S100A9 mRNA Expression Research Articles

MRNA Expression of S100A8 as a Prognostic Marker for Progression of Non-Muscle-Invasive Bladder Cancer

PurposeS100A8 is a member of the S100 protein family containing 2EF-hand calcium-binding motifs. S100 proteins are involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation. Altered expression of this protein is associated with various diseases and cancers. The present study aimed to evaluate whether S100A8 has prognostic value for non-muscle-invasive bladder cancer (NMIBC).Materials and MethodsA total of 103 primary NMIBC samples obtained by transurethral resection were evaluated. mRNA levels were examined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. The results were compared with clinico-pathological parameters. The Kaplan-Meier method was applied to plot the curves for progression-free survival. The multivariate Cox regression model was used to identify the independent prognostic factors for progression.ResultsmRNA expression levels of S100A8 were significantly related to the progression of NMIBC. Kaplan-Meier estimates demonstrated significant differences in tumor progression according to the level of S100A8 expression (log-rank test, p<0.001). The multivariate Cox regression model revealed that the S100A8 mRNA expression level (hazard ratio: 12.538; 95% confidence interval: 2.245-70.023, p=0.004) was an independent predictor for disease progression of NMIBC.ConclusionsExpression levels of S100A8 might be a useful prognostic marker for disease progression of NMIBC.

High Expression of the Ca2+-Binding Proteins S100A8 and S100A9 Cause Glucocorticoid Resistance in MLL-Rearranged Infant Acute Lymphoblastic Leukemia.

Abstract 729MLL-rearranged Acute Lymphoblastic Leukemia (ALL) in infants (i.e. children <1 year of age) represents an aggressive and difficult to treat type of leukemia, displaying cellular resistance to several chemotherapeutics, especially to glucocorticoids like prednisolone. As prednisolone response is highly predictive for clinical outcome, it is of utmost importance to unravel the mechanism underlying resistance to this drug.To gain insights in the prednisolone resistance mechanism, we compared gene expression profiles (Affymetrix HU133plus2) from prednisolone-resistant and prednisolone-sensitive MLL-rearranged infant ALL patients. This gene signature revealed that multiple genes involved in calcium signaling were up-regulated in prednisolone-resistant samples. Most pronounced up-regulation was observed for the S100 protein family members S100A8 and S100A9, which are calcium-binding proteins, that function in a complex. Quantitative RT-PCR (TaqMan) analyses confirmed that S100A8 and S100A9 mRNA expression was ∼100-fold higher in prednisolone-resistant cells compared to patients sensitive to this drug (p=0.008).Glucocorticoids are known to induce apoptosis by releasing Ca2+ from the endoplasmic reticulum (ER) into the cytosol and towards the mitochondria. Within mitochondria, elevated Ca2+ levels induce cytochrome c release, triggering apoptosis. Since S100A8/A9 are capable of binding free cytosolic Ca2+, we hypothesized that over-representation of these cytosolic proteins may prevent Ca2+ to reach the mitochondria and forestall apoptosis.To test our hypothesis, we first co-incubated prednisolone-sensitive MLL-rearranged ALL cells with prednisolone and EGTA or BAPTA-AM. Both agents represent Ca2+chelators and scavenge free cytosolic Ca2+, thereby mimicking the Ca2+binding by S100A8/A9. Flow cytometry analyses showed that both EGTA and BAPTA-AM inhibited the free cytosolic calcium released by prednisolone. Cytotoxicity tests demonstrated that these prednisolone-sensitive cells became more resistant to prednisolone after co-incubation with either EGTA or BAPTA-AM. Next we asked whether enforced over-expression (using retroviral expression vectors) of S100A8 and/or S100A9 in prednisolone-sensitive MLL-rearranged ALL cells could also inhibit free cytosolic Ca2+and induce prednisolone resistance. Indeed, both S100A8 and S100A9 were capable of binding free cytosolic Ca2+ released by prednisolone, accompanied by a 30-40% increase in cell survival when either S100A8 or S100A9 were over-expressed alone. Simultaneous over-expression of both S100A8 and S100A9 almost completely reversed the prednisolone-sensitive into a prednisolone-resistance phenotype.In conclusion, these findings implicate that high expression of S100A8 and S100A9 contributes to prednisolone resistance in MLL-rearranged infant ALL. Disclosures:No relevant conflicts of interest to declare.

S100A4 mRNA is a Diagnostic and Prognostic Marker in Pancreatic Carcinoma

ObjectiveThe aim of this study is to evaluate the clinical significance of S100A4 mRNA expression in pancreatic cancer. Materials and MethodsWe obtained invasive ductal carcinoma (IDC) cells from ten lesions, intraductal papillary mucinous neoplasm (IPMN) cells from 20 lesions, and normal ductal cells from 20 normal pancreatic tissues by laser microdissection of frozen tissues. S100A4 expression was examined in the microdissected cells and in formalin-fixed paraffin-embedded (FFPE) samples of 87 pancreatic cancers by quantitative reverse transcription-polymerase chain reaction. ResultsIDC cells expressed higher levels of S100A4 than IPMN cells (P = 0.002) and normal ductal cells (P < 0.001), although the difference between IPMN cells and normal ductal cells was not statistically significant (P = 0.070). Analysis of FFPE samples revealed that high S100A4 expression was significantly associated with a shorter overall survival (P = 0.023). In immunohistochemical analysis, the extent of S100A4 mRNA expression was significantly correlated with the expression of S100A4 protein (P = 0.028). ConclusionS100A4 could be a marker for malignancy in pancreatic tumors and for poor prognosis in patients with pancreatic cancer.

Effect of cobalt chloride on &lt;I&gt;S100A4&lt;/I&gt; expression in human gastric cancer cell BGC823

S100A4 is an important metastasis-associated gene. Researches have confirmed the close correlation between overexpression of S100A4 gene and gastric cancer's infiltration, lymph node metastasis and in vitro invasiveness of gastric cancer cells. In order to investigate the mechanism of overexpression of S100A4 gene, hypoxia mimetic cobalt chloride (CoCl2) was used to treat gastric cancer cell BGC823, and then the expression of S100A4 mRNA and protein in BGC823 cells were detected by RT-PCR, immunohistochemistry, immunofluorescence, and Western blotting analysis. After treatment with CoCl2, the expression of S100A4 mRNA and protein in BGC823 cell was increased. These results suggested that hypoxia mimetic cobalt chloride could increase the expression of S100A4 gene in gastric cancer cell BGC823.

The link between metastasis-associated protein S100A4 and rheumatoid arthritis

The metastasis-associated protein S100A4 is a member of a large family of calcium-binding proteins that appears to play regulatory roles in cancer promoting activities. S100A4 protein regulates cell motility, proliferation, apoptosis, and stimulation of angiogenesis as well as remodeling of the extracellular matrix. It has been demonstrated that proliferating synovial fibroblasts in rheumatoid arthritis (RA) up-regulate expression of S100A4 mRNA. We have previously shown increased expression of S100A4 protein at the sites of joint destruction in RA synovial tissue. The aim of our current study was to examine the involvement of S100A4 in the pathogenesis of RA. Immunohistochemical and immunofluoresence studies were performed to analyze cell-specific distribution of S100A4 protein in RA and osteoarthritis (OA) synovial tissues. Western blotting was used to quantify the activation of the tumor suppressor protein p53 in synovial fibroblasts. Plasma concentrations of S100A4 from RA and OA patients were measured by sandwich ELISA. As we previously shown that S100A4 oligomer is involved in the regulation of several matrix degrading enzymes, in this study we could demonstrate that it can also modulate the transcriptional activation function of the tumor suppressor protein p53 in RA synovial fibroblasts. Moreover, we have identified increased expression of S100A4 in RA compared to OA synovial tissue and several S100A4 producing cells such as synovial fibroblasts, macrophages, mast cells, neutrophils, fraction of T-cells, endothelial cells and pericytes, but not B-cells, within the synovium. The local up-regulation of S100A4 was accompanied by high plasma and synovial fluid concentrations of the S100A4 protein in RA patients. Unlike other S100 proteins including S100A8/9 and S100A12, S100A4 plasma concentrations were not modified by TNF blocking therapy in active RA patients. Taken together, it can be speculated that increased S100A4 protein in circulation and locally at sites of inflammation, particularly at sites of joint destruction, might be implicated in the process of aggressive fibroblast behavior contributing to the pathogenesis of rheumatoid arthritis.

The metastasis associated protein S100A4: a potential novel link to inflammation and consequent aggressive behaviour of rheumatoid arthritis synovial fibroblasts

The metastasis-associated protein S100A4 belongs to the large family of S100 calcium-binding proteins that appear to play regulatory roles in diverse biological activities. Moreover, a prognostic role of S100A4 has...

S100A4 is upregulated in injured myocardium and promotes growth and survival of cardiac myocytes

The multifunctional Ca2+-binding protein S100A4 (also known as Mts1 and Fsp1) is involved in fibrosis and tissue remodeling in several diseases including cancer, kidney fibrosis, central nervous system injury, and pulmonary vascular disease. We previously reported that S100A4 mRNA expression was increased in hypertrophic rat hearts and that it has pro-cardiomyogenic effects in embryonic stem cell-derived embryoid bodies. We therefore hypothesized that S100A4 could play a supportive role in the injured heart. Here we verify by quantitative real-time PCR and immunoblotting that S100A4 mRNA and protein is upregulated in hypertrophic rat and human hearts and show by way of confocal microscopy that S100A4 protein, but not mRNA, appears in cardiac myocytes only in the border zone after an acute ischemic event in rat and human hearts. In normal rat and human hearts, S100A4 expression primarily colocalizes with markers of fibroblasts. In hypertrophy elicited by aortic banding/stenosis or myocardial infarction, this expression is increased. Moreover, invading macrophages and leucocytes stain strongly for S100A4, further increasing cardiac levels of S100A4 protein after injury. Promisingly, recombinant S100A4 protein elicited a robust hypertrophic response and increased the number of viable cells in cardiac myocyte cultures by inhibiting apoptosis. We also found that ERK1/2 activation was necessary for both the hypertrophy and survival effects of S100A4 in vitro. Along with proposed angiogenic and cell motility stimulating effects of S100A4, these findings suggest that S100A4 can act as a novel cardiac growth and survival factor and may have regenerative effects in injured myocardium.

Alcohol Intoxication Inhibits Pulmonary S100A8 and S100A9 Expression in Rats Challenged With Intratracheal Lipopolysaccharide

Alcohol is known to inhibit the recruitment of polymorphonuclear leukocytes (PMNs) into tissue sites including the lung. During infection and inflammation, recruited neutrophils (PMNs) release S100 proteins that function to promote the recruitment of additional phagocytes. This study investigated the effects of alcohol intoxication on S100 protein production in the lung in response to lipopolysaccharide (LPS). Animals were administered alcohol (5.5 g/kg) or saline 30 minutes before intratracheal challenge with LPS (100 microg/rat). Alcohol suppressed PMN recruitment into the lung following intratracheal LPS, which was associated with an inhibition of increase in S100A8 levels in both the bronchoalveolar lavage (BAL) fluid and lysates of cells recovered by BAL at 90 minutes and 4 hours post-LPS challenge. S100A8 and S100A9 mRNA expression in cells recovered by BAL was significantly up-regulated at both 90 minutes and 4 hours after the LPS challenge, and alcohol also suppressed this response. In addition, intratracheal LPS caused a transient increase in S100A8 mRNA expression in circulating leukocytes at 90 minutes after the challenge. Similarly, this LPS-induced up-regulation of S100A8 mRNA expression was inhibited in rats intoxicated with alcohol. These data show that alcohol inhibits the S100 protein response in the lung, which may serve as a mechanism underlying alcohol-induced suppression of PMN recruitment into the terminal airways during pulmonary infection.

A Protective Function of the Calcium Binding Protein S100A4 in Rat Hepatic Injury Due to Carbon Tetrachloride

F The role of the S100A4 in rat hepatocites was clarified by measuring detailed changes in serum ALT values and intracellular Ca2+ after CCl4 administration. In this study, we further showed that the calcium binding protein S100A4 might be a protein signal involved in the response to protect cells against CCl4 intoxication. S100A4 mRNA expression levels abruptly increased to a high level in normal liver at 1 day after CCl4 administration, and then gradually reduced to the control level by day 3. This change is the opposite to that of intracellular Ca2+.

Colchicine induces membrane-associated activation of matrix metalloproteinase-2 in osteosarcoma cells in an S100A4-independent manner

Like the metastasis-associated protein S100A4, matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are important in physiological and pathological conditions. Previously, we showed that S100A4 is involved in the regulation of MMPs and TIMPs, and in the present work we have investigated whether the anti-inflammatory and microtubule-disrupting drug colchicine has an effect on the expression of these proteins in osteosarcoma cell lines (OHS) with high and low levels of S100A4. Colchicine treatment of the various OHS cells resulted in an increased expression of MT1-MMP and TIMP-2 mRNA, and a corresponding increase of these two proteins in isolated cell membranes. Colchicine-treated cells produced more of the activated form of MMP-2 than control cells. However, the drug did not affect the amount of MMP-2 and TIMP-1 mRNA or protein, and it reduced the S100A4 mRNA expression. Isolated cell membranes from the colchicine-treated cells were more effective in activating exogenous proMMP-2 than membranes from control cells, and inhibitory studies indicated that it was the colchicine-induced increase in MT1-MMP that caused the increased activation of endogenous MMP-2. A peptide inhibitor of nuclear factor κB nuclear translocation, SN50, blocked the colchicine-induced activation of proMMP-2 and reduced the synthesis of MMP-2 in colchicine-treated cells, but not in control cells. It can be concluded that colchicine modulates the expression of MT1-MMP and TIMP-2 and hence the activation of proMMP-2 independently of the S100A4 level in osteosarcoma cells.

Suppression of metastasis-associated S100A4 gene expression by gamma-interferon in human colon adenocarcinoma cells.

S100A4 belongs to the S100 subfamily of calcium-binding proteins and has been suggested to be directly involved in invasion and metastasis of rodent and human tumour cells. The present study demonstrates that interferon gamma (IFN-γ), but not IFN-α and IFN-β, down-regulates the S100A4 mRNA level in colon adenocarcinoma WiDr cells in time- and dose-dependent manners. The effect was not associated with any cytotoxicity and was specific for the S100A4 mRNA, since the levels of the S100A6 and GAPDH mRNAs were not significantly affected by the treatment. IFN-γ also strongly suppressed the S100A4 mRNA expression in HT-29 cells, but weakly in Colo201 cells. Flow cytometric analysis revealed that the level of the IFN-γ receptor expression in Colo201 cells was lower than that in WiDr and HT-29 cells, suggesting that the suppression of the S100A4 expression by IFN-γ depends on the amount of cell surface IFN-γ receptor protein. IFN-γ had no effect on the transcription rate of the S100A4 gene but reduced the stability of the S100A4 mRNA. WiDr cells treated with IFN-γ showed reduced motile ability, further supporting the assumption that the S100A4 gene product is involved in controlling cell motility. © 1999 Cancer Research Campaign

Effects of mechanical stress on the mRNA expression of S100A4 and cytoskeletal components by periodontal ligament cells.

The periodontal ligament (PDL) functions under constant mechanical stress, and PDL cells obviously control PDL functions under such conditions. We have previously found that the mRNA expression of the Ca2+-binding protein S100A4 and beta-actin is higher in the PDL from erupted teeth than in the PDL from teeth under eruption. This suggested a role for S100A4 in the response of PDL cells to mechanical stress, possibly by coupling Ca2+ and the cytoskeletal system. In the present study, we investigated the direct effects of cyclical stretching on the mRNA expression of S100A4 and two cytoskeletal components (beta-actin and alpha-tubulin) by PDL cells. In Northern blotting analysis, the expression of S100A4, beta-actin, and alpha-tubulin mRNAs was higher in the PDL from fully erupted and functional bovine teeth than in partially erupted ones. Similarly, when bovine PDL cells were mechanically stimulated by means of the Flexercell Strain Unit, the expression of S100A4, beta-actin, and alpha-tubulin mRNAs increased over the control levels. The results of our present study indicate that S100A4 is involved in the responses of PDL cells to mechanical stress possibly by coupling Ca2+ to the cytoskeletal system in these cells.

Value of liver tissue dosimetry data to design and interpret in vitro (hepatocyte) gap junction studies with peroxisomal proliferating agents

COPD patients have increased risk of pneumonia when treated with fluticasone propionate (FP), whereas this is generally not the case with budesonide (BUD) treatment. We hypothesized that BUD and FP differentially affect the expression of immune defense genes.Human bronchial epithelial 16HBE cells and air-liquid interface (ALI)-cultured primary bronchial epithelial cells (PBECs) were pre-treated with clinically equipotent concentrations of BUD or FP (0.16–16 nM BUD and 0.1–10 nM FP), and the expression of immune defense genes was studied at baseline and after exposure to rhinovirus (RV16).Using microfluidic cards, we observed that both BUD and FP significantly suppressed CXCL8, IFNB1 and S100A8 mRNA expression in unstimulated 16HBE cells. Interestingly, BUD, but not FP, significantly increased lactotransferrin (LTF) expression. The difference between the effect of BUD and FP on LTF expression was statistically significant and confirmed by qPCR and at the protein level by western blotting. RV16 infection of ALI-cultured PBECs significantly increased the expression of CCL20, IFNB1 and S100A8, but not of LTF or CAMP/LL-37. In these RV16-exposed cells, LTF expression was again significantly higher upon pre-treatment with BUD than with FP. The same was observed for S100A8, but not for CCL20, IFNB1 or CAMP/LL-37 expression.Treatment of human bronchial epithelial cells with BUD results in significantly higher expression of specific immune defense genes than treatment with FP. The differential regulation of these immune defense genes may help to explain the clinical observation that BUD and FP treatment differ with respect to the risk of developing pneumonia in COPD.