Wenda Shurety

Localization and post-Golgi trafficking of tumor necrosis factor-alpha in macrophages.

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine secreted by activated macrophages. In this study, we examined the intracellular distribution and trafficking of TNF-alpha. Immunofluorescence and immunogold localization demonstrated that in lipopolysaccharide (LPS)-stimulated RAW264 macrophages, the greatest concentration of TNF-alpha is found in the perinuclear Golgi complex. Staining of the Golgi complex appeared 20 min after activation of cells and persisted for 2-12 h, and TNF-alpha appeared on the cell surface only transiently during this time. The rate of disappearance of Golgi staining correlated with the release of the cleaved, mature TNF-alpha into the medium. Pulse chase labeling and subcellular fractionation studies indicated that both 26-kDa and 17-kDa forms of TNF-alpha may be present at the level of the Golgi complex. Post-Golgi trafficking of TNF-alpha was modulated by agents that disrupt the cytoskeleton. Interferon-gamma (IFN-gamma), which primes macrophages for TNF-alpha-dependent cellular cytotoxicity, potentiated the effect of LPS by sustaining enhanced intracellular pools of TNF-alpha and also promoted redistribution of TNF-alpha into post-Golgi vesicular compartments. We propose that the primary pool of biologically active TNF-alpha in activated macrophages is held in the Golgi complex and that the cytokine is recruited directly from this intracellular pool for release in response to tumor cells or pathogens.

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages.

Activated monocytes and macrophages secrete the inflammatory cytokine tumor necrosis factor-α (TNF-α). TNF-α is produced as a 26 kd transmembrane protein that is cleaved to release a 17 kd soluble protein. TNF-α in both forms is biologically active. The intracellular trafficking of membrane-associated TNF-α in lipopolysaccharide-activated mouse macrophages was assessed after treatment with the metalloprotease inhibitor BB-3103, which prevents the cleavage of pro–TNF-α. Immunoprecipitation and immunofluorescence studies showed sustained expression of cell-associated TNF-α in the presence of the inhibitor. Cell immunoreactivity and surface biotinylation revealed that uncleaved TNF-α accumulated on the cell surface and was endocytosed, appearing in intracellular vesicles. Perturbation of post-Golgi traffic blocked the surface expression of 26 kd TNF-α. Tracking a bolus of TNF-α over time in cycloheximide-treated cells confirmed that uncleaved TNF-α is first transported to the cell surface and subsequently endocytosed. Vesicular structures immunoreactive for TNF-α were identified as endosomes by double labeling. The secretory and membrane-associated endocytic trafficking of TNF-α provides a mechanism for modulating the quantity of biologically active 26 kd TNF-α expressed on macrophages, allowing regulation of paracrine and autocrine responses.

A putative role for endogenous FGF-2 in FGF-1 mediated differentiation of human preadipocytes

The defining characteristic of obesity is increased adipose tissue (AT) mass following chronic positive energy supply. AT mass is determined by adipocyte number and size, which reflect proliferation and differentiation of preadipocytes and hypertrophy of pre-existing adipocytes. The molecular pathways governing AT expansion are incompletely defined. We previously reported that FGF-1 primes proliferating primary human preadipocytes (phPA), thereby increasing adipogenesis. Here we examined whether FGF-1s adipogenic actions were due to modulation of other FGFs. Treatment of phPA with FGF-1 reduced FGF-2 mRNA/protein by 80%. To examine a putative functional role we performed siRNA knockdown studies. Following FGF-2 knockdown preadipocyte proliferation was decreased and expression of adipogenic genes (PPARγ, G3PDH and adiponectin) was increased at day 1 of differentiation. These results suggest that changes in endogenous FGF-2 levels contribute to FGF-1s early adipogenic effects and highlight the complexity of the paracrine interplay between FGFs within human AT.