BAG-1 Expression Research Articles

Glucocorticoid sensitizers Bag1 and Ppid are regulated by adolescent stress in a sex-dependent manner

Early life stress precipitates dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis and this effect is most pronounced in females. The mechanisms that mediate female sensitivity to stress-induced HPA axis dysregulation are unknown. The purpose of this study was to determine whether sex moderates the effects of chronic adolescent stress on glucocorticoid receptor (GR) translocation and moderators of the GR system. Female adolescent rats with a history of chronic stress exposure demonstrated a delayed resolution of the plasma corticosterone response to an acute stressor and this delay was accompanied by attenuated GR translocation compared to control adolescent females. The chronic stress-induced phenotype in females was similar to the baseline phenotype in male adolescent rats. Conversely, the expression patterns of GR moderators/co-chaperones became more sexually dimorphic following chronic stress, suggesting divergent function of the GR system between male and female adolescent rats. Gene expression of Ppid, a positive regulator of the GR, was predicted by plasma estradiol and 34% lower in control adolescent females than males, indicating that sex steroids may play a role in the sexually dimorphic response. After chronic adolescent stress, females displayed elevated hippocampal expression of Bag1 and Ppid genes that was not observed in males. Overall, the GR output to an acute stressor, illustrated by transcription of Nr3c1 (encoding the GR), Bag1, Fkbp5, Ppid, and Src1, was significantly upregulated and differed in a sex-specific and chronic stress-dependent manner. This study provides new evidence for sex differences during development and adaptation of the glucocorticoid receptor chaperone system.

Cloning and Functional Analysis of FLJ20420: A Novel Transcription Factor for the BAG-1 Promoter

BAG-1 is an anti-apoptotic protein that interacts with a variety of cellular molecules to inhibit apoptosis. The mechanisms by which BAG-1 interacts with other proteins to inhibit apoptosis have been extensively explored. However, it is currently unknown how BAG-1 expression is regulated at the molecular level, especially in cancer cells. Here we reported to clone a novel down-regulated BAG-1 expression gene named FLJ20420 using hBAG-1 promoter as a probe to screen Human Hela 5′ cDNA library by Southernwestern blot. The FLJ20420 gene encodes a ∼26-kDa protein that is localized in both the cytoplasm and nucleus. We proved that FLJ20420 protein can specially bind hBAG-1 promoter region by EMSA in vivo and ChIP assay in vivo. Northern blot analysis revealed a low level of FLJ20420 transcriptional expression in normal human tissues (i.e., brain, placenta, lung, liver, kidney, pancreas and cervix), except for heart and skeletal muscles, which showed higher levels. Furthermore, enhanced FLJ20420 expression was observed in tumor cell lines (i.e., MDA468, BT-20, MCF-7, C33A, HeLa and Caski). Knockdown of endogenous FLJ20420 expression significantly increased BAG-1 expression in A549 and L9981 cells, and also significantly enhanced their sensitivity to cisplatin-induced apoptosis. A microarray assay of the FLJ20420 siRNA –transfectants showed altered expression of 505 known genes, including 272 upregulated and 233 downregulated genes. Finally, our gene array studies in lung cancer tissue samples revealed a significant increase in FLJ20420 expression in primary lung cancer relative to the paired normal lung tissue controls (p = 0.0006). The increased expression of FLJ20420 corresponded to a significant decrease in BAG-1 protein expression in the primary lung cancers, relative to the paired normal lung tissue controls (p = 0.0001). Taken together, our experiments suggest that FLJ20420 functions as a down-regulator of BAG-1 expression. Its abnormal expression may be involved in the oncogenesis of human malignancies such as lung cancer.

Increased expression of BAG-1 in rat brain cortex after traumatic brain injury

BAG-1 protein was initially identified as a Bcl-2-binding protein. It was reported to enhance Bcl-2 protection from cell death, suggesting that BAG-1 represents a new type of anti-cell death gene. Moreover, recent study has shown that BAG-1 can enhance the proliferation of neuronal precursor cells, attenuate the growth inhibition induced by siah1. However, its function and expression in the central nervous system lesion are not been understood very well. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of BAG-1 expression in the brain cortex. Double immunofluorescence staining revealed that BAG-1 was co-expressed with NEURON and glial fibrillary acidic protein (GFAP). In addition, we detected that proliferating cell nuclear antigen had the co-localization with GFAP, and BAG-1. All our findings suggested that BAG-1 might involve in the pathophysiology of brain after TBI.

Expression of BAG-1 and PARP-1 in Precursor Lesions and Invasive Cervical Cancer Associated with Human Papillomavirus (HPV)

Cervical cancer remains persistently the second most common malignancies among women worldwide, responsible for 500,000 new cases annually. Only in Brazil, the estimate is for 18,430 new cases in 2011. Several types of molecular markers have been studied in carcinogenesis including proteins associated with apoptosis such as BAG-1 and PARP-1. This study aims to demonstrate the expression of BAG-1 and PARP-1 in patients with low-grade squamous intraepithelial lesions (LSILs), high-grade squamous intraepithelial lesions (HSILs) and invasive squamous cell carcinomas (SCCs) of the uterine cervix and to verify a possible association with HPV infection. Fifty samples of LSILs, 50 samples of HSILs and 50 samples of invasive SCCs of the uterine cervix were analyzed by immunohistochemistry for BAG-1 and PARP-1 expression. PCR was performed to detect and type HPV DNA. BAG-1 expression levels were significantly different between LSILs and HSILs (p = 0,014) and between LSILs and SCCs (p = 0,014). In regards to PARP-1 expression, we found significant differences between the expression levels in HSILs and SCCs (p = 0,022). No association was found between BAG-1 expression and the presence of HPV. However, a significant association was found between PARP-1 expression and HPV positivity in the HSILs group (p = 0,021). In conclusion our research suggests that BAG-1 expression could contribute to the differentiation between LSIL and HSIL/SCC whereas PARP-1 could be useful to the differentiation between HSIL HPV-related and SCC. Further studies are needed to clarify the molecular aspects of the relationship between PARP-1 expression and HPV infection, with potential applications for cervical cancer prediction.

Combined analysis of mRNA expression of ERCC1, BAG-1, BRCA1, RRM1 and TUBB3 to predict prognosis in patients with non-small cell lung cancer who received adjuvant chemotherapy

BackgroundThe aim of this study was to investigate prognostic value of excision repair cross-complementing 1 (ERCC1), BCL2-associated athanogene (BAG-1), the breast and ovarian cancer susceptibility gene 1 (BRCA1), ribonucleotide reductase subunit M1 (RRM1) and class III β-tubulin (TUBB3) in patients with non-small cell lung cancer (NSCLC) who received platinum- based adjuvant chemotherapy.MethodsMessenger RNA expressions of these genes were examined in 85 tumor tissues and 34 adjacent tissue samples using semi-quantitative RT-PCR. The expressions of these five genes were analyzed in relation to chemotherapy and progression-free survival (PFS) and overall survival (OS). Seventy-four patients were enrolled into chemotherapy.ResultsPatients with ERCC1 or BAG-1 negative expression had a significantly longer PFS (P = 0.001 and P = 0.001) and OS (P = 0.001 and P = 0.001) than those with positive expression. Patients with negative ERCC1 and BAG-1 expression benefited more from platinum regimen (P = 0.001 and P = 0.002). Patients with BRCA1 negative expression might have a longer OS (P = 0.052), but not PFS (P = 0.088) than those with BRCA1 positive expression. A significant relationship was observed between the mRNA expression of ERCC1 and BAG-1 (P = 0.042). In multivariate analysis, ERCC1 and BAG-1 were significantly favorable factors for PFS (P = 0.018 and P = 0.017) and OS (P = 0.027 and P = 0.022).ConclusionsERCC1 and BAG-1 are determinants of survival after surgical treatment of NSCLC, and its mRNA expression in tumor tissues could be used to predict the prognosis of NSCLC treated by platinum.

Expression and Clinical Significance of BAG-1 in Human Colorectal Carcinomas

Expression and Clinical Significance of BAG-1 in Human Colorectal Carcinomas

Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts

AbstractDiamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site–mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

Original article Expression of genes FOLR1, BAG1 and LAPTM4B in functioning and non-functioning pituitary adenomas

The mechanism of pathogenesis of adenomas pituitary is still unknown; differences between pituitary cells of different origin are observed. Identification of genes specific to pituitary adenomas should give better understanding of differences in their response to therapy, especially to radiotherapy. The aim of our study was to independently validate differences in the expression of FOLR1, BAG1, LAPTM4B between functioning (FA) and non-functioning (NFA) pituitary adenomas reported by microarray-based studies. Analysis of gene expression was performed by real-time quantitative PCR (QPCR) in 76 pituitary adenomas, 25 functioning and 51 non-functioning ones. The expression of the examined genes was normalized to the reference index, obtained by calculation of the geometric mean of reference genes expression: GUS-B, B2M, ACTB, EIF3S10, UBE2D2 and ATP6V1E. Two genes showed significant differences in expression between non-functioning adenomas and functioning ones (FA) (FOLR1 32.4 x greater p = 0.022, BAG1 2.2 x lower p = 0.0002). The expression of LAPTM4B (1.1 x lower) was only insignificantly changed. The expression of FOLR1 in all tumours (functioning and non-functioning) was higher in older patients (over 50 years of age) (p = 0.018). Expression of BAG1 was significantly lower in older patients (p = 0.015). In a subgroup of pure non-functioning adenomas there was a higher expression of FOLR1 in older patients (p = 0.006). Analysis of expression profiles and invasiveness of tumours did not reveal any significant differences both in non-functioning and functioning tumours. Among pituitary adenomas, the highest level of expression FOLR1 is seen in NFA which are negative by immunohistochemistry to all pituitary hormones while GH-producing adenomas are the only class of pituitary tumours where FOLR1 expression is virtually absent. For BAG1 we confirm a significantly higher expression in functioning (both PRL and GH producing) adenomas than non-functioning ones, while LAPTM4B does not exhibit any expression changes between different classes of pituitary tumours.

P2-12-01: Immunohistochemical (IHC) BAG1 Expression Improves the Estimation of Residual Risk (RR) by IHC4 in Postmenopausal Patients Treated with Anastrozole or Tamoxifen: A TransATAC Study.

Abstract Aim: To determine whether the incorporation of BAG1 staining improves the estimate of RR after endocrine therapy in postmenopausal patients with ER+ve tumours treated with endocrine therapy. Background: BAG1 encodes a protein (BCL2-associated athanogene 1) that binds to BCL2 and enhances its anti-apoptotic effects. BAG1 is included as a separate subgroup in the 21-gene OncotypeDx Recurrence Score (RS) that is used to assess RR after endocrine therapy in primary ER+ breast cancer. IHC4 is a 4-panel set of IHC markers (ER, PgR, HER2, Ki67) that was shown to provide as much prognostic accuracy as RS in the translational arm of the ATAC trial (TransATAC) of anastrozole versus tamoxifen alone or combined and subsequently independently validated (Cuzick et al, JCO, 2011, in press). Addition of extra markers such as BAG1 to IHC4 may improve the accuracy of the IHC4 and provide extra discriminatory power for oncologists. Methods: Samples in triplicate TMAs from the TransATAC cohort were stained for BAG1 using the Genetex 3.10G3E2 antibody after validation using siRNA knockdown. Staining was scored separately as nuclear or cytoplasmic and categorized by intensity as 0, 1, 2 or 3. BAG1 IHC values were assessed for their correlation with BAG1 mRNA levels. The statistical analysis plan was pre-specified and tested possible additional information from BAG1 expression to the IHC4 in patients not treated with chemotherapy by change in the likelihood ratio chi-square (ΔLR-X2). Results were included only if there was also complete data for ER, PgR, Ki67 and HER2. Primary analysis was on the HER2−ve node-negative (N-neg) population; secondary analysis was on all N-neg patients. Follow-up was to 10 years and the primary end-point was time to distant recurrence (TTDR). Results: Data on both nuclear and cytoplasmic BAG1 as well as the other 4 IHC parameters was available on 961 cases of which 855 were HER2−ve. There was a significant correlation between cytoplasmic and nuclear BAG1 (p=0.23, p<0.0001) but the nuclear staining correlated better with mRNA levels and was therefore considered further. Weak but significant correlations were also seen with ER, PgR and tumour grade. In the univariate analysis nuclear BAG1 was significantly associated with worse TTDR in HER2−ve and all N-neg cases (X2=7.91, p=0.005 and X2=10.63, p=0.001 respectively). Nuclear BAG1 also contributed significantly in multivariate analyses in the 2 populations firstly when added to the clinical model (X2=4.99, p=0.02 and X2=5.93, p=0.015 respectively) and secondly when subtracted from clinical plus the IHC4 parameters (X2=5.55, p=0.02 and X2=4.50, p=0.03 respectively). Conclusions: Nuclear BAG1 expression has significant value for estimating RR that is independent of standard clinical and IHC parameters and it improves the prediction of TTDR in the TransATAC population beyond that with the validated IHC4 score. Unlike IHC4 markers, BAG1 is not commonly measured in pathology work-up of breast cancers. The clinical utility of its addition to IHC4 will be tested by measuring its discrimination of high and low risk patients in clinical practice. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-12-01.

The role of expression and polymorphism of the BAG-1 gene in response to platinum-based chemotherapeutics in NSCLC

We investigated the correlation between BAG-1 expression and sensitivity to platinum-based chemotherapeutics in patients with non-small cell lung cancer (NSCLC). mRNA and protein expression of BAG-1 in lung tissue of NSCLC postoperative patients (I–IIIA stage) or healthy subjects were detected using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. Cox regression analysis was used to quantify the association of prognostic factors with survival in NSCLC patients. Venous blood samples from patients newly diagnosed with advanced NSCLC (IIIB–IV stage) were collected before chemotherapy to analyze allelic frequency and gene polymorphisms. Compared to healthy controls (11.67%, 14 cases), levels of mRNA and protein of BAG-1 in lung tissues was significantly higher in NSCLC patients (61.67%, 74 cases) (χ2=5.601, P<0.05). Moreover, BAG-1 expression was identified as an independent prognostic factor for survival in NSCLC patients. As time to progression and survival rate was dramatically increased, patients with a positive expression of BAG-1 exhibited a prolonged survival period (TTP, 49.3 months; 5-year survival rat, 16.21%) compared with those without BAG-1 expression (χ2=7.243, P<0.05). Two BAG-1 digestion patterns (CC and CT) were identified and confirmed. Patients (77.46%) had a C/C genotype at BAG-1 codon 324, while 22.54% had the C/T genotype. The T/T genotype was not present in these patients. The progression risk of patients carrying the C/C genotype at Bag-1 codon 324 was 1.87 times higher than that of patients carrying the C/T genotype (P<0.001). Follow-up examination showed that the chemotherapeutic sensitivity of patients carrying the C/C genotype was 2.852 times higher than that of patients carrying the C/T genotype (95% CI, 1.133–7.182; P=0.026). Significant differences were found in the median progression-free survival (PFS) and overall survival (OS) of these two cohorts of patients. Compared to patients carrying the C/T genotype of BAG-1, patients carrying the C/C genotype at Bag-1 codon 324 exhibited better responses to platinum-based chemotherapy. Hence, the expression of BAG-1 was closely associated with the sensitivity to platinum-based chemotherapeutics in NSCLC patients.

Inhibition of the Single Downstream Target BAG1 Activates the Latent Apoptotic Potential of MYC

Aberrant MYC expression is a common oncogenic event in human cancer. Paradoxically, MYC can either drive cell cycle progression or induce apoptosis. The latent ability of MYC to induce apoptosis has been termed "intrinsic tumor suppressor activity," and reactivating this apoptotic function in tumors is widely considered a valuable therapeutic goal. As a transcription factor, MYC controls the expression of many downstream targets, and for the majority of these, it remains unclear whether or not they play direct roles in MYC function. To identify the subset of genes specifically required for biological activity, we conducted a screen for functionally important MYC targets and identified BAG1, which encodes a prosurvival chaperone protein. Expression of BAG1 is regulated by MYC in both a mouse model of breast cancer and transformed human cells. Remarkably, BAG1 induction is essential for protecting cells from MYC-induced apoptosis. Ultimately, the synthetic lethality we have identified between MYC overexpression and BAG1 inhibition establishes a new pathway that might be exploited to reactivate the latent apoptotic potential of MYC as a cancer therapy.

A new type of pterocarpanquinone that affects Toxoplasma gondii tachyzoites in vitro

Toxoplasma gondii, the agent of Toxoplasmosis, is an obligate intracellular protozoan able to infect a wide range of vertebrate cells, including nonprofessional and professional phagocytes. Therefore, drugs must have intracellular activities in order to control this parasite. The most common therapy for Toxoplasmosis is the combination of sulfadiazine and pyrimethamine. This treatment is associated with adverse reactions, thus, the development of new drugs is necessary. In previous studies, naphthoquinone derivatives showed anti-cancer activity functioning as agents capable of acting on groups of DNA, preventing cancer cells duplication. These derivatives also display anti-parasitic activity against Plasmodium falciparum and Leishmania amazonensis. The derivative pterocarpanquinone tested in this work resulted from the molecular hybridization between pterocarpans and naphtoquinone that presents anti-tumoral and anti-parasitic activities of lapachol. The aim of this work was to determine if this derivative is able to change T. gondii growth within LLC-MK2 cells. The drug did not arrest host cell growth, but was able to decrease the infection index of T. gondii with an IC50 of 2.5μM. Scanning and transmission electron microscopy analysis showed morphological changes of parasites including membrane damage. The parasite that survived tended to encyst as seen by Dolichos biflorus lectin staining and Bag-1 expression. These results suggest that pterocarpanquinones are drugs potentially important for the killing and encystment of T. gondii.

BAG-1 interacts with the p50-p50 homodimeric NF-κB complex: implications for colorectal carcinogenesis.

Understanding the mechanisms that promote aberrant tumour cell survival is critical for the determination of novel strategies to combat colorectal cancer. We have recently shown that the anti-apoptotic protein BAG-1, highly expressed in pre-malignant and colorectal cancer tissue, can potentiate cell survival through regulating NF-κB transcriptional activity. In this study, we identify a novel complex between BAG-1 and the p50-p50 NF-κB homodimers, implicating BAG-1 as a co-regulator of an atypical NF-κB pathway. Importantly, the BAG-1-p50 complex was detected at gene regulatory sequences including the epidermal growth factor receptor [EGFR] and COX-2 [PTGS2] genes. Suppression of BAG-1 expression using siRNA was shown to increase EGFR and suppress COX-2 expression in colorectal cancer cells. Furthermore, mouse embryonic fibroblasts derived from the NF-κB1 [p105/p50] knockout mouse were used to demonstrate that p50 expression was required for BAG-1 to suppress EGFR expression. This was shown to be functionally relevant as attenuation of BAG-1 expression increased ligand activated phosphorylation of EGFR in colorectal cancer cells. In summary, this paper identifies a novel role for BAG-1 in modulating gene expression through interaction with the p50-p50 NF-κB complexes. Data presented led us to propose that BAG-1 can act as a selective regulator of p50-p50 NF-κB responsive genes in colorectal tumour cells, potentially important for the promotion of cell survival in the context of the fluctuating tumour microenvironment. As BAG-1 expression is increased in the developing adenoma through to metastatic lesions, understanding the function of the BAG-1-p50 NF-κB complexes may aid in identifying strategies for both the prevention and treatment of colorectal cancer.

Correlation between the expression of the BAG-1 gene and clinicopathologic factors in colorectal cancer

The present study aims to investigate the expression and significance of the anti-apoptotic gene Bag-1 in colorectal cancer and to evaluate the relationship between the gene and the disease. Bag-1 expression was examined in 320 colorectal cancer and 30 normal colorectal tissue samples using reverse transcriptase polymerase chain reaction (RT-PCR) and the immunohistochemical staining (streptavidin-biotin-peroxidase complex method. Using RT-PCR, Bag-1 was observed to be expressed in colorectal cancer tissues, but not in normal colorectal tissues. The expression of Bag-1 in colorectal cancer was closely correlated with pathologic grade, distant metastasis, Dukes stage, and prognosis, but it was not correlated with the pathologic type, tumor diameter, depth of invasion, and lymph node metastasis. Bag-1 protein was found to be overexpressed in colorectal cancer. They might be regarded as a biomarker for the diagnosis of the early stages of colorectal cancer. In addition, they have particular significance for the prognosis of colorectal cancer.

Involvement of a Toxoplasma gondii Chromatin Remodeling Complex Ortholog in Developmental Regulation

The asexual cycle of the parasite Toxoplasma gondii has two developmental stages: a rapidly replicating form called a tachyzoite and a slow growing cyst form called a bradyzoite. While the importance of ATP-independent histone modifications for gene regulation in T. gondii have been demonstrated, ATP-dependent chromatin remodeling pathways have not been examined. In this study we characterized C9, an insertional mutant showing reduced expression of bradyzoite differentiation marker BAG1, in cultured human fibroblasts. This mutant contains an insertion in the gene encoding TgRSC8, which is homologous to the Saccharomyces cerevisiae proteins Rsc8p (remodel the structure of chromatin complex subunit 8) and Swi3p (switch/sucrose non-fermentable [SWI/SNF]) of ATP-dependent chromatin-remodeling complexes. In the C9 mutant, TgRSC8 is the downstream open reading frame on a dicistronic transcript. Though protein was expressed from the downstream gene of the dicistron, TgRSC8 levels were decreased in C9 from those of wild-type parasites, as determined by western immunoblot and flow cytometry. As TgRSC8 localized to the parasite nucleus, we postulated a role in gene regulation. Transcript levels of several markers were assessed by quantitative PCR to test this hypothesis. The C9 mutant displayed reduced steady state transcript levels of bradyzoite-induced genes BAG1, LDH2, SUSA1, and ENO1, all of which were significantly increased with addition of TgRSC8 to the mutant. Transcript levels of some bradyzoite markers were unaltered in C9, or unable to be increased by complementation with TgRSC8, indicating multiple pathways control bradyzoite-upregulated genes. Together, these data suggest a role for TgRSC8 in control of bradyzoite-upregulated gene expression. Thus chromatin remodeling, by both ATP-independent and dependent mechanisms, is an important mode of gene regulation during stage differentiation in parasites.

WNT5A is a novel regulator of cytokine and chemokine expression in bone marrow stromal cells

WNT5A is a novel regulator of cytokine and chemokine expression in bone marrow stromal cells

Haploinsufficiency of Bag-1 affects cartilage development and osteogenic differentiation in vitro

Event Abstract Back to Event Haploinsufficiency of Bag-1 affects cartilage development and osteogenic differentiation in vitro RS Tare1*, PA Townsend2, GK Packham3 and ROC Oreffo1 1 University of Southampton, Bone and Joint Research Group, United Kingdom 2 University of Southampton, Human Genetics, United Kingdom 3 University of Southampton, Cancer Sciences, United Kingdom The Bcl-2-associated athanogene-1 (Bag-1) co-chaperone is expressed by chondrocytes and osteoblasts, and functions as a transcriptional regulator mediating chondrocyte differentiation and apoptosis in vitro (1). By utilising cell populations from a mouse model of Bag-1 haploinsufficiency (i.e. one Bag-1 null allele and one normal Bag-1 allele), this study aims to define effects of Bag-1 heterozygosity on formation of cartilage templates and osteoblast development. {BR}Micromass cultures of E11.5 Bag-1+/+ and Bag-1+/- mouse embryonic fibroblasts (MEFs), differentiated in presence of BMP-2 (100 ng/ml), ascorbate and beta-glycerophosphate, were utilised to model in vivo developmental stages in the formation of cartilaginous primordia. Cultures were analysed at days 7, 14, 21, 28, 35 and 42 for expression of Bag-1 and development of cartilage nodules. Bone marrow cells (BMCs) from 14-week Bag-1+/+ and Bag-1+/- mice were cultured for 21 days in media supplemented with either 10 nM Dexamethasone, 25 nM Calcitriol or 100 ng/ml BMP-2, to analyse their osteogenic response. {BR}During the initial 14-day period, significantly high Bag-1 expression was observed in micromass cultures of Bag-1+/+ MEFs compared to Bag-1+/- MEFs. The 14-day period of micromass culture is comparable to in vivo murine embryonic limb development at E10.5 and E11.5, associated with robust Bag-1 expression and formation of vital mesenchymal condensations prefiguring future skeletal elements. Significantly, days 7 and 14 of Bag-1+/+ MEF micromass cultures were characterised by the presence of cellular condensations and mineralised cartilaginous nodules were observed by day 42 of culture. Bag-1 haploinsufficiency compromised the ability of Bag-1+/- MEFs to undergo condensations and organise into defined cartilaginous nodules by day 42 of micromass culture. Moreover, morphologically distinct hypertrophic chondrocytes (characterised by increase in cell volume and hence, enlarged cell size) in lacunae were absent in day 42 Bag-1+/- micromass cultures. Bag-1 heterozygosity also compromised the ability of BMCs to undergo robust osteogenic differentiation, as demonstrated by a decreased osteogenic response of Bag-1+/- BMCs compared to Bag-1+/+ BMCs in presence of the supplemented osteogenic factors. {BR}Our results illustrate a role for Bag-1 in the critical phases of skeletal development, namely formation of the cartilage templates of endochondral bones and osteoblast differentiation.{BR}(1) Bone 2008;42:113-28 Keywords: Bones, Bone Research Conference: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society, Cambridge, United Kingdom, 27 Jun - 29 Jun, 2011. Presentation Type: Oral Poster Topic: Abstracts Citation: Tare R, Townsend P, Packham G and Oreffo R (2011). Haploinsufficiency of Bag-1 affects cartilage development and osteogenic differentiation in vitro. Front. Endocrinol. Conference Abstract: 2011 joint meeting of the Bone Research Society & the British Orthopaedic Research Society. doi: 10.3389/conf.fendo.2011.02.00066 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2011; Published Online: 30 Sep 2011. * Correspondence: Dr. RS Tare, University of Southampton, Bone and Joint Research Group, Southampton, SO16 6YD, United Kingdom, rt2@soton.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers RS Tare PA Townsend GK Packham ROC Oreffo Google RS Tare PA Townsend GK Packham ROC Oreffo Google Scholar RS Tare PA Townsend GK Packham ROC Oreffo PubMed RS Tare PA Townsend GK Packham ROC Oreffo Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Bag1 directly routes immature BCR-ABL for proteasomal degradation

AbstractDegradation of BCR-ABL oncoproteins by heat shock protein 90 (Hsp90) inhibitors in chronic myelogenous leukemia is expected to overcome resistance to ABL tyrosine kinase inhibitors. However, the precise mechanisms still remain to be uncovered. We found that while c-Cbl E3 ligase induced ubiquitin-dependent degradation of mature and phosphorylated BCR-ABL proteins, another E3 ligase CHIP (carboxyl terminus of the Hsc70-interacting protein) degraded immature BCR-ABL proteins and efficiently suppressed BCR-ABL–dependent leukemic growth. Interestingly, Bag1 (Bcl-2-associated athanogene-1), a nucleotide exchange factor for Hsc70, directly bound BCR-ABL with a high affinity, which was enhanced by CHIP and Hsp90 inhibitors, inhibited by imatinib and competed with Hsc70. Bag1 knockdown abrogated Hsp90 inhibitor-induced BCR-ABL degradation. Bag1 induced binding of immature BCR-ABL to proteasome. Expression of Bag1 induced BCR-ABL degradation and growth suppression in Ba/F3 cells when Hsc70 was knocked down with or without CHIP induction. CHIP appears to sort newly synthesized Hsp90-unchaperoned BCR-ABL to the proteasome not only by inhibiting Hsc70 and thereby promoting Bag1 to bind BCR-ABL, but also by ubiquitinating BCR-ABL. Bag1 may direct CHIP/Hsc70-regulated protein triage decisions on BCR-ABL immediately after translation to the degradation pathway.

Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of IκBα and nuclear accumulation of NF-κB

Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulate the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-κB. Furthermore phosphorylation and degradation of the inhibitor protein IκBα and nuclear accumulation of p65 and p50 NF-κB proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored IκBα and NF-κB activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.

Expressions of the anti-apoptotic genes Bag-1 and Bcl-2 in colon cancer and their relationship

Background The aims of this study were to investigate the expressions and significance of the antiapoptotic genes Bag-1 and Bcl-2 in colon cancer and to evaluate their relationship. Methods The expressions of Bag-1 and Bcl-2 were examined in 128 colon cancer and 20 normal colon tissue samples by reverse-transcription polymerase chain reaction and immunohistochemical technique (streptavidin-biotin-peroxidase complex method). Results Bag-1 and Bcl-2 were expressed in colorectal cancer tissues but not in normal colorectal tissues by reverse-transcription polymerase chain reaction. The expression of Bag-1 in colon cancer was closely correlated with pathologic grade, distance metastasis, Duke stage, and prognosis, but it had no effect on the pathologic type, tumor diameter, depth of invasion, and lymphoid node metastasis of the cancer. By contrast, Bcl-2 had no significant correlation with all the clinical and pathologic factors. There was a positive correlation between Bag-1 and Bcl-2 in the development of colon cancer. Conclusions High expressions of Bag-1 and Bcl-2 proteins in colon cancer were found. They might be regarded as biomarkers for the diagnosis of the early stage of colon cancer. In addition, they have significant relevance for the prognosis of colon cancer.