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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 14, 2005 as doi:10.1096/fj.05-3934fje. |
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,
,,1
* Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza," Rome, Italy;
Azienda Ospedaliera Sant’Andrea, Rome, Italy;
Istituto Dermatologico San Gallicano, Rome, Italy; and
Istituto Neurologico Mediterraneo "Neuromed, " Pozzilli, Italy
1 Correspondence: Dip. Medicina Sperimentale e Patologia, Viale Regina Elena 324, Roma 00161, Italy. E-mail: mara.torrisi{at}uniroma1.it
SPECIFIC AIMS
Receptor tyrosine kinases (RTKs) play a role in the cellular response to UV radiation. Among the RTKs expressed on the epidermal keratinocytes, we recently described that the keratinocyte growth factor receptor (KGFR) is tyrosine phosphorylated, activated, and internalized after exposure to UVB, whereas other groups have reported conflicting results on the epidermal growth factor receptor (EGFR) regarding receptor phosphorylation and ubiquitination, pathways of endocytosis, and mechanisms of degradation. Thus, aims of the present study were to 1) analyze the ligand-independent endocytic pathway of KGFR induced by UVB; 2) compare the effects of UVB-mediated and ligand-mediated activation and endocytosis of KGFR; and 3) evaluate the response to UVB of KGFR and EGFR expressed in the same cell types and exposed to equal doses and source of radiation.
PRINCIPAL FINDINGS
1. UVB exposure causes endocytosis of KGFR, but not of EGFR, and this UVB-induced internalization requires tyrosine kinase activity
We previously showed that exposure to UVB (30–50 mJ/sq cm) caused tyrosine phosphorylation and activation of KGFR, evident after 5–10 min of treatment and similar to that induced by KGF. To determine whether both KGFR and EGFR would undergo tyrosine phosphorylation after UVB irradiation, we first performed immunoprecipitation/Western blot experiments on NIH3T3 KGFR transfected cells left untreated or exposed to UVB (40 mJ/sq cm) or treated with KGF (100 ng/mL) for 10 min at 37°C: both exposure to UVB and treatment with KGF induced receptor tyrosine phosphorylation. To compare directly the possible effect induced by UVB on EGFR phosphorylation with the effect induced on KGFR, NIH3T3 EGFR cells were exposed to UVB or treated with EGF (100 ng/mL) for 10 min at 37°C: UVB exposure resulted in no tyrosine phosphorylation of EGFR, whereas receptor phosphorylation appeared to be triggered by parallel treatment with EGF.
To analyze whether KGFR and EGFR would both be internalized in response to UVB exposure under our experimental conditions, we performed parallel immunofluorescence experiments of receptor endocytosis in NIH3T3 cells expressing by transfection KGFR or EGFR, exposed to UVB (40 mJ/sq cm), then warmed to 37°C for 30 min or treated with the corresponding growth factor for 1 h at 4°C for binding, before warming to 37°C for 30 min to allow receptor internalization. Immunostaining of KGFR was performed with anti-Bek antibodies that primarily detect the exogenously overexpressed KGFRs. While UVB irradiation showed to induce KGFR endocytosis with efficiency and timing similar to the ligand KGF, the same exposure was not able to trigger EGFR internalization like the ligand EGF. The UVB failure to induce EGFR internalization was observed at higher dose of radiation (60 mJ/sq cm) and in HeLa epithelial cells expressing EGFR endogenously. In contrast, KGFR appeared internalized after UVB exposure not only in NIH3T3 KGFR fibroblasts but also in HeLa KGFR transfectants and KGFR internalization induced by both UVB or KGF was efficiently blocked by pretreatment with the tyrosine kinase inhibitor genistein, indicating that receptor phosphorylation is required for its internalization. Thus, the UVB exposure that induces tyrosine phosphorylation and internalization of KGFR is not able to trigger parallel phosphorylation and internalization of EGFR, suggesting a differential role of the two receptors in the cellular response to UVB. To evaluate whether receptor kinase activity is required for UVB or KGF-induced internalization of KGFR, we generated a kinase negative mutant KGFR in which the two tyrosine residues 656 and 657 were substituted with phenilalanine: immunofluorescence analysis revealed that the kinase negative KGFR is not internalized after either UVB exposure or KGF treatment. Thus, receptor kinase activity is required for UVB-induced KGFR internalization.
2. The UVB-induced KGFR endocytosis is mediated by ROS production
We have previously shown that the UVB induced phosphorylation of KGFR could be inhibited in the presence of antioxidants and that pro-oxidant treatment alone was sufficient to trigger receptor tyrosine phosphorylation, indicating that the UVB effects on KGFR signaling are mediated by the intracellular production of ROS. We analyzed whether the KGFR internalization could be affected by antioxidants and induced by pro-oxidant treatment. Immunofluorescence microscopic analysis was performed by exposing the cells to UVB in the presence of the antioxidant N-acetylcysteine (NAC) at the dose (10 mM), which is known to inhibit receptor phosphorylation. The UVB-dependent KGFR internalization was blocked by the treatment with NAC, indicating that ROS production is required for the UVB induced endocytosis of KGFR. Further proof that ROS are able to induce KGFR internalization is gained from experiments with pro-oxidative agents, such as cumene hydroperoxide (CUH, 200 µM) or H2O2 (5 mM), which efficiently caused KGFR endocytosis. On the contrary, parallel analysis of EGFR distribution showed, as expected, that H2O2 or CUH treatments were not able to induce receptor endocytosis.
3. Ligand-independent endocytosis of KGFR induced by UVB is clathrin-mediated
We examined whether the UVB uptake of the activated receptors might occur through clathrin-coated pits as observed in the case of KGF-induced endocytosis. To address this, we performed immunoelectron microscopy on irradiated or ligand-treated cells: the localization of KGFR in untreated, KGF-treated, or UVB-exposed NIH3T3 KGFR transfected cells was analyzed by immunogold electron microscopy using both surface labeling with a KGF-HFc chimeric protein or with anti-Bek antibodies and staining of ultrathin cryosections with anti-Bek antibodies followed by protein A-colloidal gold conjugates. After UVB irradiation and 5 min of incubation at 37°C, gold labeling appeared inside clearly recognizable clathrin-coated endocytic pits, as observed after incubation with the ligand. Quantitation of the immunogold labeling in clathrin-coated pits or in noncoated invaginations of the plasma membranes demonstrates that the ligand-independent receptor uptake does not involve clathrin-independent mechanisms.
4. UVB radiation induces KGFR ubiquitination and sorting of the internalized receptors to the endocytic degradative pathway
We performed a double immunofluorescence confocal analysis on NIH3T3 KGFR cells exposed to UVB or treated with KGF and warmed to 37°C for different time points before fixation. In both cells exposed to UVB or treated with KGF, confocal analysis revealed localization of KGFR after 10 min of internalization in early endosomes, identified by transferrin internalized for 10 min at 37°C (Fig. 1
), and in late endosomes and lysosomes, identified by the lysotracker marker after 30 min of internalization (Fig. 1)
. Similar results were obtained using HeLa KGFR transfected cells: in fact, confocal analysis showed localization of endocytosed KGFRs in early endosomes, identified by the EEA1 marker, after 10 min of warming to 37°C and in MVB/late endosomes, identified by the CD63 marker, after 30 min (Fig. 1)
. Thus, the UVB-induced endocytosis of KGFR appears to follow the same endocytic degradative pathway from early to late endosomes, then lysosomes induced by the ligand KGF.
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5. Apoptosis and cell cycle arrest are triggered by UVB-induced KGFR activation and counteracted by KGFR endocytosis
To evaluate the possible role of KGFR phosphorylation and activation induced by UVB in the control of cell cycle arrest and apoptosis, we first analyzed HaCaT cells, expressing endogenous KGFR, and NIH3T3 KGFR transfected cells; all cells were exposed to UVB or treated with KGF, then kept an additional 6 h before fixation. The block of KGFR phosphorylation, induced by pretreatment with the tyrosine kinase inhibitor genistein, significantly decreased apoptosis, suggesting a direct role of KGFR in the triggering of the apoptotic process. These results were strengthened by the use of HaCaT cells transiently transfected with KGFR Y769F mutant: UVB-induced apoptosis appeared highly increased in cells expressing KGFR WT and dramatically decreased in cells expressing the mutant receptor, indicating that KGFR plays a central role in the process and suggesting that PLC
and FRS2 phosphorylation as well as MAPK activation controlled by KGFR are required for the apoptotic events triggered by UVB.
To evaluate the biological significance of KGFR internalization in regulating the cellular response to UVB, we analyzed the apoptotic fate of HaCaT cells pretreated with KGF for 30 min or 1 h at 37°C to induce receptor internalization, then exposed to UVB: the percentage of apoptotic cells decreased with time of internalization, showing that the gradual reduction of surface exposed KGFR results in protection from apoptosis. To compare the functional role of KGFR activation induced by UVB or by KGF on the cell cycle, HaCaT cells were immunolabeled with antibodies directed against the Ki67 proliferation marker: while treatment with KGF induced intense Ki67 nuclear signal, exposure to UVB appeared to abolish the nuclear staining, indicating cell cycle arrest. Pretreatment with genistein drastically reduced KGF-induced proliferation indicating that KGFR activation by the ligand is responsible for triggering the mitogenic pathway.
CONCLUSIONS AND SIGNIFICANCE
Our results demonstrate that after UVB exposure, KGFRs are internalized by clathrin-coated pits and the internalized receptors are sorted to the degradative endocytic pathway. This ligand-independent receptor internalization is mediated by the production of reactive oxygen species (ROS) and requires receptor phosphorylation, activation, and ubiquitination (Fig. 2
). Since endocytosis and sorting in multivesicular bodies play a crucial role in attenuating survival signals and regulating apoptosis of the damaged cells, we propose that expression of KGFR might exert a beneficial role by limiting the detrimental effects of UVB.
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Under our experimental conditions, EGFR, differently from KGFR, does not appear to undergo tyrosine phosphorylation and internalization in response to UVB (Fig. 3). All together, these results lead to the hypothesis that, at variance with EGFR, both ligand-induced and UV-induced KGFR endocytosis are strictly dependent on receptor phosphorylation and activation. This is consistent with the recent suggestion that the molecular mechanisms involved in the regulation of KGFR and EGFR endocytosis, although involving in both cases a clathrin-mediated uptake, are not totally comparable: the eps15 protein, for example, which is known to play an essential role in EGFR endocytosis, is not required for KGFR internalization.
It is novel but not surprising that the KGFR, being expressed mainly on the suprabasal keratinocytes, may stimulate mechanisms of protection to UVB radiation, which penetrate partially into the skin reaching only in minimal part the basal layer, and that this response is different from that of EGFR expressed mainly on the basal cells.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3934fje;
This article has been cited by other articles:
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  P. Agostinis, M. Garmyn, and A. Van Laethem The Aryl Hydrocarbon Receptor: An Illuminating Effector of the UVB Response Sci. Signal., September 11, 2007; 2007(403): pe49 - pe49. [Abstract] [Full Text] [PDF]
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