Role for retinoblastoma protein family members in UV-enhance expression from the human cytomegalovirus immediate early promoter[para]

Photochemistry and Photobiology, Jun 2003 by Francis, Murray A, Rainbow, Andrew J

ABSTRACT

The expression from a reporter construct driven by a cytomegalovirus (CMV) immediate early (IE) promoter is strongly inducible by UV in human fibroblasts. This response is induced at lower UV fluences in transcription-coupled repair (TCR)-deficient fibroblasts compared with normal fibroblasts and is absent in their simian virus 40-transformed counterparts. In this study we demonstrate that expression of human papilloma virus (HPV) E7 (but not of HPV E6) can attenuate UV-induced expression from the human CMV-IE-driven reporter construct in human fibroblasts. Furthermore, UV-induced expression from the reporter construct appears impaired in murine fibroblasts harboring inactivating mutations in the retinoblastoma (Rb) gene family members p107 and pRb but not in fibroblasts harboring such mutations in the p53 gene. Taken together, these data suggest that one or more members of the pRb family (but not p53) play an essential role in mediating UV-induced expression from the CMV-IE promoter. In this study we report normal UV-upregulation of reporter expression in xeroderma pigmento-sum (XP) group E fibroblasts, consistent with normal TCR. Because XP-E cells deficient in the p48 subunit of the damaged DNA-binding protein are impaired in E2F-1-activated transcription, these results also suggest that the (pRb-regulated) transcription factor E2F-1 does not play an essential role in UV-enhanced expression from the CMV-IE promoter.

INTRODUCTION

UV irradiation induces expression from a number of cellular and viral promoters (reviewed in Sachsenmaier et al. [1] and Bender et al. [2]). This response can be divided into two components (reviewed in Bender et al. [2]). The "immediate" response arises at the cell membrane with the activation of cell surface receptors and their corresponding signaling cascades. This leads to a rapid induction of expression for a subset of UV-responsive genes. These genes primarily encode additional transcription factors, although several immediate-responsive genes (such as the genes for mitogen-activated protein kinase phosphalases [3,4]) provide a negative feedback for the immediate response.

Whereas the strength of the immediate response is directly proportional to the strength of the initial UV exposure, the strength and duration of the "delayed" (or secondary) response is inversely related to the cell's ability to repair transcriptionally active genes (5-7), which is mediated by a process known as transcription-coupled repair (TCR) (8,9). Expression of the delayed-responsive genes is not observed for several hours after UV exposure and is dependent on de novo protein synthesis (presumably of the transcription factors upregulated by the immediate response). Delayed-responsive genes include the genes encoding collagenase, metallothionein, interleukin-1, basic fibroblast growth factor and numerous viral promoters (2,5,6,10-12). Similarly, we have demonstrated that treatment of primary human fibroblasts with UV results in increased expression from a reporter driven by either the human or the murine cytomegalovirus (CMV) immediate early (IE) promoters (13). The enhancement of reporter activity after lower UV exposures in TCR-deficient fibroblasts, compared with their TCR-proficient counterparts, indicates that persistent damage in active genes plays a role in this induction.

In contrast to results with primary cell strains, no UV-induced expression of reporter activity was observed in any simian virus 40 (SV40)-transformed cell line (TCR-proficient or TCR-deficient) examined (13). Cellular transformation by SV40 results in a number of alterations of cellular metabolism, including the binding and inactivation of p53 and members of the pRb family (Rb stands for retinoblastoma) by the SV40 large tumor antigen (LT) (14-19). Both p53 and pRb accumulate in response to UV exposure (20,21) and can modulate the transcription of a large number of cellular genes (reviewed in Lakin and Jackson [22], el-Deiry [23] and Mayol and Grana [24]). Because we have previously demonstrated a role for p53 in enhanced reactivation of a UV-damaged reporter (25), it was of interest to examine the possible roles of these proteins in mediating enhanced expression from Ad5HCMVsp1 lacZ in response to UV (Ad stands for adenovirus and HCMV for human CMV). However, experiments with Li-Fraumeni syndrome cell lines, both heterozygous and hemizygous for two distinct mutations in the p53 gene, showed normal levels of reporter activity in UV-irradiated cells (13), suggesting that p53 is not essential for this response.

Like SV40, other viruses also encode proteins that target p53, pRb family members or both. The polyoma LT has considerable homology to SV40LT and binds pRb family members (26,27) but has not been observed to bind to or interfere with the transactivation activity of p53 (28). The human papilloma virus (HPV) E6 protein forms a specific complex with p53 and promotes its degradation in vitro (29,30), whereas the HPV E7 protein complexes with pRb family members (31,32). The human cervical carcinoma cell line HeLa expresses the HPV early genes (33) and has very low activity of both p53 and pRb family members.