Gilbert F. Morris

p53-Mediated Regulation of Proliferating Cell Nuclear Antigen Expression in Cells Exposed to Ionizing Radiation

ABSTRACT The proliferating cell nuclear antigen (PCNA) is a highly conserved cellular protein that functions both in DNA replication and in DNA repair. Exposure of a rat embryo fibroblast cell line (CREF cells) to γ radiation induced simultaneous expression of PCNA with the p53 tumor suppressor protein and the cyclin-dependent kinase inhibitor p21WAF1/Cip1. PCNA mRNA levels transiently increased in serum-starved cells exposed to ionizing radiation, an observation suggesting that the radiation-associated increase in PCNA expression could be dissociated from cell cycle progression. Irradiation of CREF cells activated a transiently expressed PCNA promoter chloramphenicol acetyltransferase construct through p53 binding sequences via a mechanism blocked by a dominant negative mutant p53. Electrophoretic mobility shift assays with nuclear extracts prepared from irradiated CREF cells produced four p53-specific DNA-protein complexes with the PCNA p53 binding site. Addition of monoclonal antibody PAb421 (p53-specific) or AC238 (specific to the transcriptional coactivator p300/CREB binding protein) to the mobility shift assay distinguished different forms of p53 that changed in relative abundance with time after irradiation. These findings suggest a complex cellular response to DNA damage in which p53 transiently activates expression of PCNA for the purpose of limited DNA repair. In a population of nongrowing cells with diminished PCNA levels, this pathway may be crucial to survival following DNA damage.

Cyclin-dependent Kinase 9 Is Required for Tumor Necrosis Factor-α-stimulated Matrix Metalloproteinase-9 Expression in Human Lung Adenocarcinoma Cells

The proinflammatory cytokine tumor necrosis factor-α (TNF-α) promotes tumor progression through activation of matrix metalloproteinase (MMP) activity. MMP-9 is a gelatinase secreted by both cancer cells and surrounding stromal cells, and it contributes to TNF-α-stimulated tumor invasion and metastasis. Cyclin-dependent kinase 9 (CDK9), the catalytic component of positive transcription elongation factor-b, phosphorylates serine 2 residues in the C-terminal domain of RNA polymerase II for productive transcription elongation and is up-regulated upon exposure to various stresses. This study investigated roles of CDK9 in TNF-α-stimulated MMP-9 expression in human lung adenocarcinoma cells. CDK9 activity was inhibited using three different strategies, including the CDK9 pharmacological inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), a dominant-negative CDK9, and a CDK9-specific small interfering RNA. All three approaches reduced TNF-α-mediated accumulation of MMP-9 in the conditioned media as demonstrated by gelatin zymography. In contrast, transforming growth factor-β1-induced accumulation of MMP-2 was unaffected by DRB. Expression of the MMP-9 gene was examined using reverse transcription real time PCR and using a transient transfection assay to evaluate MMP-9 promoter activity. DRB reduced the TNF-α-induced increase in MMP-9 mRNA levels but did not effect transforming growth factor-β1-induced MMP-2 mRNA expression. Consistently DRB and dominant-negative CDK9 completely abrogated TNF-α-stimulated human MMP-9 promoter activity. TNF-α did not regulate expression or localization of CDK9 or its regulatory partner Cyclin T. However, TNF-α stimulated CDK9 binding to Cyclin T and MMP-9 gene occupancy by both CDK9 and the serine 2-phosphorylated form of RNA polymerase II. Our findings indicate that CDK9 mediates TNF-α-induced MMP-9 transcription. Disruption of TNF-α signaling using CDK9 inhibitors could serve as a potential therapeutic strategy against tumor invasion and metastasis.

Induction of p53-dependent Activation of the Human Proliferating Cell Nuclear Antigen Gene in Chromatin by Ionizing Radiation

A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.

A complex promoter element mediates transactivation of the human proliferating cell nuclear antigen promoter by the 243-residue adenovirus E1A oncoprotein.

The adenovirus E1A oncoproteins interfere with the normal regulation of cellular proliferation through interactions with cell cycle regulatory proteins. In view of the essential role of proliferating-cell nuclear antigen (PCNA) in DNA replication, we performed a mutational analysis of the minimal human PCNA promoter (nucleotides -87 to +62) to define sequence elements which mediate transactivation by the 243-residue E1A protein (E1A 243R). Linker-scanning and site-directed mutants were examined for basal and E1A-induced expression of chloramphenicol acetyltransferase (CAT) from PCNA promoter-CAT reporter constructs transiently expressed in HeLa cells. The results define the cis-acting element required for induction of PCNA by E1A 243R as a region between -59 and -45 relative to the transcription initiation site. This PCNA E1A-responsive element (PERE), which is protected from DNase I digestion by nuclear extracts from 293 cells, includes the sequence AGCGTGG immediately upstream of the ATF binding site previously shown to be important for activation of PCNA by E1A 243R (G. F. Morris and M. B. Mathews, J. Virol. 65:6397-6406, 1991). Mutation of either the upstream component or the ATF site within the PERE diminishes basal promoter activity and abrogates transactivation by E1A 243R. This novel cis-acting element is also essential for both basal and E1A-induced expression in the context of the full-length PCNA promoter.

Post-transcriptional up-regulation of miR-21 by type I collagen.

Composition of extracellular matrix (ECM) is crucial to the establishment and maintenance of epithelial apical‐basolateral polarity. Increased ECM rigidity caused by deposition of fibrillar collagen, for example, collagen type I (Col‐1), promotes loss of epithelial polarity and tumor progression. microRNAs are small non‐coding RNAs that regulate gene expression and fundamental cellular processes. The current study explored a link between microRNAs and Col‐1 using organotypic three‐dimensional culture in which epithelial cells are embedded within Matrigel, a mimic of basement membrane matrix (Matrigel 3‐D). Matrigel 3‐D culture of A549, MCF‐7, and mK‐ras‐LE cells (lung and mammary epithelial cell lines) gave rise to acinus, an in vitro equivalent of apical‐basolateral polarity that consists of a polarized monolayer of epithelial cells facing a central lumen. Supplementation of Col‐1 disrupted acinus. Moreover, Col‐1 up‐regulated the expression of miR‐21, a well‐documented oncogenic microRNA, via a post‐transcriptional mechanism. Similar post‐transcriptional up‐regulation of miR‐21 correlated with deposition of Col‐1 in a murine model of lung fibrogenesis. In summary, our findings link altered ECM composition/rigidity and the expression of oncogenic microRNAs. The current study also suggests a novel post‐transcriptional mechanism for regulation of miR‐21 expression at maturation from pre‐miR‐21 to mature miR‐21. Mol. Carcinog.

Modulation of transcriptional activation of the proliferating cell nuclear antigen promoter by the adenovirus E1A 243-residue oncoprotein depends on proximal activators.

Previous analyses defined a proliferating cell nuclear antigen (PCNA) E1A-responsive element (PERE) in the PCNA promoter that is essential for transactivation by the 243-residue product of the adenovirus type 2 E1A 12S mRNA (E1A 243R). In this report, we show that the PERE activates a heterologous basal promoter and confers susceptibility to transactivation by E1A 243R, indicating that the PERE is both necessary and sufficient for the response of the PCNA promoter to this oncoprotein. Insertion of linker sequences between the PERE and the site of transcription initiation in the PCNA promoter severely impairs the promoters response to E1A 243R transactivation. GAL4 sites can replace the function of the PERE in the E1A 243R response of the PCNA basal promoter if transcriptional activators of suitable strength are supplied as GAL4 fusion proteins. Weak transcriptional activators render the PCNA basal promoter subject to transactivation by E1A 243R but do not endow the adenovirus E1B basal promoter with a similar response. Strong transcriptional activators do not support transactivation by E1A 243R, however; instead, E1A reduces the ability of the strong activators to activate both the PCNA and E1B basal promoters. Although other mechanistic differences might determine the response, the data imply a relationship between the activation strength of promoter-proximal effectors and the response of the PCNA basal promoter to E1A 243R. These experiments indicate that the PERE can function autonomously in mediating transactivation by E1A 243R and that the PCNA basal promoter is configured in a manner that permits modulation by E1A 243R of transcriptional activation by promoter-proximal effectors.

Thy-1 Attenuates TNF-α-Activated Gene Expression in Mouse Embryonic Fibroblasts via Src Family Kinase

Heterogeneous surface expression of Thy-1 in fibroblasts modulates inflammation and may thereby modulate injury and repair. As a paradigm, patients with idiopathic pulmonary fibrosis, a disease with pathologic features of chronic inflammation, demonstrate an absence of Thy-1 immunoreactivity within areas of fibrotic activity (fibroblast foci) in contrast to the predominant Thy-1 expressing fibroblasts in the normal lung. Likewise, Thy-1 deficient mice display more severe lung fibrosis in response to an inflammatory injury than wildtype littermates. We investigated the role of Thy-1 in the response of fibroblasts to the pro-inflammatory cytokine TNF-α. Our study demonstrates distinct profiles of TNF-α-activated gene expression in Thy-1 positive (Thy-1+) and negative (Thy-1−) subsets of mouse embryonic fibroblasts (MEF). TNF-α induced a robust activation of MMP-9, ICAM-1, and the IL-8 promoter driven reporter in Thy-1− MEFs, in contrast to only a modest increase in Thy-1+ counterparts. Consistently, ectopic expression of Thy-1 in Thy-1− MEFs significantly attenuated TNF-α-activated gene expression. Mechanistically, TNF-α activated Src family kinase (SFK) only in Thy-1− MEFs. Blockade of SFK activation abrogated TNF-α-activated gene expression in Thy-1− MEFs, whereas restoration of SFK activation rescued the TNF-α response in Thy-1+ MEFs. Our findings suggest that Thy-1 down-regulates TNF-α-activated gene expression via interfering with SFK- and NF-κB-mediated transactivation. The current study provides a novel mechanistic insight to the distinct roles of fibroblast Thy-1 subsets in inflammation.

Promotion of lung tumor growth by interleukin-17

Recent findings demonstrate that inhaled cigarette smoke, the predominant lung carcinogen, elicits a T helper 17 (Th17) inflammatory phenotype. Interleukin-17A (IL-17), the hallmark cytokine of Th17 inflammation, displays pro- and antitumorigenic properties in a manner that varies according to tumor type and assay system. To investigate the role of IL-17 in lung tumor growth, we used an autochthonous tumor model (K-Ras(LA1) mice) with lung delivery of a recombinant adenovirus that expresses IL-17A. Virus-mediated expression of IL-17A in K-Ras(LA1) mice at 8-10 wk of age doubled lung tumor growth in 3 wk relative to littermates that received a green fluorescent protein-expressing control adenovirus. IL-17 induced matrix metalloproteinase-9 (MMP-9) expression in vivo and in vitro. In accord with this finding, selective and specific inhibitors of MMP-9 repressed the increased motility and invasiveness of IL-17-treated lung tumor cells in culture. Knockdown or mutation of p53 promoted the motility of murine lung tumor cells and abrogated the promigratory role of IL-17. Coexpression of siRNA-resistant wild-type, but not mutant, human p53 rescued both IL-17-mediated migration and MMP-9 mRNA induction in p53 knockdown lung tumor cells. IL-17 increased MMP-9 mRNA stability by reducing interaction with the mRNA destabilizing serine/arginine-rich splicing factor 1 (SRSF1). Taken together, our results indicate that IL-17 stimulates lung tumor growth and regulates MMP-9 mRNA levels in a p53- and SRSF1-dependent manner.

Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures.

Three-dimensional organotypic culture using reconstituted basement membrane matrix (rBM 3-D) is an invaluable tool to characterize morphogenesis of epithelial cells and to elucidate the tumor-modulating actions of extracellular matrix. microRNAs (miRNA) are a novel class of tumor modulating genes. A substantial amount of investigation of miRNAs in cancer is carried out using monolayer 2-D culture on plastic substratum, which lacks a consideration of the matrix-mediated regulation of miRNAs. In the current study we compared the expression of miRNAs in rBM 3-D and 2-D cultures of two lung adenocarcinoma cell lines. Our findings revealed a profound difference in miRNA profiles between 2-D and rBM 3-D cultures of lung adenocarcinoma cells. The rBM 3-D culture-specific miRNA profile was highlighted with higher expression of the tumor suppressive miRNAs (i.e., miR-200 family) and lower expression of the oncogenic miRNAs (i.e., miR-17-92 cluster and miR-21) than that of 2-D culture. Moreover, the expression pattern of miR-17, miR-21, and miR-200a in rBM 3-D culture correlated with the expression of their targets and acinar morphogenesis, a differentiation behavior of lung epithelial cells in rBM 3-D culture. Over-expression of miR-21 suppressed its target PTEN and disrupted acinar morphogenesis. In summary, we provide the first miRNA profile of lung adenocarcinoma cells in rBM 3-D culture with respect to acinar morphogenesis. These results indicate that rBM 3-D culture is essential to a comprehensive understanding of the miRNA biology in lung epithelial cells pertinent to lung adenocarcinoma.

Modulation of lung inflammation by the Epstein-Barr virus protein Zta

Several studies have implicated gamma-herpesviruses, particularly Epstein-Barr virus (EBV), in the progression of idiopathic pulmonary fibrosis. The data presented here examine the possible role that EBV plays in the potentiation of this disease by evaluating the pulmonary response to expression of the EBV lytic transactivator protein Zta. Expression of Zta in the lungs of mice via adenovirus-mediated delivery (Adv-Zta) produced profibrogenic inflammation that appeared most pronounced by day 7 postexposure. Relative to mice exposed to control GFP-expressing adenovirus (Adv-GFP), mice exposed to Adv-Zta displayed evidence of lung injury and a large increase in inflammatory cells, predominantly neutrophils, recovered by bronchoalveolar lavage (BAL). Cytokine and mRNA profiling of the BAL fluid and cells recovered from Adv-Zta-treated mice revealed a Th2 and Th17 bias. mRNA profiles from Adv-Zta-infected lung epithelial cells revealed consistent induction of mRNAs encoding Th2 cytokines. Coexpression in transient assays of wild-type Zta, but not a DNA-binding-defective mutant Zta, activated expression of the IL-13 promoter in lung epithelial cells, and detection of IL-13 in Adv-Zta-treated mice correlated with expression of Zta. Induction of Th2 cytokines in Zta-expressing mice corresponded with alternative activation of macrophages. In cell culture and in mice, Zta repressed lung epithelial cell markers. Despite the profibrogenic character at day 7, the inflammation resolves by 28 days postexposure to Adv-Zta without evidence of fibrosis. These observations indicate that the EBV lytic transactivator protein Zta displays activity consistent with a pathogenic role in pulmonary fibrosis associated with herpesvirus infection.