Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells

Human Adipose Tissue Endothelial Cells Promote Preadipocyte Proliferation

Adipogenesis is preceded by development of a microvascular network, and optimal functioning of adipose tissue as an energy store and endocrine organ is dependent on extensive vascularization. We have examined the role of endothelial cell-derived factors that influence the proliferation of human preadipocytes. Microvascular endothelial cells and preadipocytes were isolated from human omental and subcutaneous adipose tissue biopsies by use of a developed procedure of collagenase digest, immunoselection, and differential trypsinization. Conditioned medium from microvascular endothelial cell cultures promoted the proliferation of preadipocytes (P = <0.001) and (to a lesser extent) other cell types. No depot-specific differences in mitogenic capacity of microvascular endothelial cell medium or of preadipocyte response were observed. These results indicate that adipose tissue endothelial cells secrete soluble adipogenic factor(s)

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-1's 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-1's early adipogenic effects and highlight the complexity of the paracrine interplay between FGFs within human AT

Endocytosis of uncleaved tumor necrosis factor-alpha in macrophages

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