Bayar Thimmapaya

Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis

The adenovirus E1A protein interferes with regulators of apoptosis and growth by physically interacting with cell cycle regulatory proteins including the retinoblastoma tumor suppressor protein and the coactivator proteins p300/CBP (where CBP is the CREB-binding protein). The p300/CBP proteins occupy a pivotal role in regulating mitogenic signaling and apoptosis. The mechanisms by which cell cycle control genes are directly regulated by p300 remain to be determined. The cyclin D1 gene, which is overexpressed in many different tumor types, encodes a regulatory subunit of a holoenzyme that phosphorylates and inactivates PRB. In the present study E1A12S inhibited the cyclin D1 promoter via the amino-terminal p300/CBP binding domain in human choriocarcinoma JEG-3 cells. p300 induced cyclin D1 protein abundance, and p300, but not CBP, induced the cyclin D1 promoter. cyclin D1 or p300 overexpression inhibited apoptosis in JEG-3 cells. The CH3 region of p300, which was required for induction of cyclin D1, was also required for the inhibition of apoptosis. p300 activated the cyclin D1 promoter through an activator protein-1 (AP-1) site at −954 and was identified within a DNA-bound complex with c-Jun at the AP-1 site. Apoptosis rates of embryonic fibroblasts derived from mice homozygously deleted of the cyclin D1 gene (cyclin D1 −/− ) were increased compared with wild type control on several distinct matrices. p300 inhibited apoptosis in cyclin D1+/+ fibroblasts but increased apoptosis in cyclin D1 −/− cells. The anti-apoptotic function of cyclin D1, demonstrated by sub-G1 analysis and annexin V staining, may contribute to its cellular transforming and cooperative oncogenic properties.

Regulation of IL-6 and IL-8 Expression in Rheumatoid Arthritis Synovial Fibroblasts: the Dominant Role for NF-κB But Not C/EBPβ or c-Jun

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) produce IL-6 and IL-8, which contribute to inflammation and joint damage. The promoters of both cytokines possess binding sites for NF-κB, C/EBPβ, and c-Jun, but the contribution of each to the regulation of IL-6 and IL-8 in RA FLS is unknown. We employed adenoviral-mediated gene delivery of a nondegradable IκBα, or dominant-negative versions of C/EBPβ or c-Jun, to determine the contribution of each transcription factor to IL-6 and IL-8 expression. Inhibition of NF-κB activation significantly reduced the spontaneous and IL-1β-induced secretion of IL-6 and IL-8 by RA FLS and the IL-1β-induced production of IL-6 and IL-8 by human dermal fibroblasts. Inhibition of C/EBPβ modestly reduced constitutive and IL-1β-induced IL-6 by RA FLS, but not by human dermal fibroblasts, and had no effect on IL-8. Inhibition of c-Jun/AP-1 had no effect on the production of either IL-6 or IL-8. Employing gel shift assays, NF-κB, C/EBPβ, and c-Jun were constitutively activated in RA FLS, but only NF-κB and c-Jun activity increased after IL-1β. The reduction of cytokines by IκBα was mediated through inhibition of NF-κB activation, which resulted in decreased IL-6 and IL-8 mRNA. NF-κB was essential for IL-6 expression, because fibroblasts in which both NF-κB p50/p65 genes were deleted failed to express IL-6 in response to IL-1. These findings document the importance of NF-κB for the regulation of the constitutive and IL-1β-stimulated expression of IL-6 and IL-8 by RA FLS and support the role of inhibition of NF-κB as a therapeutic goal in RA.

p300/cAMP-responsive Element-binding Protein Interactions with Ets-1 and Ets-2 in the Transcriptional Activation of the Human Stromelysin Promoter

In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complexin vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a.a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1–1097 and 1678–2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP.

Tumor Necrosis Factor Alpha Gene Regulation: Enhancement of C/EBPβ-Induced Activation by c-Jun

ABSTRACT Tumor necrosis factor alpha (TNFα) is a key regulatory cytokine whose expression is controlled by a complex set of stimuli in a variety of cell types. Previously, we found that the monocyte/macrophage-enriched nuclear transcription factor C/EBPβ played an important role in the regulation of the TNFα gene in myelomonocytic cells. Abundant evidence suggests that other transcription factors participate as well. Here we have analyzed interactions between C/EBPβ and c-Jun, a component of the ubiquitously expressed AP-1 complex. In phorbol myristate acetate (PMA)-treated Jurkat T cells, which did not possess endogenous C/EBPβ, expression of c-Jun by itself had relatively little effect on TNFα promoter activity. However, the combination of C/EBPβ and c-Jun was synergistic, resulting in greater than 130-fold activation. This effect required both the leucine zipper and DNA binding domains, but not the transactivation domain, of c-Jun, plus the AP-1 binding site centered 102/103 bp upstream of the transcription start site in the TNFα promoter. To determine if C/EBPβ and c-Jun might cooperate to regulate the cellular TNFα gene in myelomonocytic cells, U937 cells that possess endogenous C/EBPβ and were stably transfected with either wild-type c-Jun or the transactivation domain deletion mutant (TAM-67) were examined. U937 cells expressing ectopic wild-type c-Jun or TAM-67 secreted over threefold more TNFα than the control line in response to PMA plus lipopolysaccharide. Transient transfection of the U937 cells expressing TAM-67 suggested that TAM-67 binding to the −106/−99-bp AP-1 binding site cooperated with endogenous C/EBPβ in the activation of the −120 TNFα promoter-reporter. DNA binding assays using oligonucleotides derived from the TNFα promoter suggested that C/EBPβ and c-Jun interact in vitro and that the interaction may be DNA dependent. Our data demonstrate that the TNFα gene is regulated by the interaction of the ubiquitous AP-1 complex protein c-Jun and the monocyte/macrophage-enriched transcription factor C/EBPβ and that this interaction contributes to the expression of the cellular TNFα gene in myelomonocytic cells. This interaction was unique in that it did not require the c-Jun transactivation domain, providing new insight into the cell-type-specific regulation of the TNFα gene.

The c-myc Gene Is a Direct Target of Mammalian SWI/SNF–Related Complexes during Differentiation-Associated Cell Cycle Arrest

The activity of mammalian SWI/SNF-related chromatin remodeling complexes is crucial for differentiation, development, and tumor suppression. Cell cycle-regulating activities dependent on the complexes include induction of the p21(WAF1/CIP1) kinase inhibitor and repression of E2F-responsive promoters. These responses are linked through effects on pRb phosphorylation, but the direct role of the SWI/SNF-related complexes in their regulation is not fully understood. Results presented here reveal that the complexes are required for regulation of a distinct pathway of proliferation control involving repression of c-myc expression in differentiating cells. This involves direct promoter targeting of the c-myc gene by the complexes. Induction of p21(WAF1/CIP1) is specifically dependent on prior repression of c-myc, but repression of E2F-responsive genes is dissociable from the regulation of c-myc and p21(WAF1/CIP1).

Cyclin D1 represses p300 transactivation through a cyclin-dependent kinase-independent mechanism

Cyclin D1 encodes a regulatory subunit, which with its cyclin-dependent kinase (Cdk)-binding partner forms a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. In addition to its Cdk binding-dependent functions, cyclin D1 regulates cellular differentiation in part by modifying several transcription factors and nuclear receptors. The molecular mechanism through which cyclin D1 regulates the function of transcription factors involved in cellular differentiation remains to be clarified. The histone acetyltransferase protein p300 is a co-integrator required for regulation of multiple transcription factors. Here we show that cyclin D1 physically interacts with p300 and represses p300 transactivation. We demonstrated further that the interaction of the two proteins occurs at the peroxisome proliferator-activated receptor γ-responsive element of the lipoprotein lipase promoter in the context of the local chromatin structure. We have mapped the domains in p300 and cyclin D1 involved in this interaction. The bromo domain and cysteine- and histidine-rich domains of p300 were required for repression by cyclin D1. Cyclin D1 repression of p300 was independent of the Cdk- and retinoblastoma protein-binding domains of cyclin D1. Cyclin D1 inhibits histone acetyltransferase activity of p300 in vitro. Microarray analysis identified a signature of genes repressed by cyclin D1 and induced by p300 that promotes cellular differentiation and induces cell cycle arrest. Together, our results suggest that cyclin D1 plays an important role in cellular proliferation and differentiation through regulation of p300.

Stereotactic injection of adenoviral vectors that target gene expression to specific pituitary cell types: implications for gene therapy.

OBJECTIVE Gene therapy is a potentially useful strategy for the treatment of pituitary adenomas or hormone deficiency disorders. We investigated the feasibility of targeting gene expression to specific pituitary cell types in vivo, using a combination of stereotactic injection and adenoviral vectors that carry pituitary-specific promoters. METHODS Recombinant adenoviruses containing the human growth hormone promoter (AdGHGal) or the human glycoprotein hormone alpha-subunit promoter (AdalphaGal) were used to drive expression of the beta-galactosidase gene. The expression of beta-galactosidase activity in the pituitary was analyzed after the administration of recombinant adenoviruses via the peripheral vein or the carotid artery, or by stereotactic injection into the rat pituitary. Double-label histology was used to evaluate cell-type expression in the pituitary. RESULTS Intravascular injection of AdGHGal or AdalphaGal failed to deliver the marker gene to the pituitary. However, direct stereotactic injection of recombinant adenoviral vectors into the pituitary achieved a high level of transgene expression. In addition, immunohistochemical staining revealed selective expression of the AdGHGal or AdalphaGal transgenes in pituitary cells that normally produce the respective hormones. CONCLUSION These findings indicate that adenoviral vectors carrying pituitary gland-specific promoters may be useful for targeted gene therapy of pituitary diseases. However, because of low transduction after peripheral administration, stereotactic injection or local administration of viruses at the time of pituitary surgery is probably required for efficient gene expression.

Mcl-1 Is Essential for the Survival of Synovial Fibroblasts in Rheumatoid Arthritis

Mcl-1 is a Bcl-2-family, antiapoptotic molecule that is critical for the survival of T and B lymphocytes and macrophages; however, its role in nonhemopoietic cells remains to be fully elucidated. The current study focuses on the role of Mcl-1 in rheumatoid arthritis (RA). Mcl-1 was strongly expressed in the synovial lining and was increased in the sublining fibroblasts of patients with RA, compared with control synovial tissue. The expression of Mcl-1 in sublining fibroblasts correlated with the degree of inflammation and TNF-α, and IL-1β treatment of cultured synovial fibroblasts resulted in the increased expression of Mcl-1 at the mRNA and protein levels. Mcl-1 was critical for the survival of RA synovial fibroblasts, because the forced reduction of Mcl-1 using a Mcl-1 antisense-expressing adenoviral vector induced apoptotic cell death, which was mediated through Bax, Bak, and Bim. These observations document a critical role for Mcl-1 in protecting against apoptosis in RA and suggest that Mc1–1 is a potential therapeutic target in this disease.

Adenovirus-mediated tissue-targeted expression of the HSVtk gene for the treatment of breast cancer

In an effort to develop a genetic therapy for the treatment of breast cancer, we constructed adenoviral vectors containing either the beta-galactosidase (β-gal) reporter gene or the herpes simplex thymidine kinase (HSVtk) suicide gene driven by breast tissue-specific promoters. We utilized upstream regulatory sequences from either the human alpha-lactalbumin (hALA) gene, or the ovine beta-lactoglobulin (oBLG) gene in these vector constructs to target expression of heterologous genes transcriptionally to breast cancer cells both in vitro and in vivo. Data derived from breast tissue-specific reporter vectors in vitro demonstrate that expression from the hALA and oBLG promoters are indeed specific for breast cells (T47D, MCF-7, ZR75–1) when compared with non-breast cells (U2OS, HeLa). Moreover, these vectors displayed tumor cell specificity when compared with the normal MCF-10A breast cell line. These vectors also displayed breast tissue specificity when injected systemically (i.v.) into lactating Balb/c mice, which suggests that these promoters maintain their tissue- specific expression pattern within the context of the adenoviral genome in vivo. Tumors, derived from T47D human breast cancer cells, were established in nude mice and injected with either the tissue-specific reporter or suicide vectors. Results from tumors injected (i.t.) with reporter adenoviruses demonstrate that these promoters are active in T47D cells when grown as established tumors and we observed a marked regression of tumors injected with suicide vectors and treated systemically with gancyclovir (150 mg/kg/day) when compared with control animals. Moreover, mouse survival was prolonged after 35 days in mice undergoing therapy with the suicide vectors in conjunction with gancyclovir when compared with the control animals. These data suggest that the transcriptionally targeted hALA or oBLG driven expression of the HSVtk gene may be a feasible therapy for the treatment of human breast cancer.

Kruppel-like Factor 4 Regulates Laminin α3A Expression in Mammary Epithelial Cells

Laminin-5, the major extracellular matrix protein produced by mammary epithelial cells, is composed of three chains (designated α3A, β3, and γ2), each encoded by a separate gene. Laminin-5 is markedly down-regulated in breast cancer cells. Little is known about the regulation of laminin gene transcription in normal breast cells, nor about the mechanism underlying the down-regulation seen in cancer. In the present study, we cloned the promoter of the gene for the human laminin α3A chain (LAMA3A) and investigated its regulation in functionally normal MCF10A breast epithelial cells and several breast cancer cell lines. Using site-directed mutagenesis of promoter-reporter constructs in transient transfection assays in MCF10A cells, we find that two binding sites for Kruppel-like factor 4 (KLF4/GKLF/EZF) are required for expression driven by the LAMA3A promoter. Electrophoretic mobility shift assays reveal absence of KLF4 binding activity in extracts from T47D, MDA-MB 231, ZR75-1, MDA-MB 436, and MCF7 breast cancer cells. Transient transfection of a plasmid expressing KLF4 activates transcription from the LAMA3A promoter in breast cancer cells. A reporter vector containing duplicate KLF4-binding sites in its promoter is expressed at high levels in MCF10A cells but at negligible levels in breast cancer cells. Thus, KLF4 is required forLAMA3A expression and absence of laminin α3A in breast cancer cells appears, at least in part, attributable to the lack of KLF4 activity.