Farnesol prevents aging-related muscle weakness in mice through enhanced farnesylation of Parkin-interacting substrate.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis. Reduced PGC-1α abundance is linked to skeletal muscle weakness in aging or pathological conditions, such as neurodegenerative diseases and diabetes; thus, elevating PGC-1α abundance might be a promising strategy to treat muscle aging. Here, we performed high-throughput screening and identified a natural compound, farnesol, as a potent inducer of PGC-1α. Farnesol administration enhanced oxidative muscle capacity and muscle strength, leading to metabolic rejuvenation in aged mice. Moreover, farnesol treatment accelerated the recovery of muscle injury associated with enhanced muscle stem cell function. The protein expression of Parkin-interacting substrate (PARIS/Zfp746), a transcriptional repressor of PGC-1α, was elevated in aged muscles, likely contributing to PGC-1α reduction. The beneficial effect of farnesol on aged muscle was mediated through enhanced PARIS farnesylation, thereby relieving PARIS-mediated PGC-1α suppression. Furthermore, short-term exercise increased PARIS farnesylation in the muscles of young and aged mice, whereas long-term exercise decreased PARIS expression in the muscles of aged mice, leading to the elevation of PGC-1α. Collectively, the current study demonstrated that the PARIS-PGC-1α pathway is linked to muscle aging and that farnesol treatment can restore muscle functionality in aged mice through increased farnesylation of PARIS.

Association of Polymorphisms in Connective Tissue Growth Factor and Epidermal Growth Factor Receptor Genes With Human Longevity.

Growth pathways play key roles in longevity. The present study tested single-nucleotide polymorphisms (SNPs) in the connective tissue growth factor gene (CTGF) and the epidermal growth factor receptor gene (EGFR) for association with longevity. Comparison of allele and genotype frequencies of 12 CTGF SNPs and 41 EGFR SNPs between 440 American men of Japanese ancestry aged ≥95 years and 374 men of average life span revealed association with longevity at the p < .05 level for 2 SNPs in CTGF and 7 in EGFR. Two in CTGF and two in EGFR remained significant after Bonferroni correction. The SNPs of both CTGF and EGFR were in a haplotype block in each respective gene. Haplotype analysis confirmed the suggestive association found by χ2 analysis. We noted an excess of heterozygotes among the longevity cases, consistent with heterozygote advantage in living to extreme old age. No associations of the most significant SNPs were observed in whites or Koreans. In conclusion, the present findings indicate that genetic variation in CTGF and EGFR may contribute to the attainment of extreme old age in Japanese. More research is needed to confirm that genetic variation in CTGF and EGFR contributes to the attainment of extreme old age across human populations.

Two genetic variants in telomerase-associated protein 1 are associated with stomach cancer risk.

This study examined the impact of two single-nucleotide polymorphisms (SNPs) in the telomerase-associated protein 1 (TEP1) gene on the risk of breast, colorectal, hepatocellular, lung and stomach cancer. A significantly increased stomach cancer risk associated with the GG genotype at rs1760893 (odds ratio (OR)=1.64, 95% confidence interval (CI)=1.23-2.20, P=0.004) or CC genotype at rs1713423 (OR=2.40, 95% CI=1.88-3.07, P<0.0001) was observed, compared with their wild-type counterpart. The GG genotype at rs1760893 was also associated with enhanced hepatocellular cancer susceptibility (OR=1.46, 95% CI=1.05-2.03, P=0.02). In classification and regression tree analysis, individuals carrying the CC genotype at rs1713423 had 2.69-fold increased risk of stomach cancer (95% CI=2.18-3.32, P<0.0001) compared with the TT and TC genotypes. The current results suggested that genetic variants at TEP1 SNPs rs1760893 and rs1713423 may be associated significantly with increased risk of stomach cancer.

Copy Number Gains at 8q24 and 20q11-q13 in Gastric Cancer Are More Common in Intestinal-Type than Diffuse-Type.

The present study was aimed at discovering DNA copy number alterations (CNAs) involved in the carcinogenesis of stomach and at understanding their clinicopathological significances in the Korean population. DNA copy numbers were analyzed using Agilent 244K or 400K array comparative genomic hybridization (aCGH) in fresh-frozen tumor and matched normal tissues from 40 gastric cancer patients. Some of the detected CNA regions were validated using multiplex ligation-dependent probe amplification (MLPA) in six of the 40 patients and customized Agilent 60K aCGH in an independent set of 48 gastric cancers. The mRNA levels of genes at common CNA regions were analyzed using quantitative real-time PCR. Copy number gains were more common than losses across the entire genome in tumor tissues compared to matched normal tissues. The mean number of alterations per case was 64 for gains and 40 for losses, and the median aberration length was 44016 bp for gains and 4732 bp for losses. Copy number gains were frequently detected at 7p22.1 (20%), 8q24.21 (27%-30%), 8q24.3 (22%-48%), 13q34 (20%-31%), and 20q11-q13 (25%-30%), and losses at 3p14.2 (43%), 4q35.2 (27%), 6q26 (23%), and 17p13.3 (20%-23%). CNAs at 7p22.1, 13q34, and 17p13.3 have not been reported in other populations. Most of the copy number losses were associated with down-regulation of mRNA levels, but the correlation between copy number gains and mRNA expression levels varied in a gene-dependent manner. In addition, copy number gains tended to occur more commonly in intestinal-type cancers than in diffuse-type cancers. In conclusion, the present study suggests that copy number gains at 8q24 and 20q11-q13 and losses at 3p14.2 may be common events in gastric cancer but CNAs at 7p22.1, 13q34, and 17p13.3 may be Korean-specific.

Overexpression of MAPK15 in gastric cancer is associated with copy number gain and contributes to the stability of c-Jun.

This study was aimed at understanding the functional and clinicopathological significance of MAPK15 alteration in gastric cancer. Genome-wide copy number alterations (CNAs) were first investigated in 40 gastric cancers using Agilent aCGH-244K or aCGH-400K, and copy number gains of MAPK15 found in aCGH were validated in another set of 48 gastric cancer tissues. The expression of MAPK15 was analyzed using immunohistochemistry in concurrent lesions of normal, adenoma, and carcinoma from additional 45 gastric cancer patients. The effects of MAPK15 on cell cycle, c-Jun phosphorylation, and mRNA stability were analyzed in gastric cancer cells. Copy number gains of MAPK15 were found in 15 (17%) of 88 tumor tissues. The mRNA levels of MAPK15 were relatively high in the gastric cancer tissues and gastric cancer cells with higher copy number gains than those without. Knockdown of MAPK15 using siRNA in gastric cancer cells significantly suppressed cell proliferation and resulted in cell cycle arrest at G1-S phase. Reduced c-Jun phosphorylation and c-Jun half-life were observed in MAPK15-knockdowned cells. In addition, transient transfection of MAPK15 into AGS gastric cancer cells with low copy number resulted in an increase of c-Jun phosphorylation and stability. The overexpression of MAPK15 occurred at a high frequency in carcinomas (37%) compared to concurrent normal tissues (2%) and adenomas (21%). In conclusion, the present study suggests that MAPK15 overexpression may contribute to the malignant transformation of gastric mucosa by prolonging the stability of c-Jun. And, patients with copy number gain of MAPK15 in normal or premalignant tissues of stomach may have a chance to progress to invasive cancer.

Chromatin CKAP2, a new proliferation marker, as independent prognostic indicator in breast cancer.

BACKGROUND: The level of proliferation activity is a strong prognostic or predictive indicator in breast cancer, but its optimal measurement is still in debate, necessitating new proliferation markers. In the present study, the prognostic significance of the CKAP2-positive cell count (CPCC), a new proliferation marker, was evaluated, and the results were compared with those for the mitotic activity index (MAI). METHODS: This study included 375 early-stage breast cancer samples collected from two institutions between 2000 and 2006. Immunohistochemical staining was performed using a CKAP2 monoclonal antibody. Cox proportional hazard regression models were fitted to determine the association between the CPCC and relapse-free survival (RFS) amongst three groups formed on the basis of the CPCC or MAI value: groups 2 and 3 showing the middle and highest values, respectively, and group 1 the lowest. RESULTS: After adjustment for age, T stage, N stage, HER2 status, estrogen receptor status, progesterone receptor status, institution, and year of surgical resection, the CPCC was associated with a significantly worse RFS {hazard ratio [HR]  = 4.10 (95% CI: 1.64-10.29) for group 2; HR  = 4.35 (95% CI: 2.04-10.35) for group 3}. Moreover, its prognostic significance was similar to or higher than that based on the MAI {HR  = 2.05 (95% CI: 0.94-4.65) for group 2; HR  = 2.35 (95% CI: 1.09-5.10) for group 3}. In subgroup analyses, the CPCC showed a prognostic significance in the luminal A and triple-negative subgroups, but not in the HER2-positive subgroup. CONCLUSIONS: Chromatin CKAP2 is an independent prognostic marker for RFS in early-stage breast cancer, and could potentially replace the MAI in clinical evaluation of proliferation activity. Additionally, our study results suggest that the prognostic significance of proliferation activity differs among the various subgroups of breast cancer.

Recapitulation of previous genome-wide association studies with two distinct pathophysiological entities of gastric cancer in the Korean population.

Gastric cancer (GC) is the most common malignancy. The incidence rates remain remarkably high in East Asians. Although genome-wide association studies in the Han Chinese and Japanese populations have so far yielded susceptibility loci for GC, these findings need to be validated in an independent ethnic group. To identify the potential heterogeneity by histological classified subtypes (intestinal and diffuse), we examined the previously reported associations in the Korean population. PRKAA1 at 5p13.1 was found to be more strongly associated with intestinal type (odds ratio, OR=1.39, 95% CI (confidence interval) =1.22-1.58, P=3.77 × 10(-7)) than diffuse type. In addition, PSCA at 8q23.3 was significantly replicated in diffuse type (OR=1.49, 95% CI=1.32-1.67, P=2.43 × 10(-11)) but far less significant in intestinal type. In conclusion, these findings could bring additional insights into the etiologic heterogeneity in gastric carcinogenesis mechanisms.

Cohypermethylation of p14 in combination with CADM1 or DCC as a recurrence-related prognostic indicator in stage I esophageal squamous cell carcinoma.

BACKGROUND: The objective of this study was to discover molecular biomarkers associated with the recurrence of esophageal squamous cell carcinoma (ESCC). METHODS: The authors retrospectively analyzed the hypermethylation status of 11 genes using methylation-specific polymerase chain reaction (PCR) and the expression of epidermal growth factor receptor (EGFR), O-6 methylguanine-DNA methyltransferase (MGMT), tumor protein 53 (p53), and transforming growth factor ß (TGFß) using immunohistochemistry in 329 formalin-fixed, paraffin-embedded ESCCs. RESULTS: Recurrence was identified in 151 of 329 ESCCs (46%) at a median follow-up of 4.5 years. The recurrence was associated with hypermethylation of the genes cell adhesion molecule 1 (CADM1) (P = .003), deleted in colon carcinoma (DCC) (P = .04), or cyclin-dependent kinase inhibitor 2A (p14) (P = .02) in patients with stage I ESCC. Thirty-six of 37 Stage I ESCCs (97%) that had cohypermethylation of at least 2 of the 3 genes had hypermethylation of p14 plus either CADM1 or DCC or both CADM1 and DCC. The 5-year recurrence-free survival (RFS) rates were 93% in patients who had stage I disease without hypermethylation of the 3 genes and 56% in those who had cohypermethylation of p14 in combination with CADM1 and/or DCC. Patients who had stage I ESCC with cohypermethylation of p14 in combination with DCC and/or CADM1 had 7.13 times (95% confidence interval, 1.61-31.64 times; P = .009) poorer RFS compared with those who had no hypermethylation of the 3 genes after adjusting confounding factors. Hypermethylation of the other 8 genes and altered expression of 4 proteins were not associated with recurrence across pathologic stages. CONCLUSIONS: The current results suggested that cohypermethylation of p14 in combination with DCC and/or CADM1 may be an independent prognostic factor for recurrence in patients with stage I ESCC.

Association of RASSF1A and p63 with poor recurrence-free survival in node-negative stage I-II non-small cell lung cancer.

PURPOSE: This study was aimed at analyzing the recurrence-related prognostic significance of 12 candidate molecular biomarkers in node-negative stage I-II non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: We retrospectively analyzed promoter methylation of eight genes using methylation-specific PCR in formalin-fixed and paraffin-embedded tissues from 328 node-negative stage I-II NSCLCs. The expression of Bcl-2, E-cadherin, p53, and p63 proteins was also assessed by immunohistochemistry. RESULTS: Recurrence was found in 145 (44%) of 328 node-negative stage I-II NSCLCs with a median follow-up period of 6.2 years. No association was found between recurrence and alteration of individual biomarker in univariate analysis. We defined recurrently divergent groups on the basis of recursive partitioning analyses for 12 biomarkers and found a significant association of co-alteration of RASSF1A and p63 with poor recurrence-free survival (RFS). Cox proportional hazards analysis showed that hypermethylation of RASSF1A and negative expression of p63 was associated with poor RFS [HR, 1.93; 95% confidence interval (CI), 1.13-5.47; P = 0.009] compared with those without co-alteration of RASSF1A and p63, after adjusting for age, adjuvant therapy, histology, and tumor size. Random forest classifier including RASSF1A and p63 showed best performance in the prediction of recurrence in node-negative stage I-II NSCLCs: area under receiver operator characteristic curve for random forest was 0.91 and error rate for the model was 17%. CONCLUSION: The present study suggests that RASSF1A and p63 may be independent prognostic indicators for RFS in node-negative stage I-II NSCLCs.

Synergistic effect of Bcl-2 and cyclin A2 on adverse recurrence-free survival in stage I non-small cell lung cancer.

BACKGROUND: The prognostic significance of cyclin A2 overexpression in non-small cell lung cancer (NSCLC) is controversial. METHODS: To understand the effect of cyclin A2 on recurrence in NSCLC, we retrospectively analyzed the expression of Bcl-2, cyclin A2, E-cadherin, Ki-67, and p53 using immunohistochemistry in 635 NSCLCs. RESULTS: Overexpression of cyclin A2 was found in 466 (73%) of 635 NSCLCs, and recurrence occurred in 291 (46%) of 635 NSCLCs with a median follow-up of 5.4 years. The relationship between recurrence and cyclin A2 overexpression was not homogenous by pathologic stage (Breslow-Day test for homogeneity, P = 0.007). Overexpression of cyclin A2 was associated with poor recurrence-free survival (RFS) in 374 stage I NSCLCs (P = 0.02), and RFS was worse in patient with negative expression of Bcl-2 than those with positive expression of Bcl-2. Cox proportional hazard analysis showed that stage I NSCLC patients with overexpression of cyclin A2 and negative expression of Bcl-2 had poorer RFS (hazard ratio = 3.86, 95% confidence interval = 1.07-15.77; P = 0.03) than those with normal expression of cyclin A2 and Bcl-2, after adjusting for age, adjuvant radiotherapy, and histology. Neural network and generalized linear model including cyclin A2 and Bcl-2 showed best performance in the prediction of recurrence; error rates for neural network and generalized linear model were 15% and 12%, respectively. CONCLUSIONS: Negative effect of cyclin A2 on RFS in stage I NSCLC was aggravated by negative expression of Bcl-2.

Reduced expression of cyclin D2 is associated with poor recurrence-free survival independent of cyclin D1 in stage III non-small cell lung cancer.

BACKGROUND: Compared to well-known function of cyclin D1 in lung cancer, the role of cyclin D2 is not clear. This study was aimed at understanding the clinicopathological significance of cyclin D2 in primary non-small cell lung cancer (NSCLC). METHODS: We retrospectively analyzed expression statuses of cyclin D1, cyclin D2, p16, p21, p27, Ki-67, and phospho-pRb (Ser-807/811) using immunohistochemistry in 626 NSCLCs. RESULTS: Cyclin D2 was expressed in normal lung tissue, and its expression was reduced in 170 (27%) of 626 NSCLCs with a median duration of follow-up of 64 months. Mean phospho-pRb (Ser-807/811) levels were not associated with expression levels of cyclin D2 (P=0.15). The relationship between recurrence and the reduced expression of cyclin D2 was not homogenous by stage (Breslow-Day test for homogeneity, P=0.04). Reduced expression of cyclin D2 was not associated with patient's prognosis in 370 stage I, 112 stage II, and 18 stage IV NSCLCs. However, for 126 stage III NSCLCs, reduced expression of cyclin D2 was adversely associated with recurrence-free survival (RFS) (hazard ratio [HR]=3.71, 95% CI=1.54-13.17; P=0.01), independent of histology and expression of cyclin D1. The reduced expression of cyclin D2 was not associated with the overexpression of cyclin D1 (P=0.65). CONCLUSION: The present study suggests that reduced expression of cyclin D2 in stage III NSCLC may be associated with poor RFS. And, cyclin D2 may have a distinct role from cyclin D1 in NSCLC.

Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1.

CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (-110 to -104 bp) and a GC box (-41 to -32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.

Condensed chromatin staining of CKAP2 as surrogate marker for mitotic figures.

PURPOSE: Proliferation activity has long been known to be one of the strongest prognostic factors in many different cancers. Nevertheless, microscopic evaluation of mitotic figures remains time-consuming and, furthermore, is relatively subjective. As the expression of cytoskeleton-associated protein 2 (CKAP2) is closely related to the mitotic phase, CKAP2 was evaluated as a surrogate mitotic figure (MF) marker. METHODS: A monoclonal antibody specific to human CKAP2 was produced, and immunohistochemistry was performed on normal tissue array sections and 30 breast cancer tissues. RESULTS: The expression of CKAP2 in the normal human tissues was limited to well-known cell proliferation zones. Strong, readily visible, condensed chromatin staining of CKAP2 was observed specifically in mitotic cells, and the number of these cells was tightly correlated with the MF count in breast cancer tissues (P < 0.001, ρ = 0.743), suggesting its usefulness as a surrogate marker for MF counting. CONCLUSION: Immunohistochemical staining with CKAP2 monoclonal antibody can be considered to be a new, effective approach to the assessment of proliferation activity in cancer tissues.

A functional single nucleotide polymorphism at the promoter region of cyclin A2 is associated with increased risk of colon, liver, and lung cancers.

BACKGROUND: The objective of this was to identify functional single nucleotide polymorphisms (SNPs) in cyclin-dependent kinases (CDKs) and cyclins that are associated with risk of human cancer. METHODS: First, 45 SNPs in CDKs and cyclins were analyzed in 106 lung cancers and 108 controls for a pilot study. One SNP (reference SNP [rs] 769236, +1 guanine to adenine [G→A]) at the promoter region of cyclin A2 (CCNA2) also was analyzed in 1989 cancers (300 breast cancers, 450 colorectal cancers, 450 gastric cancers, 367 hepatocellular carcinomas, and 422 lung cancers) and in 1096 controls. Genotyping was performed using matrix-assisted laser desorption-ionization/time-of-flight mass spectrometry. Transcriptional activity of the SNP according to the cell cycle was analyzed by using a luciferase reporter assay and fluorescence-activated cell sorting analysis in NIH3T3 cells. RESULTS: In the pilot study, the SNP (rs769236) was associated significantly with the risk of lung cancer. In the expanded study, multivariate logistic regression indicated that the AA homozygous variant of the SNP was associated significantly with the development of lung cancer (P < .0001; codominant model), colorectal cancer (P < .0001), and hepatocellular carcinoma (P = .02) but not with breast cancer or gastric cancer. The luciferase activity of a 300-base pair construct that contained the A allele was 1.5-fold greater than the activity of a construct with the G allele in NIH3T3 cells. The high luciferase activity of constructs that contained the A allele did not change with cell cycle progression. CONCLUSIONS: The current results suggested that an SNP (rs769236) at the promoter of CCNA2 may be associated significantly with increased risk of colon, liver, and lung cancers.

Cystatin M loss is associated with the losses of estrogen receptor, progesterone receptor, and HER4 in invasive breast cancer.

INTRODUCTION: This study was aimed at understanding the clinicopathological significance of cystatin M loss, and investigating possible factors responsible for cystatin M loss in breast cancer. METHODS: The expression of estrogen receptor (ER), progesterone receptor (PR), HER2, HER4, and cystatin M was retrospectively analyzed using immunohistochemistry in 117 patients with ductal carcinoma in situ (DCIS) and in 175 patients with invasive breast cancer (IBC). The methylation status of CST6 gene encoding cystatin M was evaluated using methylation-specific polymerase chain reaction (PCR) in formalin-fixed paraffin-embedded tissues from 292 participants and using pyrosequencing in fresh-frozen tumor and matched normal tissues from 51 IBC patients. RESULTS: Cystatin M loss was found in 9 (8%) of 117 patients with DCIS and in 99 (57%) of 175 with invasive breast cancer (IBC) (P < 0.0001). Cystatin M loss was found in 58 (57%) of 101 HER2-negative IBCs and in 41 (55%) of 74 HER2-positive IBCs, and this difference was not statistically significant (P = 0.97). However, cystatin M loss was significantly associated with the loss of ER (P = 0.01), PR (P = 0.002), and HER4 (P = 0.003) in IBCs. Cystatin M loss occurred in 34 (76%) of the 45 HER4-negative IBCs and in 65 (50%) of the 130 HER4-positive IBCs. Multivariate analysis showed that cystatin M loss occurred at a 3.57 times (95% CI = 1.28 to 9.98; P = 0.01) higher prevalence in the triple-negative IBCs of ER, PR, and HER4 than in other subtypes, after adjusting for age. The quantity of CST6 methylation was associated with ER loss (P = 0.0002) in IBCs but not with the loss of PR (P = 0.64) or HER4 (P = 0.87). CONCLUSIONS: The present study suggests that cystatin M loss may be associated with the losses of ER, PR, and HER4 in IBC.

Specific primary sequence requirements for Aurora B kinase-mediated phosphorylation and subcellular localization of TMAP during mitosis.

During mitosis, regulation of protein structures and functions by phosphorylation plays critical roles in orchestrating a series of complex events essential for the cell division process. Tumor-associated microtubule-associated protein (TMAP), also known as cytoskeleton-associated protein 2 (CKAP2), is a novel player in spindle assembly and chromosome segregation. We have previously reported that TMAP is phosphorylated at multiple residues specifically during mitosis. However, the mechanisms and functional importance of phosphorylation at most of the sites identified are currently unknown. Here, we report that TMAP is a novel substrate of the Aurora B kinase. Ser627 of TMAP was specifically phosphorylated by Aurora B both in vitro and in vivo. Ser627 and neighboring conserved residues were strictly required for efficient phosphorylation of TMAP by Aurora B, as even minor amino acid substitutions of the phosphorylation motif significantly diminished the efficiency of the substrate phosphorylation. Nearly all mutations at the phosphorylation motif had dramatic effects on the subcellular localization of TMAP. Instead of being localized to the chromosome region during late mitosis, the mutants remained associated with microtubules and centrosomes throughout mitosis. However, the changes in the subcellular localization of these mutants could not be completely explained by the phosphorylation status on Ser627. Our findings suggest that the motif surrounding Ser627 ((625) RRSRRL (630)) is a critical part of a functionally important sequence motif which not only governs the kinase-substrate recognition, but also regulates the subcellular localization of TMAP during mitosis.

Glutamate receptor-mediated phosphorylation of ezrin/radixin/moesin proteins is implicated in filopodial protrusion of primary cultured hippocampal neuronal cells.

Previously, we reported the phosphorylation of moesin induced by electroconvulsive shock in rat brain and by glutamate in immortalized rat hippocampal cells. However, the function of phosphorylated moesin in differentiated neurons is not well understood. In this study, we observed that glutamate induces phosphorylation of ezrin/radixin/moesin proteins (ERM) in cultured hippocampal cells and that phosphorylated ERM localizes at the newly formed filopodia of neurites. The glutamate-induced phosphorylation of ERM is calcium-dependent, and inhibition of protein kinase C abolishes ERM phosphorylation as well as RhoA activation. The inhibitions of RhoA and RhoA kinase also diminishes the glutamate-induced ERM phosphorylation in cultured hippocampal cells. The knock-down of moesin or the inhibition of ERM phosphorylation results in the reduction of glutamate-induced filopodia protrusion and diminishes the increase in active synaptic boutons induced by glutamate treatment. These results indicate that glutamate-induced phosphorylation of ERM proteins in primary cultured differentiated hippocampal neurons is mediated by calcium-dependent protein kinase C, RhoA and RhoA kinase, and the phosphorylated ERM protein is necessary for the formation of filopodial protrusion and may be involved in pre-synaptic trafficking.

NGF-induced moesin phosphorylation is mediated by the PI3K, Rac1 and Akt and required for neurite formation in PC12 cells.

Moesin is a member of ERM family proteins which act as the cross-linkers between plasma membrane and actin-cytoskeleton and is activated by phosphorylation at Thr-558. In neurons, suppression of radixin and moesin alters the growth cone morphology. However, the significance of phosphorylation of ERM proteins in neuronal cells has not been fully investigated. In this study, we studied the signaling pathways responsible for moesin phosphorylation and its functional importance in NGF-treated PC12 cells. NGF rapidly induced the phosphorylation of moesin at Thr-558 in PC12 cells which was dependent on PI3K and Rac1. We found that Akt interacted and phosphorylated with moesin both in vitro and in vivo. Inhibition of PI3K and Rac1 abolished the NGF-induced Akt activation, indicating that Akt is at the downstream of PI3K and Rac1. To examine the functional role of phosphorylated ERM proteins, a dominant negative mutant form of moesin (T558A) was introduced into PC12 cells. The mutant significantly reduced the frequency of cells with neurites following NGF treatment. Our results indicate that PI3K, Rac1 and Akt-dependent phosphorylation of moesin is required for the NGF-induced neurite formation in differentiating PC12 cells.

Chloride channel conductance is required for NGF-induced neurite outgrowth in PC12 cells.

We have previously shown that in PC12 cells: (1) high extracellular KCl induces moesin phosphorylation, an event which was dependent on chloride channel activation, and (2) NGF induces moesin phosphorylation which is required for neurite outgrowth. These results suggest that NGF-induced intracellular signaling and neurite outgrowth is also mediated by activation of anion channels. Using a patch-clamp technique, we found that NGF treatment increased anionic conductance in PC12 cells, an effect which was completely blocked by NPPB, a chloride channel inhibitor. Also, the NGF-induced moesin phosphorylation was suppressed by NPPB. Additionally, NPPB and SITS, another chloride channel blocker, suppressed NGF-induced TrkA phosphorylation and subsequent PI3K/Akt phosphorylation and Rac1 activation in PC12 cells. Moreover, the chloride channel inhibitors also suppressed the neurite outgrowth and decreased the cell viability in response to long-term treatment of NGF. In summary, our results suggest that chloride ion flux plays an important role in TrkA-mediated signaling pathway during NGF-induced differentiation of PC12 cells.

Association of global levels of histone modifications with recurrence-free survival in stage IIB and III esophageal squamous cell carcinomas.

This study was aimed at understanding the effects of histone modifications on recurrence-free survival (RFS) after esophagectomy in esophageal squamous cell carcinoma (ESCC). The acetylation of histone H3 lysine (H3K9Ac), histone H3 lysine 18 (H3K18Ac), and histone H4 lysine 12 (H4K12Ac), and the dimethylation of histone H3 lysine 9 (H3K9diMe) and histone H4 arginine 3 (H4R3diMe) were analyzed by immunohistochemistry in 237 ESCCs. The K-means clustering algorithm was used to identify unique patterns of histone modifications. At a median follow-up of 5.1 years, 109 (46%) of 237 patients had developed recurrence of disease. Mean global levels of H3K9Ac, H3K18Ac, H3K9diMe, H4K12Ac, and H4R3diMe were 81.5%, 65.1%, 80.3%, 45.9%, and 27.4%, respectively. In the analysis of individual histones, a 1% increase in the global level of H3K18Ac in pathologic stage III worsened RFS at 1.009 times [95% confidence interval (CI), 1.001-1.016; P = 0.03], after adjusting for age, sex, and operative method. Cluster analysis also showed significant effects of histone modifications on RFS. For stage IIB cancers, Cox proportional hazards analysis showed that RFS of cluster 1, with high global levels of H3K18Ac and H4R3diMe, was 2.79 times poorer (95% CI, 1.14-6.27; P = 0.008) than that of cluster 2, with low levels. RFS for stage III cancers was also poorer in cluster 1 than cluster 2 (adjusted hazard ratio, 2.42; 95% CI, 1.10-5.34; P = 0.02). In conclusion, the present study suggests that global levels of histone modifications in ESCC may be an independent prognostic factor of RFS.