P19 Expression Research Articles

Polypyrimidine Tract-Binding Protein Induces p19Ink4d Expression and Inhibits the Proliferation of H1299 Cells

The expression of polypyrimidine tract-binding protein (PTB) is up-regulated in many types of cancer. Here, we studied the role of PTB in the growth of non small cell lung cancer cells. Data showed that PTB overexpression inhibited the growth of H1299 cells at least by inhibiting DNA synthesis. Quantitative real-time PCR and Western blot analyses showed that PTB overexpression in H1299 cells specifically induced the expression of p19Ink4d, an inhibitor of cyclin-dependent kinase 4. Repression of p19Ink4d expression partially rescued PTB-caused proliferation inhibition. PTB overexpression also inhibited the growth and induced the expression of p19Ink4d mRNA in A549 cells. However, Western blot analyses failed to detect the presence of p19Ink4d protein in A549 cells. To address how PTB induced p19Ink4d in H1299 cells, we showed that PTB might up-regulate the activity of p19Ink4d gene (CDKN2D) promoter. Besides, PTB lacking the RNA recognition motif 3 (RRM3) was less effective in growth inhibition and p19Ink4d induction, suggesting that RNA-binding activity of PTB plays an important role in p19Ink4d induction. However, immunoprecipitation of ribonuclearprotein complexes plus quantitative real-time PCR analyses showed that PTB might not bind p19Ink4d mRNA, suggesting that PTB overexpression might trigger the other RNA-binding protein(s) to bind p19Ink4d mRNA. Subsequently, RNA electrophoretic mobility-shift assays revealed a 300-base segment (designated as B2) within the 3′UTR of p19Ink4d mRNA, with which the cytoplasmic lysates of PTB-overexpressing cells formed more prominent complexes than did control cell lysates. Insertion of B2 into a reporter construct increased the expression of the chimeric luciferase transcripts in transfected PTB-overexpressing cells but not in control cells; conversely, overexpression of B2-containing reporter construct in PTB-overexpressing cells abolished the induction of p19Ink4d mRNA. In sum, we have shown that PTB plays as a negative regulator in H1299 cell proliferation at least by inducing p19Ink4d expression at transcriptional and post-transcriptional levels.

Efficient and specific gene knockdown by small interfering RNAs produced in bacteria

Synthetic small interfering RNAs (siRNAs) are an indispensable tool to investigate gene function in eukaryotic cells1,2 and may be used for therapeutic purposes to knockdown genes implicated in disease3. Thus far, most synthetic siRNAs have been produced by chemical synthesis. Here we present a method to produce highly potent siRNAs in E. coli. This method relies on ectopic expression of p19, a siRNA-binding protein found in a plant RNA virus4, 5. When expressed in E. coli, p19 stabilizes ~21 nt siRNA-like species produced by bacterial RNase III. Transfection of mammalian cells with siRNAs, generated in bacteria expressing p19 and a hairpin RNA encoding 200 or more nucleotides of a target gene, at low nanomolar concentrations reproducibly knocks down gene expression by ~90% without immunogenicity or off-target effects. Because bacterially produced siRNAs contain multiple sequences against a target gene, they may be especially useful for suppressing polymorphic cellular or viral genes.

RNAi suppressor P19 can be broadly exploited for enhanced adenovirus replication and microRNA knockdown experiments

RNA interference (RNAi) is a key regulator of various biological systems including viral infection. Within a virus life cycle gene products can be modulated by the RNA interference (RNAi) pathway which can crucially impact productive virus replication. Herein we explored the RNA interference suppressor protein P19 derived from a plant virus and we found that P19 enhanced adenovirus replication up to 100-fold. Critical factors responsible for this observation were overexpression of adenovirus encoded genes on mRNA and protein levels. To investigate the impact of this phenomenon on recombinant viruses, we exploited its feasibility for therapeutic and genomic applications. We found that P19 significantly increased recombinant adenovirus yields enabling up-scaling for preclinical and clinical studies. Moreover, adenoviruses possessed significantly higher oncolytic activity by expression of P19. Finally, we show that introducing a p19 expression cassette into high-capacity adenovirus provides a strategy to analyze RNAi knockdown in a tissue-specific manner.

Expression of p110, phosphorylation-protein kinase B and protein kinase B in hemangioma vascular endothelial cell in vitro

Objective To study the expression of phosphatidy linositol-3-kinase(PI3K) p110 catalytic subunit and phosphorylation-protein kinase B(p-Akt), protein kinase B(Akt) in cultured vascular endothelial cells, then the level of PI3K p110, p-Akt and Akt expression and the correlation with hemangioma vascular endothelial cell cycle and apoptosis were analyzed. Methods The hemangioma vascular endothelial cells which came from the sterile hemangioma were cultured by using tissue culture method and then synchronized cultured endothelial cells, PI3K p110, p-Akt and Akt of them were detected by way of Western blot, meanwhile cell cycle and cell apoptosis were examined by using flow cytometry technique, then the level of PI3K p110, p-Akt and Akt expression and the correlation with hemangioma vascular endothelial cell cycle and apoptosis were analyzed. Results The percentage of vascular endothelial cell was (94.00±3.00)% in the G0/G1 phase and the apoptosis rate was (11.24±2.34)% when p110 and p-Akt protein expression level were lower (0.19±0.01, 0.09±0.02), and they were (61.00±6.00)% and (0.51±0.03)% when p110 and p-Akt protein expression level were higher (0.37±0.04, 0.22±0.01). The percentage of vascular endothelial cell in the G0/G1 phase and the apoptosis rate were different in 2 groups(t=-8.42, -35.48, all P<0.01). But Akt protein expression level was not changed(0.72±0.00). Conclusions p110, p-Akt and Akt protein were expressed in the cultured hemangioma vascular endothelial cells in vitro.PI3K p110, p-Akt may be involved in cultured hemangioma vascular endothelial cells apoptosis in vitro. Key words: Hemangioma; Vascular endothelial cell; Cell culture; p110; Phosphorylation-protein kinase B; Protein kinase B; Cell apoptosis

An in vitro mouse model of congenital cytomegalovirus‐induced pathogenesis of the inner ear cochlea

Congenital human cytomegalovirus (CMV) infection is the leading nongenetic etiology of sensorineural hearing loss (SNHL) at birth and prelingual SNHL not expressed at birth. The paucity of temporal bone autopsy specimens from infants with congenital CMV infection has hindered the critical correlation of histopathology with pathogenesis. Here, we present an in vitro embryonic mouse model of CMV-infected cochleas that mimics the human sites of viral infection and associated pathology. There is a striking dysplasia/hyperplasia in mouse CMV-infected cochlear epithelium and mesenchyme, including organ of Corti hair and supporting cells and stria vascularis. This is concomitant with significant dysregulation of p19, p21, p27, and Pcna gene expression, as well as proliferating cell nuclear antigen (PCNA) protein expression. Other pathologies similar to those arising from known deafness gene mutations include downregulation of KCNQ1 protein expression in the stria vascularis, as well as hypoplastic and dysmorphic melanocytes. Thus, this model provides a relevant and reliable platform within which the detailed cell and molecular biology of CMV-induced deafness may be studied.

Expression and clinical implication of tumor necrosis factor-alpha in the intractable epilepsy with focal cortical dysplasias

Objective To investigate the expression and clinical implication of tumor necrosis factor-alpha (TNF-α),and its receptors p55 and p75 in the intractable epilepsy with focal cortical dysplasias (FCD).Methods The expression of TNF-α,p55 and p75 was detected by suing strept avidin-biotin complex (SABC) staining in epileptic foci of refractory epilepsy group (including 29 cases of FCD) and 10 cases of normal brain tissue ( control group).Results The expression of TNF-α,p55 and p75 in the control group was negative.The expression intensity of TNF-α,p55 and p75 in the FCD type Ⅲ was stronger than the FCD type Ⅱ,but there was no significant difference between type Ⅲ and type Ⅰ,and between type Ⅱand type Ⅰ.A small fraction of glial cells expressed TNF-α,p55 and p75 in the FCD type Ⅰ and type Ⅱ,but the majority of glial cells expressed them in the type Ⅲ.The expression of TNF-α,p55 and of p75 was detectable in both ectopic neurons and activated astrocytes of the medulla,and their expression in dysmorphic neurons (DNs) and balloon cells (BCs) in the FCD type Ⅱ was weaker than in other types of neurons.The expression intensity of TNF-α,p55 and p75 in BCs was significantly weaker that in DNs.Conclusion TNF-α through binding its receptors,p55 and p75,may be involved in the generation and development of epilepsy in the FCD. Key words: Focal cortical dysplasias; Epilepsy; Tumor necrosis factor-α; p55 receptor; p75 receptor

Polycomb (PcG) Proteins, BMI1 and SUZ12, Regulate Arsenic-induced Cell Transformation

Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. However, the underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. The present study explored a potential mechanism of cell transformation induced by arsenic exposure. Exposure to a low dose (0.5 μm) of arsenic trioxide (As(2)O(3)) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenic-induced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. On the other hand, tumor suppressor p16(INK4a) and p19(ARF) mRNA and protein expression were dramatically suppressed. Introduction of small hairpin (sh) RNA-BMI1 or -SUZ12 into BALB/c 3T3 cells resulted in suppression of arsenic-induced transformation. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16(INK4a) and p19(ARF) was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure.

In vitro studies reveal that different modes of initiation on HIV‐1 mRNA have different levels of requirement for eukaryotic initiation factor 4F

Expression of the two isoforms p55 and p40 of HIV-1 Gag proteins relies on distinct translation initiation mechanisms, a cap-dependent initiation and two internal ribosome entry sites (IRESs). The regulation of these processes is complex and remains poorly understood. This study was focused on the influence of the 5'-UTR and on the requirement for the eukaryotic initiation factor (eIF)4F complex components. By using an in vitro system, we showed substantial involvement of the 5'-UTR in the control of p55 expression. This highly structured 5'-UTR requires the eIF4F complex, especially RNA helicase eIF4A, which mediates initiation at the authentic AUG codon. In addition, the 5'-UTR regulates expression in an RNA concentration-dependent manner, with a high concentration of RNA triggering specific reduction of full-length Gag p55 production. HIV-1 genomic RNA also has the ability to use a strong IRES element located in the gag coding region. We show that this mechanism is particularly efficient, and that activity of this IRES is only poorly dependent on RNA helicase eIF4A when the viral 5'-UTR is removed. HIV-1 genomic mRNA exhibits in vitro translational features that allow the expression of Gag p55 protein by different mechanisms that involve different requirements for eIF4E, eIF4G, and eIF4A. This suggests that HIV-1 could adapt to its mode of translation according to the availability of the initiation factors in the infected cell.

Black tea polyphenols induce human leukemic cell cycle arrest by inhibiting Akt signaling

Tea polyphenols have potent biological activities against human cancer cells. A major causative factor in malignancies is disregulation of cell-cycle kinetics. In this study, we observed that black tea polyphenols, theaflavins (TF) and thearubigins (TR) induced cell-cycle arrest at the G(0) /G(1) phase in human leukemic U937 and K562 cells. Our objective was to understand the underlying molecular mechanism of cell-cycle inhibition by TF and TR. During elucidation, we observed that both TF and TR treatment augmented expression of p19, p21 and p27, while ablating cylcin-dependent kinase (CDK)2, CDK4, CDK6 and cyclin D1 levels. Our experimental results further determined that Akt signaling suppression by TF and TR played a major role in this process. Moreover, suppression of glycogen synthase kinase-3β, β-catenin and amplification of forkhead transcription factor 1 (FOXO1) expression were associated with regulation of certain key components of the cell-cycle machinery. In addition, depletion of heat shock protein (Hsp) 90 by TF and TR also had a pivotal role in cell-cycle arrest. More specifically, inhibition of Akt signaling by TF and TR correlated with the depletion of its downstream targets like Wnt/β-catenin signaling, cyclin D1 and increase of FOXO1, p27 levels. Inhibition of upstream Hsp90 by TF and TR consequently attenuated Akt signaling and reduced the level of CDK2. These results suggest possible mechanisms for the chemopreventive effect of TF and TR on human leukemic cells. To our knowledge, this is the first report of such a detailed molecular mechanism for TF and the less-investigated polyphenol TR-mediated cell-cycle inhibition in human leukemic U937 and K562 cells.

Up-regulation of IL-23 p19 expression in human periodontal ligament fibroblasts by IL-1β via concurrent activation of the NF-κB and MAPKs/AP-1 pathways

Interleukin (IL)-23 is an essential cytokine involved in the expansion of a novel CD4+ T helper subset known as Th17, which has been implicated in the pathogenesis of periodontitis recently. Our previous study first identified specialized human periodontal ligament fibroblasts (hPDLFs) as an important production source of IL-23. The present study was undertaken to investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-1β on hPDLFs-mediated IL-23 p19 production, and the molecular mechanism involved. IL-23 p19 expression was in situ detected in IL-1β-stimulated hPDLFs. IL-1β was capable of stimulating the expression of IL-23 p19 mRNA and protein in cultured hPDLFs, which was attenuated by IL-1 receptor antagonist (IL-1Ra) or myeloid differentiation primary response gene 88 (MyD88) inhibitor. Meanwhile, inhibitors of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), activator protein-1 (AP-1), or nuclear factor-kappaB (NF-κB) significantly suppressed IL-23 p19 production from IL-1β-stimulated hPDLFs. Moreover, IL-1β-initiated AP-1 activation was blocked by p38 MAPK, ERK 1/2, or JNK inhibition, whereas NF-κB activity remained unaltered by all the above pathway specific inhibitors. Thus, these results provide evidence that Th17-polarizing mediator IL-1β up-regulated the expression of IL-23 p19 in hPDLFs via NF-κB signaling and MAPKs-dependent AP-1 pathways. Taken together, our findings indicate that IL-1Ra may be used therapeutically to inhibit Th17-driven inflammatory diseases including periodontitis.

Regulation of CD38 Expression in Human Airway Smooth Muscle Cells

The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca(2+)-mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I-selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α-induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α-induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, β, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110β, resulted in comparable attenuation of TNF-α-induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.

Increased Th17 cells in the tumor microenvironment is mediated by tumor-secreted PGE2 (162.42)

Abstract Tumor microenvironment can convert the host immune responses to favor tumor growth. However, the underlying mechanisms of how tumor cells educate host immunity are still largely unknown. We found that Th17 cells were increased in the peripheral blood, spleen and tumor tissues of mammary gland tumor-bearing mice. Th17 cell survival factor, IL-23 was overexpressed in tumor tissues isolated from both mouse and human breast cancer patients. Soluble molecules secreted from tumor cells but not normal breast epithelial cells induced IL-23 protein secretion in DCs via induction of the p19 mRNA expression. Our data further indicates that tumor-secreted PGE2 via its receptor EP2 and EP4 enhanced IL-23 p19 gene transcription through binding to the cAMP-response element in the p19 promoter. Blocking PGE2 synthesis by NS398, a Cox2 inhibitor, abrogated the enhancement of p19 expression both in vitro and in vivo. Furthermore, blocking PKA by H89 completely abrogated the inductive effects of tumor conditioned medium and PGE2 on p19 transcription. In line with this finding, cAMP active analog, Forskolin mimics the PGE2 effect. Taken together, our results indicate that tumor-secreted PGE2 differentially induce IL-23 but not IL-12 production in the tumor microenvironment which could promote Th17 cell expansion. This inductive effect is mediated through cAMP/PKA signaling transduction pathway by increasing p19 transcription.

P2-01-25: Truncated p110 ERBB2 (CTF611) Increases Migration and Invasion of Breast Epithelial Cells by Inhibiting STAT5b Activation.

Abstract Background: Truncated ERBB2 receptors are present in a subset of human ERBB2+ amplified/overexpressing breast tumors, and are associated with trastuzumab resistance, metastasis, and poor clinical prognosis. However, whether truncated ERBB2 receptors are drivers of metastasis has not been well defined. In this study, we examined effects of full-length (p185) and truncated (p110) ERBB2 on the migration and invasion of human mammary epithelial cells, including HMLE and MCF10A cells. Material and Methods: Recombinant p185 and p110 ERBB2 were stably expressed in human mammary epithelial cells (HMLE) and MCF10A cells via retroviral vector. Expression of comparable levels of p185 and p110 in cells was confirmed by western blot. The phosphorylation states of downstream signaling proteins including STAT5 were assayed via phosphoproteomics and Collaborative Enzyme Enhanced Reactive (CEER™) immunoassay. The effects of the p110 constructs on cell migration and invasion were investigated by transwell assays. shRNA-encoding lentivirus was used for specific silencing of STAT5b in HMLE cells, and STAT5b silencing was confirmed at the protein level using western blot. Results and Discussion: Expression of p110 ERBB2 increased cell migration (HMLE, p = 0.04; MCF10A, p&amp;lt; 0.01) and invasion (HMLE, p= 0.03) when compared to expression of p185. Furthermore, expression of p110 in HMLE cells was associated with reduced phosphorylation of STAT5b. shRNA mediated silencing of STAT5b was sufficient to increase the migration (p &amp;lt; 0.01) and invasion of HMLE cells, phenocopying the p110 driven effects on HMLE cells. In clinical studies, loss of activated STAT5 protein correlates with breast cancer progression and is a negative predictor of survival. By analyzing publicly available gene expression datasets, we found that STAT5b mRNA expression is also significantly decreased in breast cancer compared to normal breast tissues in several studies, as well as in ERBB2 amplified vs. nonamplified samples. To our knowledge, this is the first reported perturbation of STAT signaling by truncated ERBB2 receptor, and suggests a mechanism by which truncated p110 ERBB2 (CTF611) increases migration and invasion of breast epithelial cells. This study extends the available data regarding STAT5 loss in breast cancer progression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-25.

The Gap-junction inhibitor Carbenoxolone suppresses the differentiation of Th17 cells through inhibition of IL-23 expression in antigen presenting cells

Carbenoxolone (CBX) is a widely used gap-junction inhibitor. We have previously shown that treatment with CBX significantly delayed the onset of experimental autoimmune encephalomyelitis (EAE). However, the mechanism by which CBX delays the onset of EAE remains to be elucidated. Here, we show that CBX specifically inhibits the production of IL-23 by dendritic cells (DCs) and microglia in vitro. CBX treatment significantly reduced the population of Th17 cells in EAE mice. Furthermore, CBX downregulated the expression of IL-23 p19 via increased production of protein phosphatase 2A (PP2A). Thus, CBX may be an effective therapeutic strategy against Th17-mediated autoimmune diseases, such as multiple sclerosis.

Inhibition of beta1-adrenergic receptor-CaMKII activation by Insulin treatment improves prolonged post-ischemic cardiac remodeling and function

ObjectivesChronic β1-adrenergic receptor (β1-AR)-calcium/calmodulin-dependent protein kinase II (CaMKII) activation leads to Ca2+ handling disturbances and adverse cardiac remodeling. We have previously demonstrated that insulin treatment exerts anti-apoptotic and prosurvival effects...

IGF binding protein 2 supports the survival and cycling of hematopoietic stem cells

AbstractThe role of IGF binding protein 2 (IGFBP2) in cell growth is intriguing and largely undefined. Previously we identified IGFBP2 as an extrinsic factor that supports ex vivo expansion of hematopoietic stem cells (HSCs). Here we showed that IGFBP2-null mice have fewer HSCs than wild-type mice. While IGFBP2 has little cell-autonomous effect on HSC function, we found decreased in vivo repopulation of HSCs in primary and secondary transplanted IGFBP2-null recipients. Importantly, bone marrow stromal cells that are deficient for IGFBP2 have significantly decreased ability to support the expansion of repopulating HSCs. To investigate the mechanism by which IGFBP2 supports HSC activity, we demonstrated that HSCs in IGFBP2-null mice had decreased survival and cycling, down-regulated expression of antiapoptotic factor Bcl-2, and up-regulated expression of cell cycle inhibitors p21, p16, p19, p57, and PTEN. Moreover, we found that the C-terminus, but not the RGD domain, of extrinsic IGFBP2 was essential for support of HSC activity. Defective signaling of the IGF type I receptor did not rescue the decreased repopulation of HSCs in IGFBP2-null recipients, suggesting that the environmental effect of IGFBP2 on HSCs is independent of IGF-IR mediated signaling. Therefore, as an environmental factor, IGFBP2 supports the survival and cycling of HSCs.

Expression of Arf Tumor Suppressor in Spermatogonia Facilitates Meiotic Progression in Male Germ Cells

The mammalian Cdkn2a (Ink4a-Arf) locus encodes two tumor suppressor proteins (p16Ink4a and p19Arf) that respectively enforce the anti-proliferative functions of the retinoblastoma protein (Rb) and the p53 transcription factor in response to oncogenic stress. Although p19Arf is not normally detected in tissues of young adult mice, a notable exception occurs in the male germ line, where Arf is expressed in spermatogonia, but not in meiotic spermatocytes arising from them. Unlike other contexts in which the induction of Arf potently inhibits cell proliferation, expression of p19Arf in spermatogonia does not interfere with mitotic cell division. Instead, inactivation of Arf triggers germ cell–autonomous, p53-dependent apoptosis of primary spermatocytes in late meiotic prophase, resulting in reduced sperm production. Arf deficiency also causes premature, elevated, and persistent accumulation of the phosphorylated histone variant H2AX, reduces numbers of chromosome-associated complexes of Rad51 and Dmc1 recombinases during meiotic prophase, and yields incompletely synapsed autosomes during pachynema. Inactivation of Ink4a increases the fraction of spermatogonia in S-phase and restores sperm numbers in Ink4a-Arf doubly deficient mice but does not abrogate γ-H2AX accumulation in spermatocytes or p53-dependent apoptosis resulting from Arf inactivation. Thus, as opposed to its canonical role as a tumor suppressor in inducing p53-dependent senescence or apoptosis, Arf expression in spermatogonia instead initiates a salutary feed-forward program that prevents p53-dependent apoptosis, contributing to the survival of meiotic male germ cells.

The Thyroid Hormone Receptors as Modulators of Skin Proliferation and Inflammation

We have analyzed the role of the thyroid hormone receptors (TRs) in epidermal homeostasis. Reduced keratinocyte proliferation is found in interfollicular epidermis of mice lacking the thyroid hormone binding isoforms TRα1 and TRβ (KO mice). Similar results were obtained in hypothyroid animals, showing the important role of the liganded TRs in epidermal proliferation. In addition, KO and hypothyroid animals display decreased hyperplasia in response to 12-O-tetradecanolyphorbol-13-acetate. Both receptor isoforms play overlapping functional roles in the skin because mice lacking individually TRα1 or TRβ also present a proliferative defect but not as marked as that found in double KO mice. Defective proliferation in KO mice is associated with reduction of cyclin D1 expression and up-regulation of the cyclin-dependent kinase inhibitors p19 and p27. Paradoxically, ERK and AKT activity and expression of downstream targets, such as AP-1 components, are increased in KO mice. Increased p65/NF-κB and STAT3 phosphorylation and, as a consequence, augmented expression of chemokines and proinflammatory cytokines is also found in these animals. These results show that thyroid hormones and their receptors are important mediators of skin proliferation and demonstrate that TRs act as endogenous inhibitors of skin inflammation, most likely due to interference with AP-1, NF-κB, and STAT3 activation.

Human T-lymphotropic virus type 1 p30 interacts with REGγ and ATM (Ataxia Telangiectasia Mutated) to promote cell survival

Human T cell leukemia virus type 1 (HTLV-1), is complex deltaretrovirus linked to adult T-cell leukemia/lymphoma (ATL) and a variety of immune-mediated disorders. HTLV-1 encodes a nuclear localizing protein, p30, which selectively alters viral and cellular gene expression, activates G2-M cell cycle checkpoints, and is essential for viral spread. p30 interacts with key cellular proteins such as CBP/p300 and Myc/TIP60 to differentially modulate host and viral gene expression. We hypothesize that interaction of p30 with host cellular proteins modulates the cellular microenvironment to favor of viral spread. Herein we used immunoprecipitation, affinity pull-down of ectopically expressed p30 coupled with mass spectrometry to identify cellular binding partners of p30. Our data indicate that p30 specifically binds to cellular ataxia-telangiectasia mutated (ATM) and REGγ (a nuclear 20S proteasome activator). In conditions of genotoxic stress p30 expression was associated with reduced levels of ATM and increased cell survival. Knockdown or over expression of REGγ paralleled p30 expression suggesting an unexpected enhancement of p30 expression in the presence of REGγ. Finally, size exclusion chromatography revealed the presence of p30 in a high molecular weight complex along with ATM and REGγ. Current studies are focused on mapping regions critical for p30- REGγ binding and how this interaction may influence HTLV-1 transcription. Based on our findings we propose that HTLV-1 p30 interacts with ATM and REGγ to increase viral spread by facilitating cell survival.

Reply to Steinman and Wang: Involvement of the p150 isoform of the RNA editing enzyme ADAR1 in embryonic lethality

In their letter, Steinman and Wang (1) raise the possibility that p110 expression may be affected by specific gene targeting of the p150 isoform of adenosine deaminase 1 (ADAR1), similar to the concerns raised in the letter by Matthaei et al. (2). Although Steinman and Wang acknowledge that the levels of the constitutive exon 1B-containing transcript encoding the p110 isoform of ADAR1 were similar in both p150+/− and p150−/− MEF cells derived from gene-targeted embryos compared with MEF cells derived from WT embryos as shown in our article (3), they suggest that protein expression may have been affected as a result of antisense ADAR1 transcription from the PGK promoter. However, they correctly indicate that there is no evidence to support this possibility.