Macrophage activation increases the invasive properties of hepatoma cells by destabilization of the adherens junction

AbstractTumor-associated macrophages play an important role in tumor progression, but whether they exert a tumor-progressive effect remains controversial. Here, we demonstrated that activated macrophage-conditioned medium (AMCM) obtained from RAW macrophages (RAW/AMCM) induced epithelial-mesenchymal transition (EMT) and stimulated the migratory and invasive activities of HepG2 cells, whereas control conditioned media had no effect. Epithelial-cadherin (E-cadherin) and β-catenin staining patterns were altered at the adherens junctions by RAW/AMCM treatment, with an approximately 50% decrease in E-cadherin and β-catenin in the cell membrane. Importantly, levels of β-catenin-associated E-cadherin were also decreased. Following RAW/AMCM treatment, enhanced activation of c-Src was seen prior to increased tyrosine phosphorylation of β-catenin, and this led to the destabilization of adherens junctions. Pretreatment of HepG2 cells with the Src kinase inhibitor, PP2, completely abolished the effects of RAW/AMCM on the EMT, migration, invasion, and expression and association of E-cadherin and β-catenin. AMCMs obtained from human THP-1 monocytes and mouse peritoneal macrophages also caused disassembly of the adherens junctions and migration of HepG2 cells. Furthermore, inhibition of the epidermal growth factor receptor (EGFR) with gefitinib partially prevented the downregulation of E-cadherin and β-catenin at the adherens junctions and migration behavior induced by RAW/AMCM. Our results suggest that activated macrophages have a tumor-progressive effect on HepG2 cells which involves the c-Src- and EGFR-dependent signaling cascades

A lipid droplet-specific capsule is present in rat adrenal cells: evidence from a monoclonal antibody

　　 We have used a monoclonal antibody, A2, to study the structure and function on the lipid droplet capsule in steroidogenic adrenal cells . This antibody reacts with a 160-kD protein found in the rat adrenal cortex. Immunofluorescence microscopy shows a dominant rim pattern, which surrounds individual lipid droplets and is distinct from the filamentous vimentin staining. The boundary of lipid droplets in steroidgenic Leydig cells and 3T3 adipocytes is also immunostained by this antibody . The strong association of the 160-kD protein with the lipid droplet demonstrated by its resistance to Triton X- 100 extraction. Stimulation of steroid secretion by adrenocorticotropin results in the detachment of this protein from the lipid droplet and its movement to the cytosol. These findings suggest that the translocation of this 160-kD protein from lipid droplet surface to cytosol on stimulation might be important in facilitating the binding of cholesterol ester hydrolase to the surface of lipid droplet, as proposed for adipocytes, during lipolytic stimulation.#1490

Effects of a Water-Soluble Extract of Cordyceps Sinensis on Steroidogenesis and Capsular Morphology of Lipid Droplets in Cultured Rat

Cordyceps sinensis contains a factor that stimulates Cordyceps sinensis contains a factor that stimulates corticosteroid production in the animal model. However, it is not known whether this drug acts directly on the adrenal glands or indirectly via the hypothalamus-pituitary axis. In the present study, we used primary rat adrenal cell cultures to investigate the pharmacological function of a water-soluble extract of Cordyceps sinensis (CS) and the signaling pathway involved. Radioimmunoassay of corticosterone indicated that the amount of corticosterone produced by adrenal cells is increased in a positively dose- dependent manner by CS, reaching a maximum at 25 microg/ml. This stimulating effect was seen 1 h after CS treatment and was maintained for up to 24 h. Concomitantly, the lipid droplets in these cells became small and fewer in number. Immunostaining with a monoclonal antibody, A2, a specific marker for the lipid droplet capsule, demonstrated that detachment of the capsule from the lipid droplet occurs in response to CS application and that the period required for decapsulation is inversely related to the concentration of CS applied. The mechanism of CS-induced steroidogenesis is apparently different from that for ACTH, since intracellular cAMP levels were not increased in CS-treated cells. However , combined application with calphostin C, a PKC inhibitor, completely blocked the effect of CS on steroidogenesis, suggesting that activation of PKC may be responsible for the CS-induced steroidogenesis

Role of M-Line Proteins in Sarcomeric Titin Assembly during Cardiac Myofibrillogenesis

A rat polyclonal anti-M-line protein antiserum and three A rat polyclonal anti-M-line protein antiserum and three mouse monoclonal anti-titin antibodies (E2, F3, and A12) were used to study the spatiotemporal relationship between M-line proteins and titin during myofibril assembly in cultured chicken cardiomyocytes by immunofluorescence microscopy. In day 2 cultures, M-line proteins and titin were detected as punctate staining in most cardiomyocytes, which possessed many nonstriated fibrils. At a late stage ( day 3 cultures), M-line proteins were incorporated into dot- like structures along nonstriated fibrils, while titin staining was continuous on these structures. As development progressed, M -line proteins were registered in periodic pattern in the mid-A band. In cardiomyocytes from day 5 cultures, the titin bands were separated by an unstained region, and achieved their adult doublet pattern. Thus, the organization of titin in the sarcomere appears to occur later than that of M-line proteins in the M-line. Our morphological data indicate that the early registration of M-line proteins in primitive myofibrils may guide titin filament alignment via interaction between M-line proteins and titin . In order to investigate the role of M-line proteins in the assembly of titin filaments, anti-M-line protein or anti-titin antibodies were introduced into cultured cardiomyocytes by electroporation to functionally bind the respective proteins , and the profile of myofibril assembly was examined. Cardiomyocytes from day 2-3 cultures with incorporated anti- M- line protein antibodies became shrunk, and exhibited defective myofibrillar assembly, as shown by the failure of titin to assemble into a typical sarcomeric pattern. Incorporation of anti-titin antibody E2, which recognizes the M-line end domain of titin, resulted in the failure of M -line proteins organized into the M-line structure, as shown by random, sporadic staining with anti-M-line protein antibody. These studies confirm the essential role of M-line proteins in the organization of titin filaments in the sarcomere and that the interaction between titin and M-line proteins is crucial to the formation of the M-line structure