF modifies three positions, generating m⁵C1400 and m⁵C1404 in addition to m⁵C1407. These three residues are clustered near the decoding site of the ribosome, but are situated in distinct structural contexts, suggesting a requirement for flexibility in the RsmF active site that is absent from the E. coli enzyme. Two of these residues, C1400 and C1404, are sufficiently buried in the mature ribosome structure so as to require extensive unfolding of the rRNA to be accessible to RsmF. In vitro, T. thermophilus RsmF methylates C1400, C1404, and C1407 in a 30S subunit substrate, but only C1400 and C1404 when naked 16S rRNA is the substrate. The multispecificity of T. thermophilus RsmF is potentially explained by three crystal structures of the enzyme in a complex with cofactor S-adenosyl-methionine at up to 1.3 Å resolution. In addition to confirming the overall structural similarity to E. coli RsmF, these structures also reveal that key segments in the active site are likely to be dynamic in solution, thereby expanding substrate recognition by T. thermophilus RsmF.     

Changes in fecal microbiota of healthy dogs administered amoxicillin

The effect of oral amoxicillin treatment on fecal microbiota of seven healthy adult dogs was determined with a focus on the prevalence of bacterial antibiotic resistance and changes in predominant bacterial populations. After 4–7 days of exposure to amoxicillin, fecal Escherichia coli expressed resistance to multiple antibiotics when compared with the pre-exposure situation. Two weeks postexposure, the susceptibility pattern had returned to pre-exposure levels in most dogs. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations using denaturing gradient gel electrophoresis (PCR-DGGE) of the 16S V3 rRNA gene region. Much of the variation in DGGE profiles could be attributed to dog-specific factors. However, permutation tests indicated that amoxicillin exposure significantly affected the DGGE profiles after controlling for the dog effect ( P =0.02), and pre-exposure samples were clearly separated from postexposure samples. Sequence analysis of DGGE bands and real-time PCR quantification indicated that amoxicillin exposure caused a shift in the intestinal ecological balance toward a Gram-negative microbiota including resistant species in the family Enterobacteriaceae .     

Functional diversity of FGF-2 isoforms by intracellular sorting

Regulation of the subcellular localization of certain proteins is a mechanism for the regulation of their biological activities. FGF-2 can be produced as distinct isoforms by alternative initiation of translation on a single mRNA and the isoforms are differently sorted in cells. High molecular weight FGF-2 isoforms are not secreted from the cell, but are transported to the nucleus where they regulate cell growth or behavior in an intracrine fashion. 18 kDa FGF-2 can be secreted to the extracellular medium where it acts as a conventional growth factor by binding to and activation of cell-surface receptors. Furthermore, following receptor-mediated endocytosis, the exogenous FGF-2 can be transported to the nuclei of target cells, and this is of importance for the transmittance of a mitogenic signal. The growth factor is able to interact with several intracellular proteins. Here, the mode of action and biological role of intracellular FGF-2 are discussed.     

Internalization of the human CRF receptor 1 is independent of classical phosphorylation sites and of beta-arrestin 1 recruitment.

The corticotropin releasing factor receptor 1 (CRFR1) belongs to the superfamily of G-protein coupled receptors. Though CRF is involved in the aetiology of several stress-related disorders, including depression and anxiety, details of CRFR1 regulation such as internalization remain uncharacterized. In the present study, agonist-induced internalization of CRFR1 in HEK293 cells was visualized by confocal microscopy and quantified using the radioligand 125I-labelled sauvagine. Recruitment of beta-arrestin 1 in response to receptor activation was demonstrated by confocal microscopy. The extent of 125I-labelled sauvagine stimulated internalization was significantly impaired by sucrose, indicating the involvement of clathrin-coated pits. No effect on the extent of internalization was observed in the presence of the second messenger dependent kinase inhibitors H-89 and staurosporine, indicating that cAMP-dependent protein kinase and protein kinase C are not prerequisites for CRFR1 internalization. Surprisingly, deletion of all putative phosphorylation sites in the C-terminal tail, as well as a cluster of putative phosphorylation sites in the third intracellular loop, did not affect receptor internalization. However, these mutations almost abolished the recruitment of beta-arrestin 1 following receptor activation. In conclusion, we demonstrate that CRFR1 internalization is independent of phosphorylation sites in the C-terminal tail and third intracellular loop, and the degree of beta-arrestin 1 recruitment.     

Mutation-induced dimerization of transforming growth factor-β–induced protein may drive protein aggregation in granular corneal dystrophy

Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-β–induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography–small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD.     

Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth medium supplemented with serum (3T3 system) or without feeder cells in a dedicated serum-free medium (EpiLife). During the culture, the cells were maintained either at ambient oxygen tension (20%) or at different levels of hypoxia (15, 10, 5, and 2% oxygen). The effect of oxygen on cell growth, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET).