S100A1 deficiency results in prolonged ventricular repolarization in response to sympathetic activation.

S100A1 is a Ca(2+)-binding protein and predominantly expressed in the heart. We have generated a mouse line of S100A1 deficiency by gene trap mutagenesis to investigate the impact of S100A1 ablation on heart function. Electrocardiogram recordings revealed that after beta-adrenergic stimulation S100A1-deficient mice had prolonged QT, QTc and ST intervals and intraventricular conduction disturbances reminiscent of 2 : 1 bundle branch block. In order to identify genes affected by the loss of S100A1, we profiled the mutant and wild type cardiac transcriptomes by gene array analysis. The expression of several genes functioning to the electrical activity of the heart were found to be significantly altered. Although the default prediction would be that mRNA and protein levels are highly correlated, comprehensive immunoblot analyses of salient up- or down-regulated candidate genes of any cellular network revealed no significant changes on protein level. Taken together, we found that S100A1 deficiency results in cardiac repolarization delay and alternating ventricular conduction defects in response to sympathetic activation accompanied by a significantly different transcriptional regulation.

Immunocytochemical localization of S100A1 in mitochondria on cryosections of the rat heart.

Immunocytochemical localization studies of S100A1 in muscle cells have so far yielded variable and conflicting results mainly due to different sample preparation techniques for immunoelectron microscopy. To minimize denaturation by fixation and embedding, cryofixation and cryosectioning followed by immunolabelling were used in the present study. Rat hearts were gently prefixed in a mixture of paraformaldehyde and glutaraldehyde. Samples from left and right ventricles and left and right atria were cryoprotected by sucrose and shock-frozen in liquid nitrogen. Ultrathin cryosections were labelled with rabbit polyclonal antiserum against S100A1. The sections were then incubated with secondary antibody conjugated to FITC (for fluorescence microscopy) or with protein A conjugated to 5 nm gold particles (for electron microscopy). The most prominent sites immunolabelled for S100A1 were mitochondria. In the fluorescence microscope the labelling of mitochondria was intense, suppressing the labelling in other compartments. In accordance with previous studies labelling of sarcoplasmic reticulum, Z-lines, actin and myosin filaments could also be detected in the electron microscope.

Pathologies involving the S100 proteins and RAGE.

The S100 proteins are exclusively expressed in vertebrates and are the largest subgroup within the superfamily of EF-hand Ca2(+)-binding proteins Generally, S100 proteins are organized as tight homodimers (some as heterodimers). Each subunit is composed of a C-terminal, 'canonical' EF-hand, common to all EF-hand proteins, and a N-terminal, 'pseudo' EF-hand, characteristic of S100 proteins. Upon Ca2(+)-binding, the C-terminal EF-hand undergoes a large conformational change resulting in the exposure of a hydrophobic surface responsible for target binding A unique feature of this protein family is that some members are secreted from cells upon stimulation, exerting cytokine- and chemokine-like extracellular activities via the Receptor for Advanced Glycation Endproducts, RAGE. Recently, larger assemblies of some S100 proteins (hexamers, tetramers, octamers) have been also observed and are suggested to be the active extracellular species required for receptor binding and activation through receptor multimerization Most S100 genes are located in a gene cluster on human chromosome 1q21, a region frequently rearranged in human cancer The functional diversification of S100 proteins is achieved by their specific cell- and tissue-expression patterns, structural variations, different metal ion binding properties (Ca2+, Zn2+ and Cu2+) as well as their ability to form homo-, hetero- and oligomeric assemblies Here, we review the most recent developments focussing on the biological functions of the S100 proteins and we discuss the presently available S100-specific mouse models and their possible use as human disease models In addition, the S100-RAGE interaction and the activation of various cellular pathways will be discussed. Finally, the close association of S100 proteins with cardiomyopathy, cancer, inflammation and brain diseases is summarized as well as their use in diagnosis and their potential as drug targets to improve therapies in the future.

Interferon-alpha and transforming growth factor-beta co-induce growth inhibition of human tumor cells.

A hallmark of resistance to type I interferons (IFNs) is the lack of antiproliferative responses. We show here that costimulation with IFN-alpha and transforming growth factor beta-1 (TGF-beta) potentiates antiproliferative activity in a sensitive (ME15) and resistant (D10) human melanoma cell line. A DNA microarray-based search for proliferation control genes involved that are cooperatively activated by IFN-alpha and TGF-beta, yielded 28 genes. Among these are the insulin-like growth factor-binding protein 3 (IGFBP3) and the calcium-binding protein S100A2; we demonstrate, that recombinant IGFBP3 protein is a potent growth inhibitor requiring TGF-beta activity. The antiproliferative activity of S100A2 is significantly enhanced by IFN-alpha in stably transfected ME15 or D10 cell lines. We show for the first time that IFN-alpha is a potent inducer of intracellular calcium release required for activation of S100A2. Our study provides a functional link between IFN-alpha and TGF-beta signaling and extends the function of IFN signaling to calcium-sensitive processes.

Co-expression/co-location of S100 proteins (S100B, S100A1 and S100A2) and protein kinase C (PKC-beta, -eta and -zeta) in a rat model of cerebral basilar artery vasospasm.

OBJECT: The cellular events leading to cerebral vasospasm after subarachnoid haemorrhages (SAH) involve a number of members of the protein kinase C (PKC) family. However, whereas calcium is thought to play a number of major roles in the pathophysiology of SAH, a number of PKCs function independently of calcium. We recently emphasized the potential role of the calcium-binding S100 proteins in a 'double haemorrhage' rat model of SAH-induced vasospasm. A number of S100 proteins are known to interfere directly with PKC, or indirectly with PKC substrates. We therefore investigated whether specific S100 proteins and PKCs are co-expressed/co-located in a rat model of SAH-induced vasospasm. METHODS AND RESULTS: SAH-induced vasospasm in rats (by means of a double cisternal injection of autologous blood from a rat femoral artery) distinctly modified the expression levels of calcium-dependent PKC-alpha and PKC-beta and calcium-independent PKC-eta and PKC-zeta in endothelial and smooth-muscle cells. The RNA levels of these four PKC isotypes were determined by quantitative RT-PCR. The present study reveals that, in endothelial cells, the S100B expression/location correlate well with those of PKC-eta, and those of S100A1 with PKC-beta. In smooth-muscle cells S100A2 expression/location correlate with those of PKC-eta, and those of S100B with PKC-zeta. CONCLUSION: The present data argue in favour of a joint action of the S100 protein network and the PKC signalling pathway during cerebral vasospasm.

Dark-cell areas in the dog vestibular endorgans: an immunohistochemical study.

The stria vascularis in the cochlea and the dark-cell areas in the vestibular endorgans are structures involved in the production of endolymphatic fluid. This study investigated the dark-cell areas in the vestibular endorgans of the dog by classical staining and by immunohistochemistry (anti-Na,K-ATPase beta2 isoform, anti-cytokeratins (against cytokeratins 5 and 8), anti-vimentin and anti-S100A6) from birth to 110 postnatal days. Using classical staining, it was not possible to discriminate dark cells from other epithelial cells lining the vestibular endolymphatic spaces. From birth, the Na,K-ATPase beta2 isoform was expressed in the lateral and basal cell membranes of a subset of cells located in the utricular wall, at the base of the cristae ampullaris and was identified as dark cells. From birth, anti-cytokeratins labelled all the cells forming the epithelial lining, including the dark cells, while anti-vimentin labelled the underlying mesenchymal cells. From postnatal day 10, anti-S100A6 labelled subepithelial cells exclusively located underneath the dark-cell areas and were identified as vestibular melanocyte-like cells. From birth, Fontana staining evidenced fine melanin granules in the subepithelial layer. The amount of melanin granules increased during the first month. Melanin distribution was closely associated with the region where S100A6-positive cells were located. The cell-specific antigen expression in the dog dark-cell areas was clearly comparable to that of the dog stria vascularis previously described. The present investigation also suggested an earlier histological and immunohistological maturity in the dark-cell areas than in the stria vascularis of dogs. This preliminary morphological description of the normal dark-cell areas in dogs by means of immunomarkers may be instrumental in studying pathological processes involving the fluid-secreting structures in vestibular endorgans.

S100A5: a marker of recurrence in WHO grade I meningiomas.

Some WHO grade I intracranial meningiomas resected from the same sites and with the same quality of resection (Simpson's grading scale) recur, while others do not. The reasons for this variability in occurrence of recurrence have not yet been determined. We therefore investigated the prognostic recurrence value of seven biological markers on a series of completely resected WHO grade I meningiomas. For this purpose, we analysed a series of 33 WHO grade I meningiomas totally resected between 1980 and 1990 (a follow-up of 10 years), including 14 cases of recurrence. The fixed tumour material from each meningioma was submitted to histochemical analyses targeting galectin-3 and its binding sites, the S100A5, S100A6 and S100B proteins, and cathepsin-B and -D. The levels of expression were assessed semi-quantitatively (in terms of the staining intensity and the labelling index) and submitted to uni- and multivariate analyses. Of all the markers investigated, only S100A5 expression can be associated with any significant prognostic value in the matter of recurrence. More particularly, the meningiomas with high levels of S100A5 staining intensity either did not recur, or recurred later than those with a low immunopositive S100A5 intensity (P = 0.004). Cox regression analyses demonstrated that this latter marker was associated with significant prognostic values independent of the patients' ages. Furthermore, the combination of the patients' ages and S100A5 staining intensity permitted the identification of a group with a particularly high risk of recurrence, that is, the patients younger than 55 and with meningiomas exhibiting low S100A5 intensities (P = 0.001). In conclusion, the S100A5 protein could play a role in the recurrence of totally resected WHO grade I meningiomas.

Filter paper cards contaminated with EMLA cream produce artefacts on acylcarnitine analysis.

Electrospray ionization tandem mass spectrometry is a widely applied method for the analysis of acylcarnitines in blood samples spotted on filter paper cards (Guthrie cards). When the filter paper cards are contaminated by EMLA cream, highly intense signals at m/z 221 and 235 are detected under ESI-MS/MS conditions, monitoring for precursors of m/z 85. These signals correspond to the active ingredients prilocaine and lidocaine in EMLA and overlap with the signals from the isotopically labelled internal standards (2H3)propionyl carnitine and (2H3)butyrylcarnitine. This interference prevents the proper quantification of the two short-chain acylcarnitines when samples are analysed without derivatization.

Immunolocalization of S100A2 calcium-binding protein in cartilage and bone cells.

S100A2 protein, a Ca2+ binding protein, was investigated by immunocytochemistry in the epiphyseal cartilage and bone cells of growing rats, and in primary cultures of osteoblasts. S100A2 was detected in the chondrocytes and in the extracellular cartilage matrix. In the later however, its presence only in the calcifying areas of the epiphyseal cartilage suggests that it could be involved in the process of calcification of cartilage.

Differential expression of S100 calcium-binding proteins in epidermoid cysts, branchial cysts, craniopharyngiomas and cholesteatomas.

AIMS: To investigate whether epidermoid cysts, branchial cysts, craniopharyngiomas and cholesteatomas express S100 proteins differentially by immunohistochemical assaying the presence of S100A1, S100A2, S100A3, S100A4, S100A5, S100A6 and S100B. METHODS AND RESULTS: Immunopositivity/negativity was recorded for each S100 protein in a series of 52 cases consisting of 12 epidermoid cysts, 12 branchial cysts, 15 adamantinomatous craniopharyngiomas and 13 acquired cholesteatomas. Except in the case of the craniopharyngiomas, immunoreactivity was assessed independently in the basal membrane and the basal, the internal and the keratin layers. Our data show that in contrast to S100B, which was rarely expressed, S100A1, S100A2, S100A4 and S100A5 were often present in these four types of epithelial lesions. S100A3 and S100A6 and, to a lesser extent, S100A5 were the most differentially expressed proteins across the different histopathological groups analysed. These three proteins are expressed more often in craniopharyngiomas and cholesteatomas, the two more aggressive types of lesions. CONCLUSIONS: This is the first study to report data on the expression of seven S100 proteins in different histopathological groups of epithelial head and neck lesions, whose precise embryological origins are still a matter of debate. S100 proteins could possibly be used as markers to target this embryonic origin, since our results show that S100A3 and S100A6 (and, to a lesser extent, S100A5) are expressed differentially across these different groups of epithelial lesions.

The Ca2+-binding S100A2 protein is differentially expressed in epithelial tissue of glandular or squamous origin.

It has been previously shown that S100A2 is downregulated in tumor cells. The level of immunohistochemical S100A2 expression was therefore characterized in 424 normal and tumoral (benign and malignant) tissues of various origins, but mostly epithelial (with either glandular, squamous, respiratory or urothelial differentiation). We also investigated whether S100A2 could be co-localized with cytokeratin K14, an intermediate filament protein expressed in basal proliferative keratinocytes. Our data show that S100A2 has a low level of expression in non-epithelial tissue. In epithelial tissue S100A2 expression decreases remarkably in the tumors when compared to the normal specimens, and was correlated with the level of keratin K14. This decrease in S100A2 staining from normal to cancer cases is more pronounced in glandular than in squamous epithelial tissue. In addition, the patterns of S100A2 staining also differ between glandular and squamous tissue. These data suggest distinct functional roles for S100A2 in epithelial tissue of squamous or glandular origins.

An original inner ear neuroepithelial degeneration in a deaf Rottweiler puppy.

Histopathological investigation was conducted on both inner ears from a 4.5-month-old Rottweiler puppy with electrophysiologically confirmed bilateral deafness. The lesions were restricted to the organ of Corti and spiral ganglion that both displayed severe degenerative changes. The outer hair cells were less affected than the inner hair cells. The number of spiral ganglion neurons was reduced, and remaining neurons were altered. The basal and middle cochlear turns were more affected than the apical one. The vestibules were normal. Immunostaining with calbindin, calretinin, S100A1 and S100A6 polyclonal antisera was helpful in identifying different cell-types in the degenerated cochlea. The early and severe spiral ganglion cell degeneration is an uncommon finding no matter the species. Such lesions bear significance within the frame of cochlear implants technology for deaf infants.

Structural insight into human Zn(2+)-bound S100A2 from NMR and homology modeling.

The S100 subfamily of EF-hand proteins is distinguished by the binding of Zn(2+) in addition to Ca(2+). In an effort to understand the role of Zn(2+) in modulating the activity of S100 proteins, we have carried out heteronuclear NMR studies of Zn(2+)-bound S100A2 and obtained near complete resonance assignments. This analysis revealed an equilibrium between multiple isoforms due to cis-trans isomerism of proline residues in flexible regions of the protein. The secondary structure of S100A2 has been determined based on the NMR chemical shift index (CSI) technique. The protein is found to possess essentially the same secondary structure found in other S100 proteins such as S100A6 and S100B. Homology models have been built based on the high resolution three-dimensional structures of other S100 proteins. The models predict two Zn(2+) binding clusters, one involving residues His17-Cys21-Cys93 and the other Cys2-His39, and with Cys86 participating in either the N-terminal or the C-terminal binding site.

Separation of low molecular weight proteins with SDS-PAGE using taurine as a new trailing ion.

Taurine as an alternative trailing ion for tricine yields an identical resolution but reduces the running time by 15% and the power consumption by 15% compared to tricine. Therefore cooling of gels is more effective in SDS PAGE with taurine and artefacts due to oxidation of proteins can thus be reduced.

Intracellular Ca2+ and Zn2+ levels regulate the alternative cell density-dependent secretion of S100B in human glioblastoma cells.

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.

Translocation of S100A1(1) calcium binding protein during heart surgery.

Myocardial ischemia during cardiopulmonary bypass terminated by reperfusion generally leads to different degrees of damage of the cardiomyocytes induced by transient cytosolic Ca(2+) overload. Recently, much attention has been paid to the role of heart-specific Ca(2+)-binding proteins in the pathogenesis of myocardial ischemia-reperfusion injury. S100A1 is a heart-specific EF-hand Ca(2+)-binding protein that is directly involved in a variety of Ca(2+)-mediated functions in myocytes. The aim of our study was to investigate the localization and translocation of S100A1 in the human heart under normal (baseline) conditions and after prolonged ischemia and reperfusion of the myocardium. Our data suggest that S100A1 is directly involved in the transient perioperative myocardial damage caused by ischemia during open heart surgery in humans. Given its role in the contractile function of muscle cells, this S100 protein could be an important "intracellular link" in ischemia-reperfusion injury of the heart.

S100A2, a putative tumor suppressor gene, regulates in vitro squamous cell carcinoma migration.

It has been previously shown that S100A2 is down-regulated in tumor cells and can be considered a tumor suppressor. We have recently shown that this down-regulation can be observed particularly in epithelial tissue, where S100A2 expression decreases remarkably in tumors as compared with normal specimens. In the present paper we investigate whether S100A2 could play a tumor-suppressor role in certain epithelial tissues by acting at the cell migration level. To this end, we made use of five in vitro human head and neck squamous cell carcinoma lines in which we characterized S100A2 expression at both RNA and protein level. To characterize the influence of S100A2 on cell kinetic and cell motility features, we used two complementary approaches involving specific antisense oligonucleotides and the addition of S100A2 to the culture media. The different expression analyses gave a coherent demonstration of the fact that the FADU and the RPMI-2650 cell lines exhibit high and low levels of S100A2 expression, respectively. Antisense oligonucleotides (in FADU) and extracellular treatments (in RPMI) showed that, for these two models, S100A2 had a clear inhibitory influence on cell motility while modifying the cell kinetic parameters only slightly. These effects seem to be related, at least in part, to a modification in the polymerization/depolymerization dynamics of the actin microfilamentary cytoskeleton. Furthermore, we found evidence of the presence of the receptor for advanced glycation end-products (RAGE) in RPMI cells, which may act as a receptor for extracellular S100A2. The present study therefore presents experimentally based evidence showing that S100A2 could play a tumor-suppressor role in certain epithelial tissues by restraining cell migration features, at least in the case of head and neck squamous cell carcinomas.

Increased expression of S100A4 and its prognostic significance in esophageal squamous cell carcinoma.

S100A4 has been implicated in invasion and metastasis of cancer, but prognostic significance of its expression in esophageal squamous cell carcinoma remains unclear. In this study, we examined the expression of S100A4 by Western blot analysis and immunohistochemistry in surgically resected esophageal squamous cell carcinoma. The relationship between S100A4 tissue status and clinicopathological findings was analyzed to assess the prognostic significance of S100A4 in esophageal squamous cell carcinoma. The S100A4 protein level was significantly higher in tumor tissue than in corresponding normal esophageal mucosa (p<0.05) in 22 cases of esophageal carcinoma by Western blot analysis. S100A4 expression was detected in 25% of 52 cases of esophageal squamous cell carcinoma by immunohistochemistry and correlated with the depth of invasion (p<0.05). Patients with S100A4-positive carcinoma had significantly poorer prognosis than those with S100A4-negative carcinoma, which was also true in the cases with deep invasion of the primary cancer (T3, T4) (p<0.01 and p<0.05, respectively). Moreover, S100A4 tissue status remained the only independent prognostic parameter in the multivariate analysis. Our results suggest that S100A4 may play a key role in the progression of esophageal carcinoma and that immunohistochemical detection of S100A4 in the primary tumor may be useful for the prediction of a poor prognosis.

Immunolocalization of the calcium binding S100A1, S100A5 and S100A6 proteins in the dog cochlea during postnatal development.

The immunolocalization of three members of the S100 calcium-binding protein family was investigated in the dog cochlea during normal postnatal development. Sections of decalcified and paraffin-embedded cochleae from 16 beagle puppies aged from birth to 3 months were treated with polyclonal antisera raised against the human recombinant S100A1, S100A5, and S100A6 proteins. At birth, in the dog cochlea, S100A1 was expressed in the immature Deiter's cells, and slightly in the pillar cells. From the second week, S100A1 was detected in the supporting structures of the organ of Corti, i.e. the Deiter's, the pillar, the border, and the Hensen's cells, and in the reticular membrane. From birth onwards, S100A5 remained a neuronal-specific protein, only located in a subpopulation of neurons in the spiral ganglion. S100A6 was not expressed at birth. From the second week of life, the Schwann cells and nerve sheaths in the modiolus, in the spiral ganglion, and running in the direction of the organ of Corti exhibited S100A6-labeling. From the 12th postnatal day, some scattered intermediate cells started to express S100A6 protein in the stria vascularis. The number of labeled intermediate cells increased during the third week. At adult stage, the intermediate cells were S100A6-stained with cytoplasmic labeling throughout the stria vascularis from the base to the apex of the cochlea. None of the other cochlear structures expressed the S100 proteins under study during the postnatal development of the dog cochlea. The S100A1, S100A5, S100A6 immunostaining was limited to specific cell types in dog cochlea. These S100 proteins were useful markers in the study of supporting cells, neurons, nerve fibers sheaths and stria vascularis (S100A6) during the normal postnatal development of the dog cochlea.

Local nonpermissive and oriented permissive cues guide vestibular axons to the cerebellum.

Information that originates from peripheral sensory organs is conveyed by axons of cephalic sensory cranial ganglia connecting the sensory organs to appropriate central targets in the brain. Thus, the establishment of correct axonal projections by sensory afferents is one of the most important issues in neural development. Previously, we examined the development of the vestibular nerve that originates from the VIIIth ganglion using a flat whole-mount preparation of the rat hindbrain and developed an in vitro, culture preparation that can recapitulate vestibular nerve development (Tashiro, Y., Endo, T., Shirasaki, R., Miyahara, M., Heizmann, C. W. and Murakami, F. (2000) J. Comp. Neurol. 417, 491-500). Both in vivo and in vitro, the ascending branch of the VIIIth ganglion projecting to the cerebellum reaches the base of the cerebellar primordium and starts to splay out towards the rhombic lip, apparently avoiding the ventral metencephalon. We now examine the nature of cues that guide vestibulocerebellar axons by applying various manipulations to the flat whole-mount in vitro preparation. Our observations suggest that local nonpermissive cues and oriented cues play a pivotal role in the guidance of vestibular axons to their central target.