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UV induces VEGF through a TNF-alpha independent pathway¹

Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA

²Correspondence: Boston University School of Medicine, Department of Dermatology, 609 Albany St., J Bldg., Boston, MA 02118, USA. E-mail: mkosmada{at}bu.edu

SPECIFIC AIMS

Vascular endothelial growth factor (VEGF) is a broadly recognized angiogenic factor. UV irradiation as well as several growth factors and cytokines are reported to induce VEGF in keratinocytic cells. Because UV induces the synthesis and secretion of tumor necrosis factor alpha (TNF-) in epidermal keratinocytes and TNF- is a strong inducer of VEGF, we investigated the role of TNF- in the UV-induced VEGF up-regulation.

PRINCIPAL FINDINGS

1. UV irradiation increases VEGF mRNA and protein in SCC-12F cells
To determine the effect of UV irradiation, cells of the well-differentiated squamous cell carcinoma line SCC-12F were irradiated with 30 mJ/cm² of UV (metered at 285±5 nm) and harvested at different intervals after irradiation. Northern blot analysis revealed constitutive VEGF expression in sham-irradiated cells with a gradual increase in mRNA levels with time. UV irradiation induced VEGF expression above sham-irradiated control, with maximum induction observed 24 h after exposure and persisting though 48 h, when the experiment was terminated (Fig. 1 A, B).

View larger version (30K): [in this window] [in a new window]   Figure 1. UV irradiation induces VEGF expression and secretion. Cells were irradiated and total cellular RNA or protein were harvested at different intervals after UV irradiation. A) VEGF mRNA of UV-irradiated cells was higher than paired sham-irradiated control samples. B) Densitometric analysis of three independent experiments. C) SCC-12F cells were UV or sham irradiated and medium from duplicate dishes was analyzed by ELISA for VEGF levels. VEGF levels in UV-irradiated conditioned medium were consistently higher than with sham-irradiated conditioned medium. Maximal 89 ± 16% induction above those in sham-irradiated conditioned medium was observed 48 h after irradiation.

To determine UV-induced VEGF secretion, medium conditioned by UV vs. sham-irradiated SCC-12F cells was analyzed. VEGF was easily detectable in sham-irradiated medium and accumulated in medium with time, as expected. However, 16–48 h after irradiation, VEGF levels were approximately twice as high in UV-irradiated conditioned medium than in sham-irradiated conditioned medium (Fig. 1C ).

2. TNF- binding peptide abrogates TNF--induced VEGF up-regulation but has no effect on UV-induced VEGF up-regulation
SCC-12F cells were provided the peptide Ac-KWIIVW-NH₂, which has been shown to prevent TNF- binding to its p55 receptor and inhibit TNF- activation of the receptor. We treated cells with 3 µM of the peptide; this concentration was not toxic to SCC-12F cells, as determined by cell yields 72 h after peptide supplementation, and is 10-fold higher than the concentration required to inhibit 50% binding of 10 ng/mL ^[125]I TNF- to TNF- p55 receptor.

To determine peptide effects, cells were treated with 10 ng/mL of TNF- (a dose routinely used to elicit TNF- effects) or simultaneously stimulated with 3 µM of TNF- binding peptide and 10 ng/mL TNF- or diluent alone. Total cellular RNA was collected 24 h after stimulation and processed for Northern blot analysis. Incubation with TNF- consistently induced VEGF mRNA level in SCC-12F cells. Treatment of SCC-12F cells with TNF- and 3 µM of the peptide abrogated TNF--induced VEGF up-regulation that was apparent in cells treated with TNF- alone (Fig. 2 A).

View larger version (28K): [in this window] [in a new window]   Figure 2. TNF- binding peptide abrogates TNF--induced VEGF up-regulation but has no effect on UV-induced VEGF up-regulation. A) SCC-12F cells were stimulated with 10 ng/mL of TNF- and 3 µM of the TNF--blocking hexapeptide. As expected, TNF- induced VEGF mRNA. However, the TNF- effect was completely abrogated in the presence of the peptide. B) SCC-12F cells were UV irradiated as above and immediately after irradiation provided with fresh medium containing 3 µM of the TNF- blocking peptide or diluent alone as control. After correcting for loading, TNF- blocking peptide did not affect VEGF mRNA induction in UV-irradiated cells or sham-irradiated controls. C) VEGF levels in SCC-12F conditioned medium were not affected by the peptide.

To determine the role of TNF- in UV-mediated VEGF induction, SCC-12F cells were UV-irradiated with 30 mJ/cm². Immediately after irradiation, cells were provided with fresh medium containing 3 µM of TNF- binding peptide, and VEGF expression and secretion were examined 24 h after irradiation. Addition of TNF- blocking peptide had no effect on VEGF mRNA induction (Fig. 2B ) or its secretion into the medium (Fig. 2C ). Similar results were observed when UV and sham-irradiated cells were treated with a TNF- neutralizing antibody.

CONCLUSIONS AND SIGNIFICANCE

We observed that VEGF mRNA and protein levels are significantly induced in SCC-12F cells after a physiological UV dose. Induction of VEGF with different UV irradiation doses has been reported by other groups for different cell types. We show in SCC-12F cells that treatment with TNF- for 24 h induces VEGF expression, in accordance with previous reports describing TNF- as inducer of VEGF in other cells. UV irradiation is known to induce TNF- expression and secretion in normal human keratinocytes and keratinocyte-derived cell lines. Previous work from our laboratory using SCC-12F cells shows that irradiation doses similar to those that induce VEGF up-regulation also induce TNF- mRNA and protein 12–16 h before VEGF induction.

To determine whether TNF- is the major cytokine that mediates VEGF induction after UV irradiation, we provided cells with the TNF- antagonistic hexapeptide Ac-KWIIVW-NH₂. Treatment of SCC-12F cells with the peptide abrogated TNF- effect on VEGF induction, confirming that the peptide effectively blocks the TNF- effect. However, when UV-irradiated SCC-12F cultures were provided TNF- blocking peptide, there was no effect on expression and release of VEGF. Our results cannot be explained by failure of the peptide to block all TNF- present in the medium because we added sufficient peptide to block more than 10 ng/mL TNF- whereas the amount of TNF- in UV-irradiated conditioned medium is substantially lower, by nearly an order of magnitude.

Our findings differ from those published by Longuet-Perret et al., who reported that addition of TNF- neutralizing antibody inhibited UV-induced VEGF secretion from normal human keratinocytes. Different experimental conditions, particularly UVB doses four-to eightfold higher than the physiological doses used in our experiment, may account for the discrepancies between the two reports.

Our irradiation system delivers both UVA and UVB (a spectral output virtually identical to that of terrestrial sunlight). Direct UVB activation of factors that transcriptionally regulate VEGF, like NFB, AP-1, and AP-2, may have an important role in the observed UV-induction of VEGF. We doubt that in our experiments UVA substantially contributed to VEGF mRNA induction, because VEGF induction occurred much later and was not observed for at least 16 h after irradiation; UVA has previously been reported to rapidly induce VEGF in HaCaT cells.

In summary, after solar simulated irradiation multiple independent mechanisms may be involved in VEGF up-regulation. Blocking one mediator (TNF-) does not detectably affect the response. The apparent redundancy of mechanisms for VEGF up-regulation suggests that this is an important adaptive response to UV irradiation.

View larger version (9K): [in this window] [in a new window]   Figure 3. Schematic diagram. Pathway of UV-induced VEGF in cells of keratinocyte origin. Although TNF- induces VEGF in SCC-12F cells, UV-induced VEGF up-regulation is not mediated by autocrine TNF- because blocking of TNF- effect does not abrogate UV-induced VEGF up-regulation. It is possible that keratinocyte-cytokines other than TNF-, known to be secreted after UV irradiation, may play a role in VEGF induction.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0379fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0379fje

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