S100A1 Regulates Neurite Organization, Tubulin Levels, and Proliferation in PC12 Cells

Abstract

As a first step in determining what cellular processes are regulated by the calcium-modulated protein S100A1 isoform in neurons, the effects of ablated S100A1 expression on neurite organization and microtubule/tubulin levels in PC12 cells were examined. A mammalian expression vector containing the rat S100A1 cDNA in the antisense orientation with respect to a cytomegalovirus promoter was constructed and transfected into PC12 cells. Indirect immunofluorescence microscopy confirmed decreased S100A1 protein levels in all three stable transfectants (pAntisense clones) that expressed exogenous S100A1 antisense mRNA. In response to nerve growth factor, pAntisense clones extended significantly more neurites than control cells (4.01 ؎ 0.16 versus 2.93 ؎ 0.16 neurites/cell). This increase in neurite number was accompanied by an increase in total ␣-tubulin levels in untreated (4.0 ؎ 0.6 versus 1.76 ؎ 0.4 ng of ␣-tubulin/mg of total protein) and nerve growth factortreated pAntisense clones (4.15 ؎ 0.4 versus 2.04 ؎ 0.5 ng of ␣-tubulin/mg of total protein) when compared with control cells. At high cell densities, pAntisense clones exhibited a significant decrease in anchorage-dependent growth. In soft agar, pAntisense clones formed significantly more colonies (153 ؎ 8%) than control cells (116 ؎ 5%). However, the pAntisense soft agar colonies were significantly smaller than those observed in control cells (40.6 ؎ 3.0 versus 59.5 ؎ 1.2 m). These data suggest that cell density inhibits both anchorage-independent and -dependent growth of pAntisense clones. In summary, ablation of S100A1 expression in PC12 cells results in increased tubulin levels, altered neurite organization, and decreased cell growth. Thus, S100A1 may directly link the cytoskeleton and calcium signal transduction pathways to cell proliferation.The S100 protein family is a group of calcium-binding proteins that exhibit a high degree of conservation in amino acid sequence, secondary structure, and genomic organization (1-3). To date, there are 18 members of the S100 family, 13 of which are clustered in region q21 of human chromosome 1 (see Refs. 4 and 5). A new nomenclature that reflects the genomic organization of these proteins has recently been adopted, and S100␣ is now designated S100A1 and S100␤ is designated S100B (6). S100A1 and S100B were the original members of this family and have been implicated in a diverse group of cellular functions including cell-cell communication, cell growth, cell structure, energy metabolism, contraction, and intracellular calcium signal transduction (see Refs. 1 and 3).Because each S100 monomer contains two EF-hand high affinity calcium binding domains, they have been included in the S100-troponin-calmodulin superfamily of calcium-modulated proteins. These proteins have no known enzymatic activity and function by modulating the activity of other proteins termed target proteins. Calmodulin appears to be the "workhorse" in calcium signaling and to be responsible for generating the main events triggered by changes in intracell...

Calcyclin (S100A6) regulates pulmonary fibroblast proliferation, morphology, and cytoskeletal organization in vitro

“…For example, in nonmuscle cells it has been reported that PEL98 (S100A4), binds to the nonmuscle tropomyosin isoform 2, regulates tropomyosin-actin interactions and suppresses the metastatic potential of Lewis lung carcinoma cells [Takenaga et al, 1994]. In a similar manner, S100A1 expression in PC12 cells has been demonstrated by antisense inhibition experiments to be important in regulating tubulin levels, neurite organization and proliferate capacity [Zimmer et al, 1998]. S100A1 and S100A4 normally co-localize to actin stress fibers within the cell [Mandinova et al, 1998].…”

Intracellular and extracellular roles of S100 proteins

“…Inhibition of expression of S100A1 in PC12 cells also results in a decrease in cell proliferation rate (Zimmer et al, 1998), a finding that was interpreted as suggestive of a role of this protein in the modulation of the activity of an unknown transcription factor. S100A2 is markedly down-regulated in breast tumor biopsies and can be re-expressed in mammary carcinoma cells by azadeoxycytidine treatment (Lee et al, 1992).…”

Post-translational S-Nitrosylation Is an Endogenous Factor Fine Tuning the Properties of Human S100A1 Protein

“…To prove that, we used the PC12 model cell line, which is frequently used in the studies of S100A1 activity (23). We have studied the effects of S-nitrosylation on Ca 2ϩ binding and on the high resolution three-dimensional structure of human apo-S100A1, 4 aiming at the elucidation of yet unknown factors regulating various biological functions of the protein.…”

“…They are known to express S100A1 at a high level (23). PC12 cells not stimulated with nitric oxide donors or other compounds were used to determine whether endogenous S-nitrosylation of S100A1 protein might be observed in a cellular system.…”