Ultraviolet-A-Induced Transactivation of the Vascular Endothelial Growth Factor Gene in HaCaT Keratinocytes is Conveyed by Activator Protein-2 Transcription Factor

Abstract

Ultraviolet-A radiation represents a significant proportion of the ultraviolet solar spectrum that was recently shown to affect gene expression of epidermal keratinocytes by molecular mechanisms distinct from ultraviolet-B radiation. As ultraviolet-A either alone or in combination with ultraviolet-B may contribute to photocarcinogenesis, we aimed to explore the biologic effects of ultraviolet-A radiation on vascular endothelial growth factor gene expression by the immortalized keratinocyte cell line HaCaT. As keratinocyte-derived vascular endothelial growth factor not only provides the major cutaneous angiogenic activity but may also augment the malignant phenotype of tumor cells, we studied the molecular mechanisms of ultraviolet-A-induced vascular endothelial growth factor expression in HaCaT cells, serving as a transformed preneoplastic epithelial cell line. Whereas ultraviolet-B-mediated vascular endothelial growth factor expression has been previously indicated to be conveyed by indirect mechanisms, ultraviolet-A rapidly induced vascular endothelial growth factor mRNA expression in a fashion comparable to that seen with the transforming growth factor alpha, representing a direct and potent activator of vascular endothelial growth factor gene transcription. Ultraviolet-A was found to readily induce vascular endothelial growth factor promoter-based reporter gene constructs through a consensus element for activator protein-2 transcription factor. The critical role of activator protein-2 was substantiated by demonstration of ultraviolet-A-induced activator-protein-2-dependent nuclear DNA binding activity to this site, and by inhibition of ultraviolet-A-mediated vascular endothelial growth factor gene transcription through insertion of a critical mutation within the activator protein-2 sequence. Together, our data further elucidate photobiologic aspects of ultraviolet-A-induced gene expression by characterizing mechanisms of vascular endothelial growth factor upregulation at the molecular level. In addition, our experiments support the concept of a more general importance of activator protein-2 in ultra- violet-A-mediated responses by keratinocytes or keratinocyte-derived cell lines.

Loss of Activator Protein-2α Results in Overexpression of Protease-activated Receptor-1 and Correlates with the Malignant Phenotype of Human Melanoma

“…Furthermore, immunohistochemical analyses of advanced primary and metastatic melanoma clinical specimens demonstrated that the loss of AP-2 expression correlated with low p21/WAF1, E-cadherin, and c-KIT expression and poor prognosis (7,8). Functional AP-2-binding elements have been identified in other genes involved in the progression of human melanoma, including p21/WAF1 (9), intercellular adhesion molecule (10), c-erbB-2/HER-2/neu (11)(12)(13), plasminogen activator inhibitor type I (14), insulin-like growth factor-binding protein-5 (15), transforming growth factor-␣ (16), vascular endothelial growth factor (17,18), E-cadherin (19), and hepatocyte growth factor (20).…”

Role and regulation of the thrombin receptor (PAR-1) in human melanoma

“…For example, phorbol esters and signals that enhance cAMP levels induce AP-2 activity independently of protein synthesis, whereas retinoic acid treatment of teratocarcinoma cell lines results in a transient induction of AP-2 mRNA levels on the transcription level (Imagawa et al, 1987;Luscher et al, 1989;Buettner et al, 1993). Other signals demonstrated to stimulate AP-2 expression and activate transcription of target genes include ultraviolet-A radiation and singlet oxygen (Grether-Beck et al, 1996;Huang and Domann, 1998;Gille et al, 2000).…”

“…In addition, inactivation of AP-2 in primary cutaneous melanoma cells (AP-2 positive) by means of a dominant-negative AP-2 gene (AP-2B) led to deregulation of the metalloproteinase MMP-2 gene and increased the cells tumor and metastatic potential in nude mice (Gershenwald et al, 2001). Furthermore, functional AP-2-binding elements have been identified in other genes involved in the progression of human melanoma such as p21/WAF1 (Zeng et al, 1997), intercellular adhesion molecule (I-CAM) (Grether-Beck et al, 1996), c-erbB-2/HER-2/neu (Hollywood and Hurst, 1993;Bosher et al, 1995;Turner et al, 1998), plasminogen activator inhibitor type I (PAI-1) (Descheemaeker et al, 1992), insulin-like growth factor binding protein-5 (Duan and Clemmons, 1995), transforming growth factor-alpha (TGF-a) (Wang et al, 1997), vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) (Gille et al, 1997(Gille et al, , 2000, E-cadherin (Batsche et al, 1998), hepatocyte growth factor (HGF) , and c-Myc (Gaubatz et al, 1995). Loss of AP-2 was also observed in clinical specimens of advanced primary and metastatic melanoma, which was associated with malignant transformation and elevated risk of tumor progression (Karjalainen et al, 1998;Baldi et al, 2001), thus supporting the hypothesis that loss of AP-2 is a crucial event in the progression and metastasis of melanoma.…”

UV induces VEGF through a TNF‐α‐independent pathway

“…Skin hypoxia also leads to up-regulation of VEGF expression in epidermal keratinocytes, dermal fibroblasts, and dermal endothelial cells (15). The cloned 3.4-kb fragment of the VEGF promoter (2) contains consensus sequences that bind a variety of transcription factors, including hypoxia-induced factor (16), AP-1 (17), SP-1 (18), and AP-2 (19). Blocking of NFκB inhibits VEGF expression (20)(21)(22) and therefore it is likely that NFκB transcriptionally regulates VEGF mRNA.…”

“…In addition to these factors, ultraviolet-A (UVA) has been reported to up-regulate VEGF in immortalized keratinocytes (HaCaT cells), presumably through activation of AP-2 (19) and maybe other as yet unidentified mechanisms. ultraviolet-B (UVB) radiation also induces VEGF expression in normal human keratinocytes, HaCaT cells, and A431 squamous carcinoma cells (23,24) and is thought to act indirectly by inducing the synthesis and release of IL-1, TNF-α, and other cytokines (23)(24)(25).…”