Shehla Izhar

Expression of S100A6 in Cardiac Myocytes Limits Apoptosis Induced by Tumor Necrosis Factor-α

S100A6 is induced in myocardium post-infarction in vivo and in response to growth factors and inflammatory cytokines in vitro. Forced expression of S100A6 in cardiomyocytes inhibits regulation of cardiac specific gene expression in response to trophic stimulation. To define regulation and function of S100A6, we characterized the human S100A6 promoter and mapped upstream regulatory elements in rat neonatal cardiac myocytes, fibroblasts, and vascular smooth muscle cells and defined a functional role for S100A6 in tumor necrosis factor-α-induced myocyte apoptosis. The functional S100A6 promoter was localized to region -167/+134 containing 167 upstream base pairs. The S100A6 promoter is regulated by positive (-361/-167 and -588/-361) and negative (-1371/-1194) elements. Tumor necrosis factor-α induced the maximal S100A6 promoter and transcription factor NF-κB (p65 subunit). Electrophoretic mobility shift showed that tumor necrosis factor-α induced p65 binding to a potential NF-κB-binding site at -460/-451. Chromatin immunoprecipitation analysis revealed p65 is recruited to the S100A6 promoter upon tumor necrosis factor-α stimulation. The NF-κB inhibitor caffeic acid phenethyl ester and mutation of the NF-κB-binding site inhibited S100A6 promoter activation by tumor necrosis factor-α. Tumor necrosis factor-α induced cardiac myocyte apoptosis. Specific inhibition of S100A6 using a small interfering RNA directed against S100A6 potentiated tumor necrosis factor-α-induced myocyte apoptosis, whereas overexpression of S100A6 by gene transfer prevented tumor necrosis factor-α-induced myocyte apoptosis by interfering with p53 phosphorylation. These results demonstrate that S100A6 is induced by tumor necrosis factor-α via an NF-κB-dependent mechanism, serving a role in homeostasis to limit tumor necrosis factor-α-induced apoptosis by regulating p53 phosphorylation.

S100B-RAGE dependent VEGF secretion by cardiac myocytes induces myofibroblast proliferation

Post-infarct remodeling is associated with the upregulation of the receptor for advanced glycation end products (RAGE), the induction of its ligand the calcium binding protein S100B and the release of the potent endothelial-cell specific mitogen vascular endothelial growth factor (VEGF). To determine a possible functional interaction between S100B, RAGE and VEGF we stimulated rat neonatal cardiac myocyte cultures transfected with either RAGE or a dominant-negative cytoplasmic deletion mutant of RAGE with S100B for 48 h. Under baseline conditions, cardiac myocytes express low levels of RAGE and VEGF and secrete VEGF in the medium as measured by ELISA. In RAGE overexpressing myocytes, S100B (100 nM) resulted in increases in VEGF mRNA, VEGF protein, VEGF secretion, and activation of the transcription factor NF-κB. Pre-treatment of RAGE overexpressing myocytes with the NF-κB inhibitor caffeic acid phenethyl ester inhibited increases in VEGF mRNA, VEGF protein and VEGF in the medium by S100B. In myocytes expressing dominant-negative RAGE, S100B did not induce VEGF mRNA, VEGF protein, VEGF secretion or NF-κB activation. In culture, rat neonatal and adult cardiac fibroblasts undergo phenotypic transition to myofibroblasts. Treatment of neonatal and adult myofibroblasts with VEGF (10 ng/mL) induces VEGFR-2 (flk-1/KDR) tyrosine kinase phosphorylation, ERK1/2 phosphorylation and myofibroblast proliferation. Together these data demonstrate that secreted VEGF by cardiac myocytes in response to S100B via RAGE ligation induces myofibroblast proliferation potentially contributing to scar formation observed in infarcted myocardium. This article is part of a Special Issue entitled Local Signaling in Myocytes.

Peripheral blood lymphocytes from patients with bipolar disorder demonstrate apoptosis and differential regulation of advanced glycation end products and S100B

Abstract Background: This study addresses the expression of the glycosylated proteins known as advanced glycation end products (AGEs), the calcium binding protein S100B and the apoptotic parameters cytochome c and caspase-3 activity in peripheral lymphocyte cytosolic extracts from a sample of bipolar disorder (BD) patients and healthy (control) subjects. Methods: Cross-sectional study of 35 patients with a clinical diagnosis of bipolar disease (10 euthymic, 12 depressed, 13 manic) and 10 healthy control subjects. Lymphocytes were used as a surrogate model in BD diagnosis and treatment. AGEs and S100B in lymphocyte cell extracts were measured by commercially available enzyme-linked immunosorbent assay. Results: AGEs were lower in all BD patients compared to healthy subjects. Depressed patients had approximately two-fold higher S100B levels compared to healthy subjects. Manic and depressed BD patients had increased superoxide dismutase mRNA levels. Apoptosis as measured by BAX/Bcl2 ratio, cytochrome c release, caspase-3 activity was increased in manic and depressed patients compared to healthy subjects. In the depressed patients, S100B levels correlated with cytochrome c release. Conclusions: In conclusion, our study shows decreased AGEs and increased S100B levels and caspase down-stream apoptosis in peripheral lymphocytes of BD patients that may underlie disease etiopathogenesis.

S100B Modulates the Hemodynamic Response to Norepinephrine Stimulation

BACKGROUNDnWe have previously reported that S100B acts as an intrinsic negative regulator of the myocardial hypertrophic response to norepinephrine (NE).nnnMETHODSnTo examine the role of S100B in acute and chronic hemodynamic responses to NE stimulation, knockout (KO) mice devoid of the S100B gene, transgenic (TG) mice with forced overexpression of S100B, and control CD1 mice were injected subcutaneously once daily with NE (1.5 mg/kg) or vehicle for 28 days.nnnRESULTSnThe acute and chronic hemodynamic responses were not different in CD1 and TG mice. In KO mice, both the chronic and acute increase in blood pressure (BP) in response to NE was attenuated compared with CD1 mice. NE induced ventricular myocyte hypertrophy and smooth muscle proliferation in CD1 mice, responses that were augmented in KO mice. In TG mice, NE did not induce myocyte hypertrophy or smooth muscle cell proliferation. NE treatment of smooth muscle cells derived from KO mice resulted in lower cytosolic calcium concentrations compared to CD1 and TG mice. NE induced S100B in ventricular myocytes and increased S100B in arterial tissues of CD1 and TG mice. The giant phosphoprotein AHNAK is expressed in both ventricular myocytes and aortic smooth muscle cells (ASMCs). In response to NE, S100B co-immunoprecipitates with AHNAK in ventricular myocytes and ASMCs.nnnCONCLUSIONnThus, absence of S100B is associated with attenuation of the hemodynamic response to catecholamines, in contradistinction to, the augmented cardiac hypertrophy and smooth muscle cell proliferation.

Conditional Cardiac Overexpression of S100A6 Attenuates Myocyte Hypertrophy and Apoptosis Following Myocardial Infarction

S100A6, a 20 kDa, Ca2+ - binding dimer with low basal cardiac expression, is upregulated in the rat heart following infarction and forced expression of S100A6 in rat neonatal cardiac myocyte cultures, inhibited the induction of β myosin heavy chain (MHC), skeletal α actin (skACT) and myocyte apoptosis in response to diverse stimuli including tumor necrosis factor α. To define a role for S100A6 in vivo, we generated cardiac myocyte-specific transgenic mice by placing the human S100A6 cDNA downstream of a promoter responsive to a doxycycline (DOX)-regulated transcriptional activator (tTA) and breeding this line with one harboring cardiac myocyte-restricted (αMHC) expression of tTA (αMHC-tTA). We compared S100A6-αMHC-tTA mice 35 days post-myocardial infarction (MI) produced by coronary artery ligation with similar matched sham-operated controls on (S100A6 transgene overexpressed) or off (S100A6 transgene silenced) DOX. There were no differences between the sham groups on or off DOX. Thirty five days post-MI, myocardial S100A6 levels increased 12.5-fold in S100A6-α-MHC-tTA mice off DOX compared with S100A6-α-MHC-tTA mice on DOX. Hemodynamic studies, echocardiography and postmortem examination indicated that S100A6-αMHC-tTA mice on DOX 35 days post-MI mounted a hypertrophic response (20-22.5 % increase) accompanied by a program of fetal gene re-expression, fibrosis and myocardial apoptosis. Whereas the S100A6-α-MHC-tTA mice off DOX showed an attenuated myocyte hypertrophic response, less fibrosis and apoptosis which was beneficial to preservation of cardiac function. Therefore, S100A6 is a potential therapeutic target for modulation of adverse left ventricular remodeling in the early post infarct period.

Deficiency of S100B confers resistance to experimental diabetes in mice

Abstract The calcium binding protein S100B has been implicated in diabetic neuronal and vascular complications but has not been examined in the development of diabetes. S100B knock out (S100B KO) and wild‐type (WT) mice were injected with 40 mg/kg body weight streptozotocin (STZ) for 5 days. Blood and pancreatic tissue samples were obtained to examine islet structure and function, the profile of glucose and insulin and expression of glucose transporter 2 (Glut2), S100B and its receptor, the receptor for advanced glycation end products (RAGE). Primary islet &bgr;‐cells cultures from WT mice were used to test the apoptotic potential of S100B. S100B KO mice were resistant to STZ induced‐diabetes with lower urine volume, food and water intake compared to WT mice. S100B increased in the WT islet following diabetes but did not co‐localize with beta or peri‐islet Schwann cells but with CD3 + T lymphocytes. S100B KO mice exhibited enhanced glucose tolerance, insulin sensitivity, prevented &bgr;‐cell destruction and functional impairment in response to STZ treatment. S100B deficiency was associated with decreased Glut2 and RAGE. In primary &bgr;‐cell cultures from WT mice, S100B induced reactive oxygen species (ROS) and RAGE‐dependent apoptosis. In the STZ diabetic animal model, abrogation of S100B enhances insulin sensitivity and reduces pancreatic islet, and &bgr;‐cell destruction. S100B may be a promising target for pharmacological interventions aimed at repressing diabetes. HighlightsS100B KO mice are resistant to STZ induced‐diabetes.The abrogation of S100B enhances insulin sensitivity and reduces &bgr;‐cell destruction.S100B represents a promising pharmacological target aimed at repressing diabetes.