Department of Medical Biochemistry and Molecular Biology, University of Saarland Medical Center, D-66421 Homburg, Germany
Submitted 14 July 2003 ; accepted in final form 10 December 2003
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ABSTRACT |
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c-Jun; Egr-1; TrkB
In the basal and granular layers of the epidermis, another tyrosine kinase receptor has been identified, the neurotrophin receptor TrkB. This receptor is also found in hair germ keratinocytes (1). Likewise, the ligands for this receptor, brain-derived neurotrophic factor (BDNF) and neurotrophins 3 and 4, have been detected in mouse skin and cultured keratinocytes (2, 13, 38). The fact that the innervation of skin by sensory and sympathetic neurons is mediated by neurotrophins demonstrates that these regulatory molecules directly act on cells of the skin (3). Therefore, the neurotrophins have been called "epitheliotrophins" (2) to indicate that they may play important regulatory roles in the cell biology of the skin.
Recently, we reported that EGF and thrombin function as mitogens in the human cell line HaCaT (22). HaCaT cells are spontaneously immortalized keratinocytes (4) that have been extensively used as an established in vitro model system of keratinocyte cell biology. Many of these studies have analyzed keratinocyte growth and cell death, differentiation, or signal transduction. HaCaT cells are derived from an adult donor and are nontumorigenic in experimental animals (4). We have extended our previous study and analyzed the signaling pathway and the biological consequences of TrkB neurotrophin receptor stimulation in HaCaT cells. We compared the signaling cascades induced by EGF activation of the EGF receptor with that induced by BDNF stimulation of the TrkB receptor. After binding of the cognate ligand, EGF, or BDNF, the intrinsic kinase of the EGF or TrkB neurotrophin receptor is activated and the receptors are tyrosine phosphorylated. The phosphotyrosyl residues are binding sites for adapter proteins that interface with numerous downstream signaling pathways, including activation of the extracellular signal-regulated protein kinase (ERK) pathway, activation of phosphatidylinositol 3-kinase (PI3-kinase), activation of signal transducers and activators of transcription (STAT), and activation of phospholipase C-. It has been suggested that activation of ERK and/or activation of PI3-kinase is essential for controlling cell survival and proliferation of epithelial cells (11, 21, 22, 32).
The results presented in this study show that EGF and BDNF function as potent mitogens for HaCaT keratinocytes, and this activity depends on the activation of ERK. The crucial role of ERK was further demonstrated in HaCaT cells expressing a conditionally active form of A-Raf. Finally, the results show that EGF and BDNF, as well as activation of A-Raf, rapidly induced biosynthesis of the transcription factors Egr-1 and c-Jun.
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MATERIALS AND METHODS |
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Retroviral gene transfer. The retroviral vector pMSCVpac (17) was a kind gift of R. G. Hawley (Sunnybrook Health Science Centre, Toronto, ON, Canada). Plasmid TrkB.TK(+)flag-pEF/BOS (16), encoding FLAG-tagged TrkB receptor, was a kind gift of A. Haapasalo and E. Castrén (A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland). The coding region for the FLAG-tagged TrkB receptor was excised with BamHI and HpaI and cloned into the BglII/HpaI sites of pMSCVpac, generating the retroviral expression plasmid pMSCV-FLAG-TrkB.TK. Plasmid pBabepuro3A-Raf:ER, encoding an activated form of the protein kinase A-Raf as a fusion protein with the hormone binding domain of the murine estrogen receptor (ERTM, estrogen receptor tamoxifen mutant), was kindly provided by M. McMahon (Cancer Research Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA) (29). The packaging cell line Phoenix-Ampho was obtained from G. Nolan (Stanford University, Palo Alto, CA). Cells were transfected with retroviral vectors according to the protocol developed by G. Nolan (www.stanford.edu/group/nolan/NL-helper.html) using the calcium coprecipitation procedure. Viral supernatants were harvested 72 h after transfection, filtered through a 0.45-µm filter, and used to infect HaCaT cells in the presence of 8 µg/ml polybrene at 37°C. After 6 h, the medium was removed, and the cells were supplied with fresh complete medium and cultured for 72 h before addition of selection medium containing 0.6 µg puromycin/ml. Mass pools of stable transfectants were selected and used for all experiments to eliminate the possibility of specific clonal effects.
Proliferation assays. Cells were seeded in 96-well plates at a density of 3 x 104 cells/well and incubated for 24 h. The serum concentration was lowered to 0.05%, and the cells were incubated for another 24 h. The cells were stimulated with BDNF and EGF for 24 or 40 h and incubated with 4-OHT for 48 h. Induction of DNA synthesis was measured by incorporation of the pyrimidine analog 5-bromo-2'-deoxyuridine (BrdU), instead of thymidine, into the DNA of proliferating cells using the cell proliferation ELISA kit (catalog no. 1647229, Roche Diagnostics, Mannheim, Germany). The assay was performed according to the instruction manual with minor modifications. The labeling time with BrdU was 2 h, and incubation with the anti-BrdU peroxidase antibody was 90 min. Peroxidase activity was determined spectrophotometrically as described in the instruction manual. Each experiment was performed in quadruplicate, and the mean ± SD is depicted.
The mitochondrial reduction capacities were determined by quantification of the level of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) reduction to formazan dye crystals (MTT assay). HaCaT cells were plated in quadruplicate in 96-well plates at a density of 3 x 104 cells/well and incubated for 24 h. The serum concentration was reduced to 0.05%. After 24 h, cells were stimulated with BDNF, EGF, 4-OHT, or vehicle for 48 h. MTT solution (0.5 mg/ml final concentration/well, dissolved in PBS) was added to the cultures, which were then incubated for 4 h at 37°C in 5% CO2. Crystals were solubilized in 10 mM HCl containing 10% SDS, and the plates were incubated overnight at 37°C. Absorbance was quantified on a microplate reader (model 550, Bio-Rad) using a test wavelength of 595 nm. MTT reduction was expressed as a percentage of controls. All experiments represent at least two independent replications performed in quadruplicate.
Transient transfections and reporter gene assays. Plasmids pColl(-517/+63)luc and pEBS14luc have been described elsewhere (6, 43). Plasmid pColl(-517/+63)luc contains the human collagenase regulatory sequence from -517 to +63 upstream of the luciferase open reading frame. The minimal Egr-1-responsive reporter plasmid pEBS14luc contains four binding sites for Egr-1 derived from the Egr-1 promoter upstream of a minimal promoter consisting of the human immunodeficiency virus TATA box and the adenovirus major late promoter initiator element. The expression vector of murine Egr-1, pCMVEgr-1, formerly termed pCMVzif, has been described elsewhere (44).
HaCaT cells were seeded at a density of 5 x 105 cells/plate onto 35-mm plates. Cells were transfected using FuGENE 6 (Roche Molecular Biochemicals) according to the manufacturer's protocol. Fu-GENE 6 was diluted with DMEM and mixed with DNA in the ratio 1 µg of DNA to 3 µl of FuGENE 6. The mixture was incubated for 45 min at room temperature and then added to the cells and incubated for 24 or 48 h in medium containing 10% serum. The serum concentration was lowered to 0.05%, and the cells were incubated for a further 24 h. Cells were stimulated with EGF and BDNF for 6 h and with 4-OHT for 16 h. Transfection experiments involving expression vectors encoding Egr-1 or C2/c-Jun were performed in the presence of pRSV (0.8 µg/plate) to correct for variations in the transfection efficiencies. Cell extracts were prepared and reporter gene activity was determined as described elsewhere (43), except chlorophenol red-
-D-galactopyranoside (Roche Molecular Biochemicals) was used as a substrate for
-galactosidase.
Preparation of cell extracts. Whole cell extracts, nuclear extracts, and crude membranes were prepared as described elsewhere (22).
Antibodies and immunoblot analysis. To detect the phosphorylated form of ERK or c-Akt, 50 µg of proteins derived from whole cell extract preparations were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Blots were probed with an antibody directed against ERK (catalog no. sc-153, Santa Cruz Biotechnology, Heidelberg, Germany), the phosphorylated form of ERK (catalog no. V8031, Promega), or a mixture of two antibodies directed against the phosphorylated residues Ser473 and Thr308 of c-Akt (catalog nos. 9271 and 9275, New England Biolabs). To analyze Egr-1 synthesis, 20 µg of nuclear proteins were separated by a 10% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The blots were incubated with antibodies directed against human Egr-1 (catalog no. sc-110, Santa Cruz Biotechnology) or human c-Jun (catalog no. sc-1694, Santa Cruz Biotechnology). To analyze TrkB receptor expression, 10 µg of proteins from a crude membrane preparation were separated by a 7.5% SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The blot was incubated with M2 monoclonal antibody (catalog no. F3165, Sigma) directed against the FLAG epitope present at the NH2 terminus of the expressed TrkB receptor. Blots were developed using horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (catalog nos. 111-035-003 and 115-035-003, Dianova, Hamburg, Germany) and enhanced chemiluminescence (Amersham, Freiburg, Germany).
Statistical analysis. P values were determined using one-way ANOVA.
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RESULTS |
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BDNF and EGF activate phosphorylation of ERK and c-Akt in HaCaT-TrkB and HaCaTpac cells. Activation of the EGF receptor triggers the phosphorylation and activation of the protein kinases ERK1 and ERK2 in HaCaT cells (22). We used an antibody directed against the phosphorylated p42 isoform of ERK, termed ERK2, to analyze the effect of BDNF stimulation of HaCaT-TrkB cells on the activation state of ERK. Administration of BDNF induced phosphorylation, i.e., activation of ERK2, in HaCaT-TrkB cells (Fig. 2A). Similarly, EGF induced an activation of ERK in HaCaTpac cells (Fig. 2B, top). BDNF- and EGF-triggered phosphorylation of ERK was completely blocked by preincubation of the cells with PD-98059 (Fig. 2, A and B, right lanes). This compound inhibits phosphorylation of MEK, thus blocking the activation of ERK (10). No activation of ERK was observed in BDNF-stimulated HaCaTpac cells that lacked TrkB receptor expression (Fig. 2B, bottom). Activation of ERK by BDNF or EGF was robust but transient (Fig. 2C). Both ligands stimulated a rapid phosphorylation of ERK. Within 15 min after stimulation, phosphorylated ERK could be detected. In BDNF-treated HaCaT-TrkB cells, ERK remained phosphorylated for 1 h. In EGF-treated HaCaTpac cells, ERK was already dephosphorylated and inactivated 1 h after stimulation.
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The PI3-kinase pathway can be directly activated by recruitment to tyrosine-phosphorylated receptors or, indirectly, through an activated Ras. Active PI3-kinase catalyzes the synthesis of 3'-phosphorylated inositol lipids, which control the intracellular localization and activity of a key molecule of PI3-kinase signaling, the protein kinase c-Akt. Phosphorylation of c-Akt serves as an indicator for a previous PI3-kinase activation. Incubation of HaCaT-TrkB cells with BDNF or EGF induced the phosphorylation and activation of c-Akt, as detected with phosphospecific antibodies (Fig. 2D, left lanes). BDNF- and EGF-triggered c-Akt phosphorylation was prevented by preincubation of the cells with the PI3-kinase inhibitor wortmannin (Fig. 2D, right lanes). Wortmannin also caused a slight reduction of EGF- or BDNF-triggered ERK activation (Fig. 2E). Taken together, activation of the EGF or BDNF receptor signaling pathways leads to the phosphorylation and activation of the protein kinases ERK and c-Akt.
BDNF and EGF stimulate biosynthesis of the transcription factors Egr-1 and c-Jun in HaCaT-TrkB and HaCaTpac cells. To follow the BDNF and EGF signaling cascade in HaCaT-TrkB and HaCaTpac cells, we analyzed the expression of the transcription factors Egr-1 and c-Jun. Transcription of the Egr-1 gene has been shown to be induced by stimulation with EGF or neurotrophins (14, 22, 30). Similarly, c-Jun gene expression has been reported to be controlled by growth factors (15, 28). To test the effects of BDNF and EGF on Egr-1 and c-Jun biosynthesis, HaCaT-TrkB and HaCaTpac cells were serum starved for 24 h and then incubated with BDNF or EGF for 15 min, 1 h, 4 h, or 8 h. The cells were harvested, and nuclear extracts were prepared and analyzed by Western blotting using antibodies directed against Egr-1 or c-Jun. BDNF (Fig. 3A) and EGF (Fig. 3B) strikingly increased the biosynthesis of Egr-1, with a peak of expression at 1 h after stimulation. An enhancement of c-Jun biosynthesis was also observed, with high levels of c-Jun immunoreactivity detectable 1 h after stimulation. However, the induction was not as strong as that observed for Egr-1 and lasted longer. The results show that stimulation of HaCaTpac cells with EGF or of HaCaT-TrkB cells with BDNF induced Egr-1 biosynthesis with very similar kinetics. Likewise, c-Jun synthesis was activated very similarly by EGF or BDNF in HaCaTpac and HaCaT-TrkB cells, respectively. Thus, in terms of phosphorylation and activation of ERK and stimulation of Egr-1 and c-Jun biosynthesis, the signaling cascades initiated by the BDNF/TrkB or EGF/EGF receptor system are largely comparable.
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EGF and BDNF increase the transcriptional activation potential of Egr-1 and c-Jun in HaCaT cells. The ability of Egr-1 to activate transcription depends on the concentrations of the Egr-1 negative cofactors NAB1 and NAB2. These proteins bind to Egr-1 and block transcriptional activation via Egr-1 (36, 41, 43). Thus elevated Egr-1 protein levels do not automatically indicate an increased transcription of Egr-1 target genes. Using an Egr-1-responsive reporter, we determined the activation potential of Egr-1 in HaCaT cells. The plasmid pEBS14luc contained luciferase as the reporter gene. Immediately upstream of the TATA box, four binding sites for Egr-1 derived from the Egr-1 promoter were present as a "minimal promoter" (Fig. 4A). The responsiveness of the reporter plasmid was shown in a transfection experiment using overexpressed Egr-1 (Fig. 4B). Next, we tested whether EGF or BDNF induces reporter gene transcription. The reporter plasmid pEBS14luc was transfected into HaCaT cells, and cells were maintained in complete medium for 48 h and in serum-reduced medium for 24 h. Cells were stimulated with EGF or BDNF for 6 h, and cellular extracts were prepared and analyzed for luciferase activity. Figure 4C shows that EGF and BDNF significantly stimulated reporter gene transcription, indicating that biologically active Egr-1 had been synthesized in the cells. The collagenase promoter has frequently been used to monitor c-Jun activity, because this promoter contains the classic 12-O-tetradecanoylphorbol-13-acetate response element, which functions as a binding site for c-Jun homodimers and heterodimers. The collagenase reporter plasmid is depicted in Fig. 4D. Figure 4E shows that transcription of the collagenase promoter/luciferase reporter gene was strongly induced in HaCaT cells after expression of a constitutively active c-Jun mutant containing the activation domain of cAMP response element-binding protein (CREB2) fused to the DNA-binding and dimerization domain of c-Jun. Similarly, stimulation of the cells with EGF or BDNF elevated collagenase promoter activity, indicating that transcriptionally active c-Jun had been synthesized in the cells (Fig. 4F).
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BDNF and EGF induce proliferation of HaCaT cells expressing their cognate receptors. The fact that the signaling pathway initiated via the neurotrophin BDNF/TrkB system is very similar to that induced by EGF led us to analyze the mitogenic activity of BDNF in HaCaT-TrkB cells. As a molecular indicator for proliferation, DNA synthesis was measured by incorporation of BrdU into the DNA. Incorporation of BrdU was used as a measure of DNA replication and served as an indicator of cellular activity in the S phase of the cell cycle. HaCaT-TrkB cells were serum starved for 24 h and then treated with BDNF or EGF for 24 h. Figure 5A, left, shows that EGF and BDNF induced a significant increase in BrdU incorporation in HaCaT-TrkB cells, indicating that EGF and BDNF function as mitogens for these cells. In contrast, BDNF did not show any effect on cell proliferation in HaCaTpac cells that did not express TrkB neurotrophin receptors (Fig. 5B, left). We repeated these experiments using the reduction of tetrazolium salts by mitochondrial NAD(P)H-dependent dehydrogenases to formazan (MTT assay). Here, the overall metabolic activity of the cells is measured and used as an indirect indicator of the viable cell number. Previously, neurotrophin-induced cell growth had been measured with the MTT assay (18). HaCaT-TrkB cells were serum starved for 24 h and then treated with BDNF or EGF for 48 h, and cell growth was measured by the MTT assay. The results of the MTT assay are compared with the amount of formazan formed in the absence of EGF or BDNF. Figure 5A, right, shows that EGF and BDNF induced a significant increase in the mitochondrial reduction capacities. BDNF did not show any effect in TrkB-lacking HaCaTpac cells (Fig. 5B, right). EGF, however, was a potent mitogen for HaCaT-TrkB and HaCaTpac cells, both expressing functional EGF receptors.
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To confirm the role of ERK or PI3-kinase in EGF- and BDNF-induced cell proliferation, we preincubated HaCaT-TrkB cells with the MEK inhibitor PD-98059 or the PI3-kinase inhibitor wortmannin before stimulating the cells with EGF or BDNF. DNA synthesis was measured by incorporation of BrdU into DNA 24 or 40 h after stimulation. Figure 6, A and B, middle, shows that PD-98059 efficiently blocked BrdU incorporation into DNA in HaCaT-TrkB cells that had been stimulated with EGF or BDNF. PD-98059 did not significantly affect cell viability (data not shown), in agreement with published data (20). The PI3-kinase inhibitor wortmannin delayed the DNA synthesis of EGF- or BDNF-stimulated HaCaT-TrkB cells (Fig. 6, A and B, right) but was unable to finally block the proliferation of the cells. These data were confirmed using the MTT assay (Fig. 6C). Taken together, these results indicate that the mitogenic activity of EGF and BDNF is mediated by an activated ERK, and not by activation of the PI3-kinase pathway.
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Conditional activation of the ERK signaling pathway in human HaCaT keratinocytes by expression of a A-Raf:ER fusion protein. In the previously described experiments, we distinguished between the receptor tyrosine kinase-induced activation of ERK or PI3-kinase pathway using pharmacological inhibitors. To specifically activate the ERK pathway in HaCaT cells and to confirm the importance of the ERK signaling pathway for HaCaT cell proliferation, we generated HaCaT cells expressing a
A-Raf:ER fusion protein. The modular structure of the Raf protein kinase is depicted in Fig. 7A. The A-Raf protein kinase contains three domains: CR1, CR2, and CR3. CR1 is a cysteine-rich region and functions as a binding site for activated Ras-GTP at the cell membrane. CR2 is rich in serine and threonine residues and negatively regulates the biological activity of the catalytic domain, perhaps by direct protein-protein interaction with the kinase domain. CR3 encompasses the protein kinase domain. Expression of this catalytic domain of A-Raf as a fusion protein with the ligand binding domain of the murine ER keeps the protein kinase in an inactive state in the absence of hormone but allows induction of the Raf-1 protein kinase by the addition of hormone (31). The use of the ER mutant ERTM allowed us to utilize the synthetic ligand 4-OHT for induction. The encoded ligand binding domain of the ER contained a glycine residue at position 525, instead of an arginine. As a result, the receptor is largely insensitive to 17
-estradiol but can be readily activated by 4-OHT (26).
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To study the signaling induced by A-Raf:ER, we incubated HaCaT-
A-Raf:ER cells with 4-OHT. Whole cell extracts were prepared and analyzed for phosphorylated, i.e., activated, ERK using a phosphospecific antibody. Figure 7B shows that ERK is phosphorylated in these cells after stimulation with 4-OHT. However, the kinetics are very different from the kinetics of HaCaT-TrkB cells stimulated with BDNF or HaCaTpac cells treated with EGF (cf. Fig. 7B with Fig. 2C). Whereas BDNF and EGF induced a strong phosphorylation of ERK in HaCaT-TrkB and HaCaTpac cells, respectively, within 1 h after stimulation, phosphorylated ERK was barely detected in HaCaT-
A-Raf:ER cells incubated for 1 h with 4-OHT. Phosphorylated ERK was, however, detected in these cells 4 and 8 h after induction of A-Raf. Despite the moderate activation of ERK in HaCaT-
A-Raf:ER cells, stimulation of Egr-1 biosynthesis occurred 1 h after treatment of the cells with 4-OHT (Fig. 7C). In contrast to the robust but transient synthesis of Egr-1 in EGF- or BDNF-treated HaCaTpac or HaCaT-TrkB cells, a sustained synthesis of Egr-1 was detected in HaCaT-
A-Raf:ER cells incubated with 4-OHT (Fig. 7C) that lasted for
24 h (data not shown). Induction of c-Jun biosynthesis was delayed in HaCaT-
A-Raf:ER cells treated with 4-OHT compared with growth factor-stimulated HaCaT-TrkB or HaCaTpac cells. Higher concentrations of c-Jun were detected 48 h after stimulation. In contrast, EGF and BDNF induced c-Jun biosynthesis, with highest amounts of c-Jun measured 14 h after stimulation. Transfection experiments of the Egr-1 or c-Jun responsive reporter plasmids pEBS14luc or pColl(-517/+63)luc revealed that the transactivation potentials of Egr-1 and c-Jun were elevated in 4-OHT-treated HaCaT-
A-Raf:ER cells (Fig. 7, D and E), indicating that biologically active Egr-1 and c-Jun had been synthesized in the cells. Taken together, activation of the ERK signaling pathway in HaCaT cells by activation of the
A-Raf:ER fusion protein induced a sustained phosphorylation of ERK and a delayed but long-lasting synthesis of biologically active Egr-1 and c-Jun that differs very clearly from the robust and transient activation of ERK and Egr-1/c-Jun biosynthesis triggered by EGF or BDNF.
Activation of ERK is essential for cell proliferation conferred by A-Raf:ER. HaCaT-
A-Raf:ER cells were serum starved for 24 h and then incubated with 4-OHT for 48 h as described previously (23). Figure 8A, left, shows that activation of the catalytic function of A-Raf induced DNA synthesis in HaCaT-
A-Raf:ER cells. In contrast, 4-OHT was without effect on BrdU incorporation in HaCaTpac cells, which express puromycin acetyltransferase, instead of the conditionally activated form of A-Raf (Fig. 8B, left). We confirmed these data with the MTT assay as a measure for the number of viable cells. HaCaT-
A-Raf:ER cells were starved for 24 h and then incubated with 4-OHT for 48 h. The mitochondrial reduction capacities were determined and are depicted in comparison with untreated cells. Figure 8, A and B, right, shows that activation of the catalytic function of A-Raf increased the mitochondrial reduction capacities of HaCaT-
A-Raf:ER, but not HaCaTpac, cells.
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The relevance of ERK activation for A-Raf:ER-mediated proliferation was studied with the MEK inhibitor PD-98059. The cells were preincubated with PD-98059 and then incubated with 4-OHT for 48 h. DNA synthesis was measured by incorporation of BrdU into DNA. Figure 8C, left, shows that PD-98059 efficiently blocked BrdU incorporation into DNA of HaCaT-
A-Raf:ER cells that had been stimulated with 4-OHT. Moreover, the increase of the number of viable cells by activation of
A-Raf:ER was also dependent on the ERK signaling pathway, as shown by the lack of stimulation by 4-OHT in the presence of the MEK inhibitor PD-98059 (Fig. 8C, right). In contrast, we were unable to impair proliferation with the PI3-kinase inhibitor wortmannin (Fig. 8D). These data indicate that the mitogenic activity of
A-Raf:ER is mediated by the sustained activation of ERK.
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DISCUSSION |
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The generation of human HaCaT keratinocytes expressing the TrkB neurotrophin receptor is reported here. We wanted to analyze the effect of BDNF in a keratinocyte cell line that does not express the cognate TrkB neurotrophin receptor. Similar experiments have been done with fibroblasts expressing the TrkA or TrkB neurotrophin receptor. These fibroblasts, engineered to express the neurotrophin receptors TrkA or TrkB, proliferated as a result of stimulation with their cognate ligands BDNF and nerve growth factor (NGF) (12). However, a growth-inhibitory activity of NGF on TrkA-expressing NIH 3T3 fibroblasts was also described (9). Our results show that BDNF functions as a mitogen for TrkB-expressing HaCaT keratinocytes. Thus HaCaT-TrkB cells show a very similar physiological response to BDNF compared with primary keratinocytes. This indicates that we have generated a cellular model that may be very valuable as an easily accessible system to study the cell biology of BDNF in a keratinocyte environment. The creation and validation of this model system represent the second objective of our work. HaCaT cells have been described to exhibit deficiencies in organotypic cocultures with fibroblasts, representing an in vitro skin equivalent model, although essentially all epidermal differentiation markers are expressed. A recent study showed that supplementation of HaCaT cells with transforming growth factor- (TGF-
), an EGF receptor ligand, restored the capacity of the cells to form structured epithelia in organotypic cocultures (27). TGF-
enhanced expression of interleukin-1 and the receptors for keratinocyte growth factor and granulocyte macrophage-colony-stimulating growth factor in HaCaT cells, thereby restoring the delayed and deficient growth and differentiation capacities of the cells. Epidermal tissue differentiation of TGF-
-stimulated HaCaT cells was shown to be comparable to cultures of normal human skin keratinocytes (27), indicating that TGF-
-stimulated HaCaT cells are a regular skin equivalent that may be very useful as a highly standardized in vitro tissue model.
The signaling pathways and the biological effects induced by BDNF in HaCaT-TrkB cells or by EGF in HaCaT or HaCaTpac cells were shown to be very similar, indicating that activation of the TrkB or the EGF receptor tyrosine kinase function is connected with the same or a very similar signaling cascade in keratinocytes. The fact that supplementation of HaCaT cells with EGF also normalized regeneration of epidermal tissue differentiation suggests that BDNF stimulation of HaCaT-TrkB cells may also be sufficient to restore the capacity of the cells to form structured epithelia in organotypic cocultures. Activation of the ERK signaling pathway by Ras and Raf and activation of PI3-kinase have been described to be essential for the induction of cell growth of epithelial cells. Here, we have shown that the activation of ERK is crucial for EGF- and BDNF-mediated the induction of HaCaT keratinocyte proliferation. These assumptions are based on experiments performed with the MEK inhibitor PD-98059, which prevents activation of this kinase by the "upstream" protein kinase Raf. PD-98059 has a very impressive selectivity profile, as demonstrated by the fact that no other protein kinase was inhibited by this compound when used at a concentration of 50 µM (8). PD-98059 inhibited EGF- or BDNF-induced cell proliferation in HaCaT-TrkB cells, indicating that activation of ERK is required for the growth-promoting activity of both ligands.
The biological role of the ERK signaling pathway in the control of keratinocyte proliferation was further investigated using a conditionally active form of A-Raf. Analysis of the signaling cascade induced by activation of A-Raf showed an activation of ERK. The kinetics of ERK phosphorylation were, however, quite distinct from that observed in BDNF- or EGF-treated HaCaT-TrkB or HaCaTpac cells. While the naturally occurring ligands triggered a robust, but transient, activation of ERK, we observed a sustained phosphorylation of ERK in A-Raf:ER-expressing HaCaT cells. Despite those differences, growth factor stimulation of HaCaT-TrkB cells and 4-OHT stimulation of HaCaT-
A-Raf:ER cells induced cellular growth. These results further indicate that the kinetics of ERK activation (transient vs. sustained) are of less importance for the induction of the mitogenic program of keratinocytes. This observation is in contrast to the role of ERK in neuronal survival, where a sustained activation of ERK is required for neuroprotection (35). Similarly, neuronal differentiation of PC12 cells has been connected with a sustained activation of ERK (46).
Activation of PI3-kinase has been linked to many key cellular functions in mammalian cells, including cell survival and cell proliferation. In Mv1Lu mink lung epithelial cells, for example, PI3-kinase activity has been shown to be essential for hepatocyte growth factor-induced mitogenic signals (32). Likewise, insulin-like growth factor I-induced DNA synthesis and cell division of human breast cancer cells were blocked by the PI3-kinase inhibitor LY-294002, but not by PD-98059, indicating that mitogenic signaling of these cells requires PI3-kinase and is independent of ERK (11). The results described in this study reveal that EGF-, BDNF-, and A-Raf-induced cell growth uses the ERK signaling pathway, independent of PI3-kinase activation. The PI3-kinase inhibitor wortmannin delayed the mitogenic response to EGF or BDNF, but 40 h after stimulation with the growth factors, similar proliferation rates were measured in the presence or absence of wortmannin. The delay of EGF- or BDNF-induced DNA synthesis observed after treatment of the cells with wortmannin may be due to the slight reduction of ERK activation. Thus wortmannin did not block EGF- or BDNF-induced activation of ERK but reduced the levels of active ERK (Fig. 2E). Similar observations have been reported for CCl39 fibroblasts and COS cells (5, 45). How wortmannin reduces ERK activation is not yet clear, but the fact that strong elevation of PI3-kinase did not activate ERK in COS cells indicates that PI3-kinase does not have an upstream regulatory role in the Raf-MEK-ERK signaling pathway (45). PI3-kinase may have a major role as a key regulator of early-phase differentiation of keratinocytes, as shown by the fact that blockage of PI3-kinase triggered differentiation, whereas activation of PI3-kinase prevented it (37). Thus EGF- and BDNF-activated PI3-kinase may support the mitogenic program by preventing the differentiation of the cells.
Induction of proliferation by extracellular signaling molecules involves the activation of gene transcription. Here, we have analyzed the EGF-, BDNF-, or A-Raf-induced biosynthesis of the transcription factors Egr-1 and c-Jun. Similar to the transient or sustained activation of ERK, we observed transient stimulation of Egr-1 biosynthesis by EGF or BDNF in HaCaTpac or HaCaT-TrkB cells, whereas a sustained synthesis of Egr-1 was detected in 4-OHT-treated HaCaT-A-Raf:ER cells. Nevertheless, the biological consequences of transient vs. sustained synthesis of Egr-1 were identical: the proliferation of HaCaT cells. Since the discovery of the Egr-1 gene as an "early growth response gene," research has been directed toward elucidating the function of Egr-1 in growth and proliferation. Induction of Egr-1 gene transcription was monitored in many cell types in response to mitogens (42), and a direct role of Egr-1 during multistage carcinogenesis in the skin has been proposed (33). The fact that genes encoding growth factors, such as insulin-like growth factor II, platelet-derived growth factors A and B, and TGF-
1, have been identified as target genes of Egr-1 (24, 25, 40) indicates that Egr-1 may prolong the mitogenic signaling cascade by stimulation of growth factor synthesis. The proposed role for Egr-1 in controlling cell growth is, however, largely based on the correlation between mitogenic response and Egr-1 biosynthesis. Gain-of-function and loss-of-function experiments are required to decipher the exact role of Egr-1 in keratinocyte growth control. Moreover, the identification of Egr-1 target genes in keratinocytes should provide clues about how Egr-1 is performing its biological function.
The basic region leucine zipper protein c-Jun, one of the proteins that constitute the activator protein AP-1 transcription factor complex, plays an essential role in many cell types in controlling cell growth, survival, or death (19). The biological activity of c-Jun is regulated on several levels, including transcription of the c-jun gene, the turnover rate of the mRNA and protein, and posttranslational modifications, as well as the interaction and dimerization with other basic region leucine zipper proteins. Here, we have shown that the c-Jun concentration is increased as a result of BDNF or EGF stimulation or activation of the A-Raf protein kinase. The growth factor-mediated increase in the c-Jun concentration is more moderate than the striking enhancement of Egr-1. However, the elevated c-Jun concentration is persistent compared with the short and transient synthesis of Egr-1. Gain-of-function and loss-of-function experiments may clarify whether BDNF, EGF, or A-Raf requires c-Jun as a positive regulator of proliferation in HaCaT cells.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 530.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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