Cytoplasmic Nuclear Translocation Research Articles

Decreased Stathmin-1 Expression Inhibits Trophoblast Proliferation and Invasion and Is Associated with Recurrent Miscarriage

Fetal trophoblasts invade endometrium and establish a complex interaction with the maternal microenvironment during early pregnancy. However, the molecular mechanisms regulating trophoblast migration and invasion at the maternal-fetal interface remain poorly understood. Immunohistochemistry and immunoblotting have shown that stathmin-1 (STMN1) was down-regulated significantly in placental villi tissue and trophoblasts from patients with recurrent miscarriage. Invitro, overexpression of STMN1 promoted human trophoblast proliferation, migration, and invasion, whereas knockdown of STMN1 inhibited these processes. In addition, knockdown of STMN1 down-regulated N-cadherin and up-regulated E-cadherin in trophoblasts, whereas E-cadherin was up-regulated and N-cadherin was down-regulated in recurrent miscarriage villi tissue. Knockdown of STMN1 attenuated cytoplasmic-nuclear translocation of β-catenin and in turn down-regulated trophoblast matrix metalloproteases. Furthermore, tumor necrosis factor-α (TNF-α) down-regulated STMN1 expression, and serum TNF-α expression correlated inversely with trophoblast STMN1 levels. Interestingly, M1 macrophage-derived TNF-α reduced trophoblast migration and invasion, and an anti-TNF-α antibody reversed this effect. Collectively, this study indicated that STMN1 may play a key role in regulating trophoblast invasion, and that impaired STMN1 expression may lead to abnormal trophoblast invasion and result in recurrent miscarriage.

FRI0351 The Feedback Loop between Long Noncoding RNA NRON and NFAT5 Regulates the Inflammatory Response of Rheumatoid Arthritis Synovial Fibroblasts

BackgroundLong noncoding RNA (lncRNA) are novel RNA transcripts, emerging as master regulators of gene expression. The lncRNA NRON, noncoding repressor of nuclear factor of activated T cells (NFAT) can repress...

SEPT9_i1 is required for the association between HIF-1α and importin-α to promote efficient nuclear translocation

Septin 9 isoform 1 (SEPT9_i1) protein associates with hypoxia-inducible factor (HIF)-1α to augment HIF-1 transcriptional activity. The first 25 amino acids of SEPT9_i1 (N25) are unique compared with other members of the mammalian septin family. This N25 domain is critical for HIF-1 activation by SEPT9_i1 but not essential for the protein-protein interaction. Here, we show that expression of N25 induces a significant dose-dependent inhibition of HIF-1 transcriptional activity under normoxia and hypoxia without influencing cellular HIF-1α protein levels. In vivo, N25 expression inhibits proliferation, tumor growth and angiogenesis concomitant with decreased expression levels of intratumoral HIF-1 downstream genes. Depletion of endogenous SEPT9_i1 or the exogenous expression of N25 fragment reduces nuclear HIF-1α levels accompanied by reciprocal accumulation of HIF-1α in the cytoplasm. Mechanistically, SEPT9_i1 binds to importin-α through N25 depending on its bipartite nuclear localization signal, to scaffold the association between HIF-1α and importin-α, which leads to facilitating HIF-1α nuclear translocation. Our data explore a new and a previously unrecognized role of a septin protein in the cytoplasmic-nuclear translocation process. This new level in the regulation of HIF-1α translocation is critical for efficient HIF-1 transcriptional activation that could be targeted for cancer therapeutics.

Su1001 Circulating Scca-IgM Complex Is an Useful Biomarker to Monitor HCC Therapy

Background and Aims: Using in vitro cell culture and in vivo xenograft mouse models, we have previously shown that the heparan sulfate-degrading endosulfatase SULF1 functions as a tumor suppressor in established hepatocellular carcinoma cell lines by abrogating receptor tyrosine kinase signaling. In contrast, whole genome gene expression analysis of resected human HCCs showed increased expression of SULF1 in 70% of primary HCCs; and SULF1 expression in primary HCCs has been associated with activation of the oncogenic Wnt/β-catenin pathway. Here we report for the first time the results of DEN-induced hepatocarcinogenesis in a hepatocyte-specific transgenic Sulf1 mouse model in which the Sulf1 gene is driven by the transthyretin promoter. The effect of Sulf1 transgenesis on Wnt/ β-catenin activation was assessed. Methods: Liver carcinogenesis was induced in wild-type or hepatocyte-specific Sulf1 transgenic mice by intraperitoneal injection of 15 mg/kg of the liver carcinogen diethylnitrosamine (DEN) at 14 days after birth. Mice were sacrificed at 8 months of age and the harvested livers were examined for weight, tumor size and number. Cell proliferation was assessed using the BrdU proliferation index and Wnt/ β-catenin activation was measured by assessment of β-catenin staining and cytoplasmic-nuclear translocation of β-catenin. Results: Consistent with the observation of increased SULF1 in primary human HCCs, Sulf1 transgenic mice showed increased liver tumorigenesis as demonstrated by tumor number, tumor volume, liver weight/body weight ratio, and BrdU proliferation index. To investigate the mechanism for this, we performed immunohistochemistry for β-catenin. Wnt-β-catenin signaling was increased in Sulf1 transgenic mice compared to wild-type mice, as assessed by both increased β-catenin immunostaining and increased cytoplasmic-nuclear translocation of β-catenin. Conclusions: In contrast to the tumor suppressor effects of SULF1 in established human cell lines, which are mediated by inhibition of the co-receptor function of heparan sulfate in receptor tyrosine kinase signaling, primary HCCs in an in vivo model of DEN-induced HCC in Sulf1 transgenic mice show increased liver tumorigenesis associated with activation ofWnt-β-catenin signaling. This suggests that the effect of Sulf1 in carcinogenesis is context-dependent, but appears to be mediated through oncogenic Wntβ-catenin signaling in the majority of HCCs.

Su1004 High Mobility Group Box-1 Can Increase miR-21 Expression in Hepatocellular Carcinoma Through STAT3 Signaling

Background and Aims: Using in vitro cell culture and in vivo xenograft mouse models, we have previously shown that the heparan sulfate-degrading endosulfatase SULF1 functions as a tumor suppressor in established hepatocellular carcinoma cell lines by abrogating receptor tyrosine kinase signaling. In contrast, whole genome gene expression analysis of resected human HCCs showed increased expression of SULF1 in 70% of primary HCCs; and SULF1 expression in primary HCCs has been associated with activation of the oncogenic Wnt/β-catenin pathway. Here we report for the first time the results of DEN-induced hepatocarcinogenesis in a hepatocyte-specific transgenic Sulf1 mouse model in which the Sulf1 gene is driven by the transthyretin promoter. The effect of Sulf1 transgenesis on Wnt/ β-catenin activation was assessed. Methods: Liver carcinogenesis was induced in wild-type or hepatocyte-specific Sulf1 transgenic mice by intraperitoneal injection of 15 mg/kg of the liver carcinogen diethylnitrosamine (DEN) at 14 days after birth. Mice were sacrificed at 8 months of age and the harvested livers were examined for weight, tumor size and number. Cell proliferation was assessed using the BrdU proliferation index and Wnt/ β-catenin activation was measured by assessment of β-catenin staining and cytoplasmic-nuclear translocation of β-catenin. Results: Consistent with the observation of increased SULF1 in primary human HCCs, Sulf1 transgenic mice showed increased liver tumorigenesis as demonstrated by tumor number, tumor volume, liver weight/body weight ratio, and BrdU proliferation index. To investigate the mechanism for this, we performed immunohistochemistry for β-catenin. Wnt-β-catenin signaling was increased in Sulf1 transgenic mice compared to wild-type mice, as assessed by both increased β-catenin immunostaining and increased cytoplasmic-nuclear translocation of β-catenin. Conclusions: In contrast to the tumor suppressor effects of SULF1 in established human cell lines, which are mediated by inhibition of the co-receptor function of heparan sulfate in receptor tyrosine kinase signaling, primary HCCs in an in vivo model of DEN-induced HCC in Sulf1 transgenic mice show increased liver tumorigenesis associated with activation ofWnt-β-catenin signaling. This suggests that the effect of Sulf1 in carcinogenesis is context-dependent, but appears to be mediated through oncogenic Wntβ-catenin signaling in the majority of HCCs.

Su1003 Role of Activated Hepatic Stellate Cells in Metastasis of Hepatocellular Carcinoma

Background and Aims: Using in vitro cell culture and in vivo xenograft mouse models, we have previously shown that the heparan sulfate-degrading endosulfatase SULF1 functions as a tumor suppressor in established hepatocellular carcinoma cell lines by abrogating receptor tyrosine kinase signaling. In contrast, whole genome gene expression analysis of resected human HCCs showed increased expression of SULF1 in 70% of primary HCCs; and SULF1 expression in primary HCCs has been associated with activation of the oncogenic Wnt/β-catenin pathway. Here we report for the first time the results of DEN-induced hepatocarcinogenesis in a hepatocyte-specific transgenic Sulf1 mouse model in which the Sulf1 gene is driven by the transthyretin promoter. The effect of Sulf1 transgenesis on Wnt/ β-catenin activation was assessed. Methods: Liver carcinogenesis was induced in wild-type or hepatocyte-specific Sulf1 transgenic mice by intraperitoneal injection of 15 mg/kg of the liver carcinogen diethylnitrosamine (DEN) at 14 days after birth. Mice were sacrificed at 8 months of age and the harvested livers were examined for weight, tumor size and number. Cell proliferation was assessed using the BrdU proliferation index and Wnt/ β-catenin activation was measured by assessment of β-catenin staining and cytoplasmic-nuclear translocation of β-catenin. Results: Consistent with the observation of increased SULF1 in primary human HCCs, Sulf1 transgenic mice showed increased liver tumorigenesis as demonstrated by tumor number, tumor volume, liver weight/body weight ratio, and BrdU proliferation index. To investigate the mechanism for this, we performed immunohistochemistry for β-catenin. Wnt-β-catenin signaling was increased in Sulf1 transgenic mice compared to wild-type mice, as assessed by both increased β-catenin immunostaining and increased cytoplasmic-nuclear translocation of β-catenin. Conclusions: In contrast to the tumor suppressor effects of SULF1 in established human cell lines, which are mediated by inhibition of the co-receptor function of heparan sulfate in receptor tyrosine kinase signaling, primary HCCs in an in vivo model of DEN-induced HCC in Sulf1 transgenic mice show increased liver tumorigenesis associated with activation ofWnt-β-catenin signaling. This suggests that the effect of Sulf1 in carcinogenesis is context-dependent, but appears to be mediated through oncogenic Wntβ-catenin signaling in the majority of HCCs.

Su1000 Enhanced Liver Tumorigenesis in Sulfatase 1 (SULF1) Transgenic Mice Is Associated With Activation of the Wnt-β-Catenin Signaling Pathway

Background and Aims: Using in vitro cell culture and in vivo xenograft mouse models, we have previously shown that the heparan sulfate-degrading endosulfatase SULF1 functions as a tumor suppressor in established hepatocellular carcinoma cell lines by abrogating receptor tyrosine kinase signaling. In contrast, whole genome gene expression analysis of resected human HCCs showed increased expression of SULF1 in 70% of primary HCCs; and SULF1 expression in primary HCCs has been associated with activation of the oncogenic Wnt/β-catenin pathway. Here we report for the first time the results of DEN-induced hepatocarcinogenesis in a hepatocyte-specific transgenic Sulf1 mouse model in which the Sulf1 gene is driven by the transthyretin promoter. The effect of Sulf1 transgenesis on Wnt/ β-catenin activation was assessed. Methods: Liver carcinogenesis was induced in wild-type or hepatocyte-specific Sulf1 transgenic mice by intraperitoneal injection of 15 mg/kg of the liver carcinogen diethylnitrosamine (DEN) at 14 days after birth. Mice were sacrificed at 8 months of age and the harvested livers were examined for weight, tumor size and number. Cell proliferation was assessed using the BrdU proliferation index and Wnt/ β-catenin activation was measured by assessment of β-catenin staining and cytoplasmic-nuclear translocation of β-catenin. Results: Consistent with the observation of increased SULF1 in primary human HCCs, Sulf1 transgenic mice showed increased liver tumorigenesis as demonstrated by tumor number, tumor volume, liver weight/body weight ratio, and BrdU proliferation index. To investigate the mechanism for this, we performed immunohistochemistry for β-catenin. Wnt-β-catenin signaling was increased in Sulf1 transgenic mice compared to wild-type mice, as assessed by both increased β-catenin immunostaining and increased cytoplasmic-nuclear translocation of β-catenin. Conclusions: In contrast to the tumor suppressor effects of SULF1 in established human cell lines, which are mediated by inhibition of the co-receptor function of heparan sulfate in receptor tyrosine kinase signaling, primary HCCs in an in vivo model of DEN-induced HCC in Sulf1 transgenic mice show increased liver tumorigenesis associated with activation ofWnt-β-catenin signaling. This suggests that the effect of Sulf1 in carcinogenesis is context-dependent, but appears to be mediated through oncogenic Wntβ-catenin signaling in the majority of HCCs.

2-photon imaging of phagocyte-mediated T cell activation in the CNS

Autoreactive T cells can infiltrate the CNS to cause disorders such as multiple sclerosis. In order to visualize T cell activation in the CNS, we introduced a truncated fluorescent derivative of nuclear factor of activated T cells (NFAT) as a real-time T cell activation indicator. In experimental autoimmune encephalomyelitis, a rat model of multiple sclerosis, we tracked T cells interacting with structures of the vascular blood-brain barrier (BBB). 2-photon imaging documented the cytoplasmic-nuclear translocation of fluorescent NFAT, indicative of calcium-dependent activation of the T cells in the perivascular space, but not within the vascular lumen. The activation was related to contacts with the local antigen-presenting phagocytes and was noted only in T cells with a high pathogenic potential. T cell activation implied the presentation of an autoantigen, as the weakly pathogenic T cells, which remained silent in the untreated hosts, were activated upon instillation of exogenous autoantigen. Activation did not cogently signal long-lasting arrest, as individual T cells were able to sequentially contact fresh APCs. We propose that the presentation of local autoantigen by BBB-associated APCs provides stimuli that guide autoimmune T cells to the CNS destination, enabling them to attack the target tissue.

Actin Cytoskeleton Reorganization Correlates with Cofilin Nuclear Expression and Ultrastructural Changes in CHO AA8 Cell Line after Apoptosis and Mitotic Catastrophe Induction by Doxorubicin

The effect of doxorubicin on the expression of cofilin and actin in CHO AA8 cells was estimated by fluorescence and electron microscopy. The presence of cofilin and actin was observed particularly in the nuclei of cells by different modes after treatment by doxorubicin. Cells undergoing mitotic catastrophe expressed some entirely characteristic features together with overlapping elements of other types of cell death. Additionally, the authors suggest that, as defined here, reorganization of F-actin might be involved in all cell death processes. Changes in the nuclear expression of cofilin are related to F-actin cytoplasm–nuclear translocation and its intranuclear dynamic reorganization.

14‐3‐3ζ escorts CCTα for calcium‐activated nuclear import in lung epithelia

Integrity of animal biomembranes is critical to preserve normal cellular functions and viability. Phosphatidylcholine, an indispensible membrane component, requires the enzyme CCTalpha for its biosynthesis. Nuclear expression of CCTalpha is needed for expansion of the nuclear membrane network, but mechanisms for CCTalpha nuclear import are unknown. Herein, we show that in epithelia, extracellular Ca(2+) triggers CCTalpha cytoplasmic-nuclear translocation. CCTalpha nuclear import was associated with binding to 14-3-3zeta, a key regulator of protein trafficking. 14-3-3zeta was both sufficient and required for CCTalpha nuclear import. Helix G within the 14-3-3zeta binding groove interacts with a putative molecular signature within the CCTalpha carboxyl-terminal phosphoserine motif (residues 328-343). 14-3-3zeta was critically involved in preserving phosphatidylcholine synthesis and cell viability in a model of Pseudomonas aeruginosa infection where Ca(2+) concentrations increase within epithelia. Thus, 14-3-3zeta controls CCTalpha nuclear import in response to calcium signals, thereby regulating mammalian phospholipid synthesis. Agassandian, M., Chen, B. B., Schuster, C. C., Houtman, J. C. D., Mallampalli, R. K. 14-3-3zeta escorts CCTalpha for calcium-activated nuclear import in lung epithelia.

Mechanistic insight into WEB-2170-induced apoptosis in human acute myelogenous leukemia cells: The crucial role of PTEN

This study aimed to investigate the mechanisms of action of WEB-2170, an inverse agonist of platelet-activating factor receptor, capable of inducing apoptosis in human acute myelogenous leukemia (AML) cells. Gene expression profiling followed by cytofluorimetric, morphologic, and biologic analyses were used to monitor WEB-2170 effects in AML cell lines (ie, NB4, KG1, NB4-MR4, THP1, and U937) and blasts from patients with different AML (M0-M5) subtypes. PTEN silencing with small interfering RNA was also performed. We have demonstrated that drug-mediated cytostasis/apoptosis in NB4 cells is characterized by upregulation of cyclin G2, p21/WAF1, NIX, TNF-alpha, and PTEN expression, and downregulation of cyclin D2 and BCL2 expression. We observed an increase in PTEN protein accompanied by a decrease in phospho-extracellular signal-regulated kinase 2 (ERK2) and phospho-AKT, and by forkhead box O3a (FOXO3a) cytoplasmic-nuclear translocation; the mitochondrial cytochrome C release and PARP cleavage marked the late apoptotic steps. We have found that WEB-2170 triggered apoptosis in NB4, KG1, and NB4-MR4 cells where PTEN was expressed, but not in THP1 and U937 cells where PTEN was absent. Finally, we show that PTEN silencing in NB4 cells by PTEN-specific small interfering RNA resulted in a significant reduction of drug-induced apoptosis. We demonstrated that WEB-2170 is a powerful antileukemic agent with interesting translational opportunities to treat AML and described mechanisms of drug-induced intrinsic and extrinsic apoptosis both in AML cell lines and blasts from AML patients by addressing PTEN as the master regulator of the whole process.

Bortezomib-Induced Apoptosis with Limited Clinical Response Is Accompanied by Inhibition of Canonical but not Alternative Nuclear Factor-κB Subunits in Head and Neck Cancer

Nuclear factor-kappaB (NF-kappaB)/REL transcription factors promote cancer cell survival and progression. The canonical (NF-kappaB1/RELA or cREL) and alternate (NF-kappaB2/RELB) pathways require the proteasome for cytoplasmic-nuclear translocation, prompting the investigation of bortezomib for cancer therapy. However, limited clinical activity of bortezomib has been observed in many epithelial malignancies, suggesting this could result from incomplete inhibition of NF-kappaB/RELs or other prosurvival signal pathways. To examine these possibilities, matched biopsies from 24 h posttreatment were obtained from accessible tumors of patients who received low-dose bortezomib (0.6 mg/m(2)) before reirradiation in a phase I trial for recurrent head and neck squamous cell carcinoma (HNSCC). Effects of bortezomib on apoptosis and proliferation by TUNEL and Ki67 staining were compared with nuclear staining for all five NF-kappaB subunits, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated signal transducers and activators of transcription 3 (STAT3) in tumor biopsies, and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTP) and DNA binding assay for the five NF-kappaB subunits in HNSCC cell lines. HNSCC showed increased nuclear staining for all five NF-kappaB subunits, phosphorylated ERK1/2, and phosphorylated STAT3. Bortezomib treatment significantly enhanced apoptosis with inhibition of nuclear RELA in three of four tumors, but other NF-kappaB subunits, ERK1/2, and STAT3 were variably or not affected, and tumor progression was observed within 3 months. In HNSCC cell lines, 10(-8) mol/L bortezomib inhibited cell density while inhibiting tumor necrosis factor-alpha-induced and partially inhibiting basal activation of NF-kappaB1/RELA, but not NF-kappaB2/RELB. Although low-dose bortezomib inhibits activation of subunits of the canonical pathway, it does not block nuclear activation of the noncanonical NF-kappaB or other prosurvival signal pathways, which may contribute to the heterogeneous responses observed in HNSCC.

Ser484 and Ser494 in REL Are the Major Sites of IKK Phosphorylation In Vitro: Evidence That IKK Does Not Directly Enhance GAL4-REL Transactivation

Human c-Rel (REL) is a member of the NF-kappa B family of transcription factors, and one of its primary physiological roles is in the regulation of B-cell proliferation and survival. Although REL is primarily regulated by cytoplasmic-nuclear translocation through interaction with I kappa B inhibitors, REL also undergoes several posttranslational modifications that have been proposed to modulate its transcriptional activation activity. For example, phosphorylation of C-terminal sequences of REL has been proposed to increase its transactivation activity. In this report, we have used immune complex kinase assays to identify Ser484 and Ser494 as the primary sites of IKK alpha- and IKK beta-mediated in vitro phosphorylation in the C-terminal transactivation domain of REL. However, in cotransfection studies in A293 cells we have failed to detect IKK beta-mediated phosphorylation of these sites on REL in vivo, nor does IKK beta appear to interact with REL in these cells. Ser-to-Ala mutation of Ser484 and Ser494 does not affect IKK's ability to enhance GAL4-REL transactivation in reporter gene assays in A293 cells. We also show that the previously reported effects of overexpressed IKK and tumor necrosis factor treatment on GAL4-REL transactivation are due to IKK-mediated activation of the endogenous NF-kappa B pathway, which increases transcription from kappa B sites in the promoter of a commonly used GAL4 expression vector. Taken together, these results do not support a role for IKK-mediated phosphorylation as means for regulating the activity of REL in vivo.

Structure‐based JAZ ligand‐J1 and its analogues show strong cytotoxic effects on glioma cells in vitro ‐ A new class of glioma therapy drugs?

Hypoxia/ischemia and oxidative stress has been shown to be involved in glioma tumorigenesis. The Jaz gene encodes a zinc finger protein that can respond to oxidative stress with increased expression both in vitro and in vivo. We have previously reported that JAZ exhibits a tumor grade‐dependent increased expression in gliomas with the highest levels in GBM and shows increased cytoplasm‐nuclear translocation adjacent to pseudopalisading necrosis. We hypothesized that JAZ plays a role in glioma tumorigenesis. Targeting JAZ with a molecular docking approach, we identified one small molecule compound named “J1” that exhibited strong dose‐ and time‐dependent glioma cytotoxic effects in vitro (human U87MG, U118MG, A172, and mouse GL261 glioma cell lines) with GI50 around 25microM. Several known J1 analogues also displayed differential glioma killing effects. Flow cytometry showed that J1 led to cell cycle arrest. Quantitative proteomic analysis revealed that, in both U87MG and U118MG glioma cells, J1 treatment led to a significant decrease of PLOD2 (procollagen hydroxylase 2), which is known to be induced by hypoxia, associated with cancer progression, and was reported to be increased in pseudopalisading GBM cells. These results suggest that JAZ ligand J1 and its analogues have the potential to become a new class of chemotherapy drugs for glioma treatment.

Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR‐32 neuroblastoma cells

The antioxidant-activated transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the induction of cytoprotective genes against chemical toxicity and oxidative injuries. The role of phosphorylation in Nrf2 activation has been suggested but remains elusive. We report that phenolic antioxidant/pro-oxidant tert-butylhydroquinone (tBHQ) induced two forms of the Nrf2 protein in neuroblastoma cells (IMR-32), which migrated as distinctive bands on SDS-PAGE. In vitro treatment with lambda phosphatase eliminated the slower migrating form and increased the amount of the faster migrating form of Nrf2. In vivo (32)Pi-phosphorylation resulted in (32)Pi-labeling of the Nrf2 protein in the presence of tBHQ that can be dephosphorylated by lambda phosphotase, indicating that the slower migrating form is a phosphorylated Nrf2 protein and the faster form an unphosphorylated Nrf2. Unphosphorylated Nrf2 predominated in the cytoplasm, whereas the phosphorylated form preferentially localized in the nucleus. Nuclear Nrf2 can be dephosphorylated by lambda phosphotase in vitro and be converted to the faster migrating form, implicating phosphorylation of Nrf2 in the cytoplasmic-nuclear translocation of the protein. Deletional analyses from both the carboxyl- and amino-ends revealed the transcription activation (TA) domains Neh4 (Nrf2-ECH homology 4) and Neh5 (Nrf2-ECH homology 5) as a major region necessary for the phosphorylation. The TA domains are characterized by the presence of multiple phosphorylation sites of casein kinase 2 (CK2). Moreover, CK2 phosphorylated the TA domains in vitro. Treatment with CK2 inhibitor 2-dimethylamino-4,5,6,7,-tetrabromo-1H-benzimidazole (DMAT) blocked the induction of endogenous target genes of Nrf2 in cells and inhibited the TA activities of both the full length and the TA domains of Nrf2 to a large extent. Finally, phosphorylation of the TA domains correlated with the nuclear translocation of Nrf2 that was inhibited by DMAT in a concentration-dependent manner. The findings demonstrated that phosphorylation of Nrf2 at the TA domains by CK2 is an integral component of Nrf2 activation necessary for the nuclear localization and transcription activation function of Nrf2 in neuroblastoma cells.

Antithymocyte Globulins Suppress Dendritic Cell Function by Multiple Mechanisms

The polyclonal rabbit antithymocyte and anti-T-cell immunoglobulins (ATGs) Thymoglobulin (TG) and ATG-Fresenius S (ATG-F) have been widely used for the prevention and therapy of allograft rejection and graft versus host disease in transplantation. Although immunosuppressive mechanisms of ATGs on T cells are well studied, less is known about their impact on dendritic cells (DCs). Effects of TG and ATG-F on immune functions and signaling pathways of human monocyte-derived DCs were determined by flow cytometry, enzyme-linked immunosorbent assay, Western blot, apoptosis assays, endocytosis assays, and T cell stimulation assays. TG and ATG-F bind rapidly and with high affinity to CD11c, CD80, CD86, CD40, CD36, CD38, CD206, and human leukocyte antigen-DR on DCs. TG and, to a lesser extent, ATG-F induce apoptosis in immature and mature DCs. Macropinocytotic and receptor-mediated endocytotic antigen uptake in immature DCs is inhibited by TG and ATG-F due to their binding of the C-type lectins CD206 and CD209. TG and ATG-F induce activation of the mitogen-activated protein kinases ERK1/2 and p38 that contributes to the induction of apoptosis. TG and ATG-F also induce cytoplasmic-nuclear translocation of the NF-kappaB/Rel transcription factors RelB, RelA, p50, and p52. Production of interleukin-12p70 in mature DCs is suppressed by TG and ATG-F. TG and ATG-F reduce the capacity of mature DCs to stimulate allogeneic and autologous T cells. ATGs interfere with basic DC functions, suggesting that DCs are relevant targets for the immunosuppressive action of ATGs in transplantation.

Tumor Necrosis Factor-α Activates the Human Prolactin Gene Promoter via Nuclear Factor-κB Signaling

Pituitary function has been shown to be regulated by an increasing number of intrapituitary factors, including cytokines. Here we show that the important cytokine TNF-alpha activates prolactin gene transcription in pituitary GH3 cells stably expressing luciferase under control of 5 kb of the human prolactin promoter. Similar regulation of the endogenous rat prolactin gene by TNF-alpha in GH3 cells was confirmed using real-time PCR. Luminescence microscopy revealed heterogeneous dynamic response patterns of promoter activity in individual cells. In GH3 cells treated with TNF-alpha, Western blot analysis showed rapid inhibitory protein kappaB (IkappaBalpha) degradation and phosphorylation of p65. Confocal microscopy of cells expressing fluorescence-labeled p65 and IkappaBalpha fusion proteins showed transient cytoplasmic-nuclear translocation and subsequent oscillations in p65 localization and confirmed IkappaBalpha degradation. This was associated with increased nuclear factor kappaB (NF-kappaB)-mediated transcription from an NF-kappaB-responsive luciferase reporter construct. Disruption of NF-kappaB signaling by expression of dominant-negative variants of IkappaB kinases or truncated IkappaBalpha abolished TNF-alpha activation of the prolactin promoter, suggesting that this effect was mediated by NF-kappaB. TNF-alpha signaling was found to interact with other endocrine signals to regulate prolactin gene expression and is likely to be a major paracrine modulator of lactotroph function.

Statistics of Assay Validation in High Throughput Cell Imaging of Nuclear Factor κB Nuclear Translocation

This report describes statistical validation methods implemented on assay data for inhibition of subcellular redistribution of nuclear factor kappaB (NF kappaB) in HeLa cells. We quantified cellular inhibition of cytoplasmic-nuclear translocation of NF kappaB in response to a range of concentrations of interleukin-1 (IL-1) receptor antagonist in the presence of IL-1alpha using eight replicate rows in each four 96-well plates scanned five times on each of 2 days. Translocation was measured as the fractional localized intensity of the nucleus (FLIN), an implementation of our more general fractional localized intensity of the compartments (FLIC), which analyzes whole compartments in the context of the entire cell. The NF kappaB antagonist assay (inhibition of IL-1- induced NF kappaB translocation) data were collected on a Q3DM (San Diego, CA) EIDAQtrade mark 100 high throughput microscopy system. [In 2003, Q3DM was purchased by Beckman Coulter Inc. (Fullerton, CA), which released the IC 100 successor to the EIDAQ 100.] The generalized FLIC method is described along with two-point (minimum-maximum) and multiple point titration statistical methods. As a ratio of compartment intensities that tend to change proportionally, FLIN was resistant to photobleaching errors. Two-point minimum-maximum statistical analyses yielded the following: a Z' of 0.174 with the data as n = 320 independent well samples; Z' by row data in a range of 0.393-0.933, with a mean of 0.766; by-plate Z' data of 0.310, 0.443, 0.545, and 0.794; and by-plate means of columns Z' data of 0.879, 0.927, 0.945, and 0.963. The mean 50% inhibitory concentration (IC50) for IL-1 receptor antagonist over all experiments was 213 ng/ml. The combined IC50 coefficients of variation (CVs) were 0.74%, 0.85%, 2.09%, and 2.52% for the four plates. Repeatability IC50 CVs were as follows: day to day 3.0%, row to row 8.0%, plate to plate 2.8%, and day to day 0.6%. The number of cells required for statistically resolvable differences in dose concentrations, plotted in a family of FLIN sigma/deltamicro (SD/range) curves and tabulated, demonstrated cell-by-cell assay precision with our combined sigma/deltamicro = 0.32 that required approximately 10-fold fewer cells than in a previously reported NF kappaB assay with sigma/deltamicro = 1.52. To better understand the relationship between cell-by-cell measurements and IC50 precision, 500 Monte Carlo simulations with varying cell-measurement SDs were used to explore three-, five-, seven-, and 11-point model titrations. The reductions in deltaIC50 90% confidence intervals from 11- to three-point titrations were 10-fold with the previously reported sigma/deltamicro = 1.52 and twofold with our sigma/deltamicro = 0.32. With these normalized parameters, this report provides a common statistical foundation, independent of the assay details, for evaluating the performance of imaging data on any instrument.

The role of CREB and other transcription factors in the pharmacotherapy and etiology of depression

Psychiatric diseases are genetically complex and consequently, altered programs of gene expression have been hypothesized as the molecular basis of psychopathology. Since transcription factors represent the final communicative link between receptor activation and the orchestration of programs of gene expression, they are prime targets for studies on both the pharmacotherapy and the etiology of depression. The cyclic AMP response element binding protein (CREB) and the glucocorticoid receptor (GR) are altered by chronic treatment with antidepressants. Since it is phosphorylated CREB (pCREB) that determines its transcriptional activity, it is pertinent that some antidepressants have been shown to reduce pCREB in brain in vivo and in tissue culture in vitro. Moreover, pCREB is downregulated in human fibroblasts from patients with major depression and in postmortem brain of suicide victims with a history of depression. With regard to GR, its mRNA, immunoreactivity, density and cytoplasmic-nuclear translocation are increased by antidepressants. While transcription factor mediated programs of gene expression relevant to either the pharmacotherapy or the etiology of depression are still largely elusive, studies utilizing modern technologies such as differential display and cDNA microarrays promise to lead eventually to the identification of structure and function of psychophathologically relevant target genes.

Cytoplasmic localization and the choice of ligand determine aggregate formation by androgen receptor with amplified polyglutamine stretch.

Polyglutamine tract expansion in androgen receptor is a recognized cause of spinal and bulbar muscular atrophy (SBMA), an X-linked motor neuronopathy. Similar mutations have been identified in proteins associated with other neurodegenerative diseases. Recent studies have shown that amplified polyglutamine repeat stretches form cellular aggregates that may be markers for these neurodegenerative diseases. Here we describe conditions that lead to aggregate formation by androgen receptor with polyglutamine stretch amplification. In transfection experiments, the mutant, compared with the wild-type receptor, was delayed in its cytoplasmic-nuclear translocation and formed large cytoplasmic aggregates in the presence of androgen. The cytoplasmic environment appears crucial for this aggregation, since retention of both the wild-type and mutant receptors in this cellular compartment by the deletion of their nuclear localization signals resulted in massive aggregation. Conversely, rapid nuclear transport of both receptors brought about by deletion of their ligand binding domains did not result in aggregate formation. However, androgen antagonists that altered the conformation of the ligand binding domain and promoted varying rates of cytoplasmic-nuclear translocation all inhibited aggregate formation. This demonstrates that in addition to the cytoplasmic localization, a distinct contribution of the ligand binding domain of the receptor is necessary for the aggregation. The finding that antiandrogens inhibit aggregate formation may provide the basis for in vivo determination of the role of these structures in SBMA.