Polyubiquitination of prolactin receptor stimulates its internalization, postinternalization sorting, and degradation via the lysosomal pathway

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48- and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.     

Leptin modulates the survival of autoreactive CD4+ T cells through the nutrient/energy-sensing mammalian target of rapamycin signaling pathway

Chronic inflammation can associate with autoreactive immune responses, including CD4(+) T cell responses to self-Ags. In this paper, we show that the adipocyte-derived proinflammatory hormone leptin can affect the survival and proliferation of autoreactive CD4(+) T cells in experimental autoimmune encephalomyelitis, an animal model of human multiple sclerosis. We found that myelin olygodendrocyte glycoprotein peptide 35-55 (MOG(35-55))-specific CD4(+) T cells from C57BL/6J wild-type mice could not transfer experimental autoimmune encephalomyelitis into leptin-deficient ob/ob mice. Such a finding was associated with a reduced proliferation of the transferred MOG(35-55)-reactive CD4(+) T cells, which had a reduced degradation of the cyclin-dependent kinase inhibitor p27(kip1) and ERK1/2 phosphorylation. The transferred cells displayed reduced Th1/Th17 responses and reduced delayed-type hypersensitivity. Moreover, MOG(35-55)-reactive CD4(+) T cells in ob/ob mice underwent apoptosis that associated with a downmodulation of Bcl-2. Similar results were observed in transgenic AND-TCR- mice carrying a TCR specific for the pigeon cytochrome c 88-104 peptide. These molecular events reveal a reduced activity of the nutrient/energy-sensing AKT/mammalian target of rapamycin pathway, which can be restored in vivo by exogenous leptin replacement. These results may help to explain a link between chronic inflammation and autoimmune T cell reactivity.     

Effect of gestational sex steroid exposure on limb development and endochondral ossification in the pregnant C57Bl/6J mouse: I. Medroxyprogesterone acetate

Although data supporting the teratogenic potential of intrauterine progestin exposure is lacking, concern persists among some individuals within the scientific community that these drugs have the potential for nongenital teratogenesis, especially with regard to limb reduction defects. Our laboratory has been interested in the ontogeny of steroid receptors in the developing embryo and in the role of steroid-receptor interactions in limb development, particularly the process of endochondral ossification. Since limb reduction defects can be produced from abnormal processes that are operative during organogenesis or during midgestation (vascular disruption) we have designed an animal study whereby embryos were exposed to sex steroids throughout organogenesis and fetal development. The present study assesses the effects of medroxyprogesterone acetate (MPA) on intrauterine endochrondral bone development specifically, as well as overall embryo-fetal development. Primagravid C57Bl/6J mice were treated via subdermal pellets which deliver MPA at dosages of 5.0, 50.0, and 500.0 mg/kg/day on gestational days 7 through 19. These doses were 25-, 250-, and 2,500-fold higher on a mg/kg basis than the human dose equivalent (HDE). No increases in nongenital malformations were noted at any evaluated MPA dosage level. At 25 X the HDE, MPA did not influence endochondral bone development as evidenced by a lack of significant effects on assessed bone growth parameters. In the 250- and 2,500-fold HDE dosage groups, MPA was shown to exert an embryotoxic effect inducing 48 and 100% resorptions respectively. Mean embryo weights/litter were significantly reduced by MPA exposure at 250 X the HDE. Intrauterine exposure to 250 X the MPA HDE induced reductions in humeral and femoral diaphyseal length in proportion to a reduction in overall growth. The data demonstrate that MPA, administered at dosages of up to several orders of magnitude in excess of the HDE and which permitted embryo survival, did not induce increases in the frequency of nongenital teratogenesis at any dose or gestational stage. Importantly, limb reduction defects were not noted even in instances where the dosage of MPA induced an inhibition of endochondral bone growth.     

Distribution of glutathione transferases in Gram-positive bacteria and Archaea

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.     

Interleukin-18 as a Therapeutic Target in Acute Myocardial Infarction and Heart Failure

Interleukin 18 (IL-18) is a proinflammatory cytokine in the IL-1 family that has been implicated in a number of disease states. In animal models of acute myocardial infarction (AMI), pressure overload, and LPS-induced dysfunction, IL-18 regulates cardiomyocyte hypertrophy and induces cardiac contractile dysfunction and extracellular matrix remodeling. In patients, high IL-18 levels correlate with increased risk of developing cardiovascular disease (CVD) and with a worse prognosis in patients with established CVD. Two strategies have been used to counter the effects of IL-18:IL-18 binding protein (IL-18BP), a naturally occurring protein, and a neutralizing IL-18 antibody. Recombinant human IL-18BP (r-hIL-18BP) has been investigated in animal studies and in phase I/II clinical trials for psoriasis and rheumatoid arthritis. A phase II clinical trial using a humanized monoclonal IL-18 antibody for type 2 diabetes is ongoing. Here we review the literature regarding the role of IL-18 in AMI and heart failure and the evidence and challenges of using IL-18BP and blocking IL-18 antibodies as a therapeutic strategy in patients with heart disease.     

Extracellular matrix of lymphoid tissues in the chick

We describe the immunohistochemical distribution of components of the extracellular matrix of the chick lymphoid system. In the thymus, basement membranes of epithelial cells bordering the lobules were intensely stained by laminin antibodies; fibronectin antibodies labeled the capsule and the septal matrix, and similar reactivity was seen with tropoelastin and gp 115 antibodies. No positivity was detected with any of the antibodies within the cortical parenchymal cells. Laminin was not detected in the medullary parenchyma, whereas fibronectin was present as coarse fibers. Tropoelastin and gp 115 appeared as a finer and more diffuse meshwork. In the bursa, laminin antibodies outlined the epithelial cells separating the cortex from the medulla. Fibronectin, tropoelastin, and gp 115 antibody stained the interfollicular septa and the cortical matrix, although to a different extent. Laminin was also detected in association with the interfollicular epithelium (IFE) basement membrane, whereas no staining was found underneath the follicle-associated epithelium (FAE). FAE cells not only lack a proper basement membrane but are also not separated from medullary lymphocytes by any of the other extracellular matrix components were investigated. Consequently, medullary lymphocytes are not sequestered, and can come easily into contact with antigens present in the intestinal lumen. All four antibodies stained the spleen capsule and spleen blood vessels, tropoelastin and gp 115 antibodies giving the strongest reactivity. A fine trabecular staining pattern was detected with gp 115 antibodies in the white pulp.