Intracellular Ca2+ and Zn2+ Levels Regulate the Alternative Cell Density-dependent Secretion of S100B in Human Glioblastoma Cells

Abstract

In recent years, protein translocation has been implicated as the mechanism that controls assembly of signaling complexes and induction of signaling cascades. Several members of the multifunctional Ca 2؉ -(Zn 2؉ -and Cu 2؉ )-binding S100 proteins appear to translocate upon cellular stimulation, and some are even secreted from cells, exerting extracellular functions. We transfected cells with S100B-green fluorescent fusion proteins and followed the relocation in real time. A small number of cells underwent translocation spontaneously. However, the addition of thapsigargin, which increases Ca 2؉ levels, intensified ongoing translocation and secretion or induced these processes in resting cells. On the other hand, EGTA or BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid), the Ca 2؉ -chelating agents, inhibited these processes. In contrast, relocation of S100B seemed to be negatively dependent on Zn 2؉ levels. Treatment of cells with TPEN (N,N,N,N-tetrakis(2-pyridylmethyl)ethylenediamine), a Zn 2؉ -binding drug, resulted in a dramatic redistribution and translocation of S100B. Secretion of S100B, when measured by ELISA, was dependent on cell density. As cells reached confluence the secretion drastically declined. However, an increase in Ca 2؉ levels, and even more so, a decrease in Zn 2؉ concentration, reactivated secretion of S100B. On the other hand, secretion did not decrease by treatment with brefeldin A, supporting the view that this process is independent of the endoplasmic reticulum-Golgi classical secretion pathway. S100B is a small acidic protein containing two distinct EFhands, predominantly expressed in astrocytes, oligodendrocytes, and Schwann cells. Intracellularly, S100B regulates the cytoskeletal dynamics through disassembly of tubulin filaments and binding to fibrillary proteins such as CapZ (1-4). Furthermore, S100B interacts in a Ca 2ϩ -dependent manner with the cytoplasmic domain of myelin-associated glycoprotein and inhibits its phosphorylation by protein kinase A (5). Similarly, interaction of S100B with p53 was shown to inhibit protein kinase C phosphorylation (6, 7). When secreted by astrocytes, in addition to an autocrine effect leading, for example, to the activation of extracellular signal-regulated kinase (8), S100B can have a paracrine effect on neurons, promoting their survival during development and after injury through the NF-B pathway, as well as through neurite outgrowth (1, 9 -11). However, the extracellular concentration of S100B plays a crucial role in the physiological response. Although nanomolar quantities have trophic effects on cells, high levels of this protein have been implicated in glial activation (a prominent feature in Down syndrome and Alzheimer's disease), up-regulation of nitric-oxide synthase, and apoptosis (11-13). Recently, Schmidt and colleagues (14, 15) identified the surface receptor RAGE (receptor for advanced glycation endproducts) for S100B, shedding more light on its extracellular function. Others also demonstrated that the binding of extracel...

Nuclear expression of S100A4 is associated with aggressive behavior of epithelial ovarian carcinoma: An important autocrine/paracrine factor in tumor progression

“…In addition, our immunofluorescent analysis showed that S100A4 treatment resulted in the translocation of S100A4 from the cytoplasm into the nucleus in SKOV3 cells. Although S100A4 was first identified as cytoplasmic protein, its translocation between the cytoplasm and the nucleus has been reported in human cells in vitro, such as endothelial cells, (30) fibroblasts, (31) glioblastoma cells, (32) and colorectal carcinoma.…”

The Extracellular Region of the Receptor for Advanced Glycation End Products Is Composed of Two Independent Structural Units

“…Figure 1C shows the sequence coverage obtained for both intact sRAGE ( Figure 1B, arrow a) and the ~25 kDa fragment. Notably, coverage obtained for the V and C1 domains was nearly complete in the 25 kDa band ( Figure 1B, arrow b), ranging from a peptide encoding a region of the N-terminus (23)(24)(25)(26)(27)(28)(29) to a peptide near the C-terminus of C1 (199-216).…”

“…These EF-hand Ca 2+ -binding proteins are best known as mediators of intracellular Ca 2+ signals, although they are also exported from the cell by a yet unknown mechanism (29). S100 proteins have an exposed hydrophobic binding pocket that mediates protein-protein interactions (30)(31)(32).…”

The Calcium-binding Protein S100B Down-regulates p53 and Apoptosis in Malignant Melanoma

“…There are now over twenty S100 proteins widely distributed in human tissue (13)(14)(15), including a large numbers of cancers (16 -18), making it the largest family of EF-hand calcium-binding proteins (19). S100B, a 21.5-kDa symmetric homodimer, is overexpressed in malignant melanoma (18), anaplastic astrocytomas (20), and glio-blastomas (21), and in the case of malignant melanoma, high concentrations of S100B correlate directly with poor prognosis in patients (22). More recently, S100B has gained attention, because it binds directly to the p53 tumor suppressor protein in melanoma (23)(24)(25)(26), reduces p53 protein levels (26,27), and inhibits wild-type p53 functions in malignant melanoma (26 -28).…”