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HB-EGF-induced VEGF production and eNOS activation depend on both PI3 kinase and MAP kinase in HaCaT cells
Journal of Dermatological Science 55 (2009) 170–178
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HB-EGF-induced VEGF production and eNOS activation depend on both PI3 kinase and MAP kinase in HaCaT cells Kozo Nakai a,*, Kozo Yoneda a, Tetsuya Moriue a, Junske Igarashi b, Hiroaki Kosaka b, Yasuo Kubota a a b
Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2009 Received in revised form 21 May 2009 Accepted 2 June 2009
Background: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of growth factors that have been implicated in skin patho-physiology. Although endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) appear to be involved in mitogenesis and chemotaxis in epidermal keratinocytes, the activation of eNOS and VEGF production induced by HB-EGF and its signaling mechanism remains undefined. Objective: We examined possible signal transduction pathways by which HB-EGF leads to eNOS activation and VEGF production in human epidermal keratinocyte cell line (HaCaT cells). Methods: The phosphorylation of epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK; p42/p44 MAPK), Akt and eNOS were examined by Western blotting analysis. VEGF production was determined by enzyme-linked immunosorbent assay. Various inhibitors were utilized to investigate the signaling mechanisms of eNOS activation and VEGF production. Results: HB-EGF-induced phosphorylation of EGFR with maximum phosphorylation at 1 h. HB-EGFinduced phosphorylation of p42/p44 MAPK in a few minutes. It activated Akt with maximum phosphorylation at 1 h and eNOS with maximum phosphorylation at 3 h. The HB-EGF-induced eNOS activation was significantly blocked by the p42/p44 MAPK inhibitor U0126 and the phosphatidylinositol 3-kinase (P13K) inhibitor LY294002. HB-EGF increased VEGF production. The HB-EGF-induced VEGF production was blocked by U0126 and LY294002. Finally, the HB-EGF-induced activation of Akt and eNOS was suppressed by VEGF competitive antagonist, CBO-P11. Conclusion: These results demonstrate that HB-EGF-induced eNOS activation depends on p42/p44 MAPK, PI3K/Akt pathways and endogenous VEGF in HaCaT cells. ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: HB-EGF VEGF eNOS HaCaT cell
1. Introduction The study of proliferation and migration of epidermal keratinocytes are focused in the pathogenesis of cutaneous wounds and various skin diseases such as psoriasis. Several mitogens have been implicated in these processes, one of which is heparin-binding epidermal growth factor-like growth factor (HBEGF). Studies have shown that HB-EGF is overexpressed in psoriatic lesional skin and that it is a potent mitogenic and chemotactic factor for epidermal keratinocytes [1,2], and participates in the epidermal response to cutaneous wounds [3]. The biological actions of HB-EGF are mediated through some members of the epidermal growth factor receptor (EGFR) superfamily, also known as erbB1, and erbB4 [4]. On activation, these receptors undergo homo- or heterodimerization, followed by activation of intrinsic tyrosine kinase activity, leading to a myriad of signaling
* Corresponding author. Tel.: +81 87 891 2162; fax: +81 87 891 2163. E-mail address: [email protected] (K. Nakai).
events. To this end, a variety of cellular responses occur, such as cell proliferation, migration, and differentiation. The mitogen-activated protein kinases (MAPKs) are a welldocumented family of serine/threonine kinases that include p42/ p44 MAPK, p38 MAPK and c-Jun N-terminal kinase. p42/p44 MAPK are the most characterized, and mediate proliferative and chemotactic responses in various cells, including epidermal keratinocytes [5]. Phosphoinositide 30 -OH kinase (PI3K) is a lipid kinase that phosphorylates the 3-hydroxyl of the head group of phosphatidylinositol, thereby activating several downstream protein kinases, such as Akt [6,7]. Akt is also involved in cell growth by eliciting cell survival/antiapoptotic effects [8,9]. Although the direct effects of MAPK and PI3K on cell growth and migration have been well reported, these kinases also influence endothelial nitric oxide synthase (eNOS) activation [10] and vascular endothelial growth factor (VEGF) production [11], which might partly relate to cell growth and migration, especially in vascular endothelial and smooth muscle cells. It has also been reported that eNOS is a key regulator of the proliferation of epidermal keratinocytes [12]. There is strong evidence supporting
0923-1811/$36.00 ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2009.06.002
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PI3K activation of Akt, which in turn is responsible for regulating the phosphorylation and activation of eNOS [13–15]. However, it is becoming increasingly clear that Akt is not the only protein kinase that can phosphorylate eNOS at the Ser1177 residue. Other protein kinases, including protein kinase A, protein kinase G, and AMP kinase have also been shown to phosphorylate eNOS at the Ser1177 residue [16–18]. Thus, it may be possible that eNOS at the Ser1177 residue can be phosphorylated by different protein kinases depending upon cell types and each given stimuli. The derangement of VEGF production by epidermal keratinocytes concerns various skin diseases including carcinogenesis and psoriasis [19], as well as wound healing [20]. In addition, the expression of VEGF receptor (VEGFR) and the proliferation and migration of epidermal keratinocytes by VEGF have been reported [21,22]. EGFR stimulation by EGF has been shown to increase VEGF production [23]. However, there are no reports on whether or not HB-EGF increases VEGF production, and the related signaling mechanisms have not been defined in human epidermal keratinocytes. In the present study, by using human epidermal keratinocyte cell line, HaCaT cells, we examined the signaling mechanisms required for HB-EGF-induced eNOS activation and
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VEGF production that are most likely related to various cellular functions of epidermal keratinocytes. 2. Materials and methods 2.1. Reagents Dulbecco’s modified Eagle’s medium (DMEM), HB-EGF and LNAME were obtained from Sigma (St. Louis, MO, USA). AG1478, LY294002, U0126 and VEGF competitive antagonist (CBO-P11) were purchased from Calbiochem-Novabiochem (San Diego, CA, USA). 4,5-Diaminofluorescein diacetate (DAF-2/DA) was purchased from Daiichi Pure Chemicals Co., Tokyo, Japan. eNOS antibody and activated EGFR antibody were obtained from BD Transduction Laboratories (Lexington, KY, USA). The antibodies against EGFR, p42/p44, phospho-p42/p44, Akt, phospho-AktSer473 and phospho-eNOSSer1177 were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.2. Cell culture HaCaT cells (a generous gift from Professor N.E. Fusenig, German Cancer Research Center, Germany) were cultured in
Fig. 1. HB-EGF increased NO production and promoted cell proliferation in HaCaT cells. (a) After HB-EGF (50 ng/ml) treatment for 5 h, HaCaT cells were stained with DAF-2DA for 15–30 min. The intensity of fluorescence was measured by a fluorescence microplate reader. (b) HaCaT cells were treated with HB-EGF (50 ng/ ml) for 12 h. Cell number was assessed using Cell Counting Kit-8. The results of relative levels of OD from pooled data. Values represent means S.E. (n = 6). **P < 0.005; ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HBEGF-treated group.
Fig. 2. EGFR activation, eNOS phosphorylation and related signaling pathways in HB-EGF-treated HaCaT cells. HaCaT cells were treated with HB-EGF (50 ng/ml) and lysed at specific time points for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (c) EGFR antibody or activated EGFR antibody, (d) p42/p44 antibody or phospho-p42/p44 antibody, (e) Akt antibody or phospho-AktSer473 antibody, and (f) eNOS antibody or phospho-eNOSSer1177 antibody. Similar results were obtained for more than three independent experiments. (a, b) eNOS expression in skin and HaCaT cells, and the HB-EGF-induced eNOS phosphorylation in HaCaT cells. Human umbilical vein endothelial cells and bovine aortic endothelial cells were used as positive control.
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DMEM with 10% heat-inactivated fetal calf serum (FCS) under 37 8C in a humidified atmosphere of 5% CO2 and 95% air [24]. On the treatment day, the medium was replaced with DMEM containing 1% FCS. Cells were about 50% confluent and treated with or without HB-EGF and inhibitors at the same time. 2.3. Measurement of intracellular NO production Production of intracellular NO was determined using DAF-2/ DA. DAF-2/DA is a cell-permeable compound that is converted to DAF-2 by intracellular esterases in the presence of NO. A triazole derivative of DAF-2 emits light; the intensity of this light is proportional to the amount of NO present. The fluorescence was detected using a fluorescence microplate reader, Fluoroskan Ascent FL1 (Thermo Fisher Scientific, Waltham, MA, USA), at excitation and emission wavelengths of 485 nm and 538 nm.
2.4. Cell proliferation assay Cell proliferation was assayed with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), in which WST-8 (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) is converted to formazan by cellular dehydrogenase. The absorbance of formazan, which is proportional to cell number, was determined at 450 nm. 2.5. Measurement of VEGF secretion Cell-free supernatants were collected and stored at 70 8C until required for assay. Secreted VEGF levels in the culture media of HaCaT cells were determined by sandwich ELISA by using a commercially available ELISA human VEGF kit (Quantikine Human VEGF Immunoassay, R&D Systems, Minneapolis, MN, USA). According to the manufacture’s protocol, absorption of avidin-
Fig. 3. AG1478 inhibited the HB-EGF-induced activation of EGFR, p42/p44 MAPK, Akt and eNOS. HaCaT cells were treated with HB-EGF (50 ng/ml) plus AG1478 (10 mM) and lysed for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (a) EGFR antibody or activated EGFR antibody, (b) p42/p44 antibody or phospho-p42/p44 antibody, (c) Akt antibody or phospho-AktSer473 antibody, and (d) eNOS antibody or phospho-eNOSSer1177 antibody. (Upper) Shown are the results from representative data. (Lower) The results of densitometric analysis from pooled data. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
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horseradish peroxidase color reaction was measured at 405 nm and VEGF concentrations were determined using a standard graph prepared using serial dilutions of human recombinant VEGF as a standard.
densitometry analysis using Image J software (National Institutes of Health, Bethesda, MD, USA).
2.6. Western blotting analysis
Data were given as mean S.E. A statistical analysis was performed by an analysis of variance followed by the Fisher’s protected least significant difference. P-values <0.05 were considered statistically significant.
Cells were washed with PBS. Then, lithium dodecyl sulfate sample buffer was added. An equal amount of protein was separated on 7% Tris–Acetate gel (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. The membrane was then probed with antibodies against EGFR, activated EGFR, p42/ p44, phospho-p42/p44, Akt, phospho-AktSer473, eNOS and phospho-eNOSSer1177. Membrane-bound primary antibodies were visualized by using appropriate secondary antibodies conjugated with horseradish peroxidase and chemiluminescent substrate (Pierce, Rockford, IL, USA). The images were subjected to
2.7. Statistical analysis
3. Results 3.1. HB-EGF increased NO production and promoted cell proliferation in HaCaT cells First, we examined whether HB-EGF increases NO production in HaCaT cells. By using DAF-2/DA, we have confirmed that NO
Fig. 4. The PI3K pathway and MAPK pathway involve HB-EGF-induced eNOS activations in HaCaT cells. HaCaT cells were treated with HB-EGF (50 ng/ml) plus U0126 (10 mM) or LY294002 (10 mM) and lysed for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (a) p42/p44 antibody or phospho-p42/p44 antibody, (b) Akt antibody or phospho-AktSer473 antibody, and (c) eNOS antibody or phospho-eNOSSer1177 antibody. (Upper) Shown are the results from representative data. (Lower) The results of densitometric analysis from pooled data. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
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production is slightly, but significantly, increased in HB-EGFtreated HaCaT cells at 5 h (Fig. 1a). The increase in NO production induced by HB-EGF was suppressed by L-NAME (1 mM), a NOS inhibitor. As HB-EGF increases NO production in human vascular endothelial cells and promotes angiogenesis [25], we investigated whether NO is necessary for HB-EGFinduced proliferation of human epidermal keratinocyte cell line. As shown in Fig. 1b, HB-EGF (50 ng/ml) increased proliferation of HaCaT cells at 12 h. L-NAME (1 mM) suppressed the HB-EGFinduced proliferation of HaCaT cells. These data suggest that NO production is involved in HB-EGF-induced cell proliferation in HaCaT cells. L-NAME did not inhibit the base line increase in HaCaT cell proliferation. 3.2. Time course dependent activation of EGFR, eNOS and related signaling pathways in HB-EGF-treated HaCaT cells HB-EGF elicits a variety of cellular responses that are initiated by binding to EGFR. Following ligand binding, EGFR is activated and, in turn, phosphorylates and activates several other endogenous proteins including MAPK and PI3K/Akt in keratinocytes. Subsequently, eNOS may be phosphorylated and activated. We have confirmed the eNOS expression in human skin and HaCaT cells (Fig. 2a), and the HB-EGF-induced eNOS phosphorylation in HaCaT cells (Fig. 2b). To gain an insight into the order and mechanisms of the activation of these proteins, we have investigated the time course dependant phosphorylation of these proteins in HaCaT cells. As previously demonstrated, the activation of EGFR increased rapidly 5 min after stimulation and reached a maximum at 30–60 min (Fig. 2c). The phosphorylation of p42/p44 increased rapidly and reached its peak 5 min after stimulation, and gradually decreased (Fig. 2d). Interestingly, the phosphorylation of Akt was slightly increased at 5 min, but strongly increased and reached its peak at 60 min (Fig. 2e). Similarly, the phosphorylation of eNOS was slightly increased at 5 min, but strongly increased and reached its peak at 180 min (Fig. 2f). Thus, transit activation of p42/p44 MAPK, PI3K/Akt and eNOS occurred at 5 min. However, PI3K/Akt and eNOS were activated strongly at 60 min and 180 min, respectively. We decided to investigate the phosphorylation of EGFR at 60 min, p42/p44 at 60 min, Akt at 60 min and eNOS at 180 min. To examine whether the HB-EGF-induced phosphorylation of p42/p44, PI3K/Akt and eNOS was EGFR dependent, we used an inhibitor of EGFR kinase, AG1478 and confirmed its effects on HBEGF-induced phosphorylation of the aforementioned kinases. The maximum phosphorylations of these proteins were all inhibited by AG1478 (10 mM, Fig. 3).
Akt pathway, in HB-EGF-induced activation of eNOS in HaCaT cells. 3.4. HB-EGF-induced VEGF production does not require NO in HaCaT cells EGF increases VEGF production in normal human keratinocytes [23]. As HB-EGF increases NO production in HUVEC [25], and NO stimulates VEGF production in cardiomyocytes [26], there is a possibility that HB-EGF-induced NO could increase VEGF production in HaCaT cells. Thus, we examined secreted VEGF levels in the culture media of HaCaT cells by ELISA method. As shown in Fig. 5a, HB-EGF increased VEGF production in HaCaT cells. However, L-NAME did not affect the HB-EGF-induced VEGF production in HaCaT cells. To determine the signaling pathways underlying VEGF production in response to HB-EGF, AG1478, LY294002 and U0126 were administered (Fig. 5b). HB-EGFinduced VEGF production was completely inhibited by AG1478 (10 mM) or U0126 (10 mM). LY294002 (10 mM) slightly, but significantly, suppressed the HB-EGF-induced VEGF production. These results clearly illustrate that the activation of both PI3K and p42/p44 MAPK are involved in HB-EGF-induced VEGF production in HaCaT cells, but p42/p44 MAPK is involved, more than PI3K.
3.3. The PI3K pathway and MAPK pathway involve HB-EGF-induced eNOS activations in HaCaT cells To determine the mechanisms underlying eNOS activation in response to HB-EGF, we attempted to identify the role of PI3K by using LY294002, a PI3K inhibitor. As p42/p44 MAPK pathway appeared to be activated first following the activation of EGFR, we decided to investigate the effects of U0126, a p42/p44 inhibitor. U0126 (10 mM) suppressed HB-EGF-induced phosphorylation of p42/p44 (Fig. 4a), but it did not suppress phosphorylation of Akt (Fig. 4b). LY294002 (10 mM) did not suppress HB-EGF-induced phosphorylation of p42/p44 (Fig. 4a), but it did suppress phosphorylation of Akt (Fig. 4b). Interestingly, both U0126 and LY294002 inhibited phosphorylation of eNOS (Fig. 4c). These results suggest that the activation of both PI3K/Akt pathway and p42/p44 MAPK pathway are involved, and p42/p44 MAPK pathway did not require PI3K/
Fig. 5. HB-EGF-induced VEGF production does not require NO, but depends on both PI3K pathway and MAPK pathway in HaCaT cells. HaCaT cells were treated with (a) HB-EGF (5–50 ng/ml) plus L-NAME (1 mM), and (b) HB-EGF (50 ng/ml) plus AG1478 (10 mM), U0126 (10 mM) or LY294002 (10 mM). Secreted VEGF levels in the culture media at 5 h of HaCaT cells were determined by sandwich ELISA by using a commercially available ELISA human VEGF kit. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
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Fig. 6. VEGF is required for the HB-EGF-induced activation of eNOS in HaCaT cells. HaCaT cells were treated with HB-EGF (50 ng/ml) plus VEGF inhibitor (20 mM) and lysed for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (a) EGFR antibody or activated EGFR antibody, (b) p42/p44 antibody or phospho-p42/p44 antibody, (c) Akt antibody or phospho-AktSer473 antibody, and (d) eNOS antibody or phosphoeNOSSer1177 antibody. (Upper) Shown are the results from representative data. (Lower) The results of densitometric analysis from pooled data. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
3.5. VEGF is required for the HB-EGF-induced activation of eNOS in HaCaT cells After confirming that HB-EGF increased VEGF production, we investigated whether HB-EGF-induced VEGF is involved in the activation of eNOS in HaCaT cells, since VEGFR is expressed in HaCaT cells [21]. In addition, VEGF phosphorylates the eNOS via Akt pathway in endothelial cells [27,28]. The HB-EGF-induced phosphorylations of Akt and eNOS were significantly suppressed by the VEGF competitive antagonist, CBO-P11 (20 mM) in HaCaT cells. However, the phosphorylation of p42/p44 MAPK was not suppressed (Fig. 6).
4. Discussion In this study, we demonstrated the HB-EGF-induced activation of p42/p44 MAPK, PI3K/Akt and eNOS through the EGFR in HaCaT cells. These activated signaling pathways were completely blocked by an EGFR kinase inhibitor, AG1478. Our data demonstrated that HB-EGF promoted the VEGF production in HaCaT cells. We further showed that MAPK inhibitor and PI3K inhibitor blocked the HBEGF-induced eNOS activation and VEGF production in HaCaT cells. Finally, VEGF inhibitor prevented the HB-EGF-induced Akt and eNOS activation without affecting the activity of EGFR in HaCaT cells.
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Fig. 7. Schematic representation of HB-EGF-induced VEGF production and eNOS activation in HaCaT cells. HB-EGF first activates EGFR, and subsequently activates p42/p44 MAPK and PI3K pathway. There may be a PI3K/Akt/eNOS activation pathway. The activation of p42/p44 MAPK can directly lead to the eNOS activation. The activation of both p42/p44 MAPK pathway and PI3K pathway leads to an increase in VEGF production. Then, VEGF activates eNOS via VEGFR/PI3K/Akt pathway.
In our study, it is suggested that NO is involved in the cell proliferation of HB-EGF-treated HaCaT cells. However, various factors must be involved in HB-EGF-induced proliferation of HaCaT cell. The reason why L-NAME did not inhibit the base line increase in the cell proliferation may be because cell proliferation did not totally depend on NO. As shown in Fig. 1a, the slight levels of NO production were observed in untreated HaCaT cells. Presumably, this NO production may involve the effects of internal EGF family. However, the levels of this constitutive NO production may be too low to affect cell proliferation. Thus, it is suggested that L-NAME inhibited the NO production, but did not inhibit the base line increase in the cell proliferation of HaCaT cells. The action mechanism of NO to increase the proliferation remains controversial, since NO is a signaling mediator with many diverse and often opposing biological activities. In addition, endogenous NO
and exogenous NO have different roles. Low levels of exogenous NO increase keratinocyte proliferation in vitro while higher doses inhibit proliferation [29]. Endogenous NO is synthesized by three isoforms of NOS. There are two types of NOS: constitutive NOS and inducible NOS. Constitutive NOS includes neuronal NOS (nNOS) and endothelial NOS (eNOS). All of these NOS are expressed in human epidermal keratinocytes [30]. Inducible NOS is mostly induced during cytokine-triggered inflammatory processes in skin [31]. Although the physiological and pathological roles of nNOS and eNOS in skin are still unclear, eNOS deficiency is known to impair epithelial proliferation in mice [12]. Over the years, much insight has been gained into the signaling pathways involved in the activation of eNOS, i.e. phosphorylation of eNOS at the Ser1177 residue. HB-EGF is reported to activate eNOS and NO production via a PI3K-dependent pathway through its interaction with EGFR in
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human umbilical vein endothelial cells [24]. The regulation of the phosphorylation of eNOS by PI3K activation of Akt is the most well known pathway [13–15]. However, it is important to emphasize that Akt is unlikely to be the only protein kinase that modulates eNOS phosphorylation. It is known that activation of p42/p44 MAPK also regulates eNOS phosphorylation and eNOS activity. Inhibition of p42/p44 MAPK activation has been shown to suppress estrogen-induced eNOS activation in ovine pulmonary endothelial cells [18]. Hypoxia increases eNOS phosphorylation partially via p42/p44 MAPK pathway in porcine coronary artery endothelium [32]. Our study demonstrated that HB-EGF-induced EGFR activation resulted in an early activation of p42/p44 MAPK, and inhibition of this pathway reduced eNOS phosphorylation in HaCaT cells. Interestingly, the treatment with HB-EGF also activated PI3K/Akt pathway, and inhibition of this pathway had reduced eNOS phosphorylation in HaCaT cells. Although it is difficult to determine whether both pathways are involved independently or synergistically, to our knowledge, this is the first report demonstrating the involvement of both p42/p44 MAPK and PI3K in HB-EGF-induced phosphorylation of eNOS in HaCaT cells. There are many reports about the derangement of VEGF production by epidermal keratinocytes, since VEGF may relate to various skin diseases, including psoriasis [19], as well as wound healing [20]. Previous studies have shown that EGF can increase VEGF production in human epidermal keratinocytes [23,33]. Nacetylcysteine downregulates the VEGF production, suggesting the involvement of p42/p44 MAPK. In this study, we found that HBEGF increases VEGF production in HaCaT cells. According to previous reports, it is demonstrated that insulin-like growth factor-II induces VEGF gene expression via MAPK pathway and PI3K pathways [34], and that PI3K/Akt pathway rather than p42/ p44 MAPK pathway is involved in UV-induced VEGF production in HaCaT cells [35]. We thus investigated whether or not p42/p44 MAPK and PI3K/Akt pathways are involved in HB-EGF-induced VEGF production in HaCaT cells. Our results showed that HB-EGFinduced VEGF production was suppressed by U0126, a specific p42/p44 MAPK inhibitor, and slightly suppressed by LY294002, a specific PI3K inhibitor. Thus, p42/p44 MAPK pathway is mostly involved, but PI3K/Akt pathway is also involved in HB-EGFstimulated VEGF production in HaCaT cells. Interestingly, HaCaT cells have been reported to express VEGFR. Among three major VEGFR (VEGFR-1, VEGFR-2 and VEGFR-3), VEGFR-1 and VEGFR-2 are primarily expressed in human epidermal keratinocytes [22]. VEGFR-2 stimulation through PI3K activation is well known [36]. Although VEGF activates p42/p44 MAPK in uterine artery endothelial cells, this is not necessary for eNOS activation since U0126 blocks p42/p44 MAPK phosphorylation but not eNOS activation [37]. In HaCaT cells, our results showed that U0126 suppressed the HB-EGFinduced phosphorylations of p42/p44 MAPK and eNOS. In addition, VEGF inhibitor suppressed the HB-EGF-induced phosphorylations of Akt and eNOS without affecting the phosphorylation of p42/p44 MAPK. Thus, we speculate that HB-EGF activated eNOS via p42/p44 MAPK pathway, and that the HB-EGF-induced VEGF also activated PI3K/Akt and eNOS via VEGFR in HaCaT cells. However, the exact nature of this discrepancy may require further study. To summarize our results is difficult, since a considerable number of multiple signaling pathways are involved in HB-EGFinduced eNOS activation and VEGF production in HaCaT cells. However, we suggest a possible mechanism for HB-EGF-induced VEGF production and eNOS activation in HaCaT cells (Fig. 7, Scheme). HB-EGF first activates EGFR, and subsequently activates p42/p44 MAPK and PI3K pathway. There may be a PI3K/Akt/eNOS activation pathway. The activation of p42/p44
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MAPK can directly lead to the eNOS activation, since U0126 did not suppress the Akt activation. The activation of both p42/p44 MAPK pathway and PI3K pathway leads to an increase in VEGF production, because both U0126 and LY294002 suppressed the increase in VEGF production. VEGF inhibitor suppressed the activation of Akt but not p42/p44MAPK. At least a few hours may be needed for HB-EGF-induced VEGF production in HaCaT cells. By viewing the strong activation of eNOS at 180 min, this activation might depend on VEGFR/PI3K/Akt pathway in HaCaT cells. In conclusion, we have demonstrated the HB-EGF-induced VEGF production and eNOS activation, and suggested possible signaling pathways in HaCaT cells. Acknowledgements This work was supported by grants-in-aid for scientific research to K. Nakai from the Ministry of Education, Science, Sports and Culture of Japan. We thank Ms. Fumiko Nishiyama for technical help and Mr. Ian Willey for editorial help in the preparation of this manuscript.
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[17] Butt E, Bernhardt M, Smolenski A, Kotsonis P, Frohlich LG, Sickmann A, et al. Endothelial nitric-oxide synthase (type III) is activated and becomes calcium independent upon phosphorylation by cyclic nucleotide-dependent protein kinases. J Biol Chem 2000;275:5179–87. [18] Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME, Shaul PW. Estrogen receptor alpha mediates the nongenomic activation of endothelial nitric oxide synthase by estrogen. J Clin Invest 1999;103:401–6. [19] Detmar M, Brown LF, Claffey KP, Yeo KT, Kocher O, Jackman RW, et al. Overexpression of vascular permeability factor/vascular endothelial growth factor and its receptors in psoriasis. J Exp Med 1994;180:1141–6. [20] Brown LF, Yeo KT, Berse B, Yeo TK, Senger DR, Dvorak HF, et al. Expression of vascular permeability factor (vascular endothelial growth factor) by epidermal keratinocytes during wound healing. J Exp Med 1992;176:1375–9. [21] Yang XH, Man XY, Cai SQ, Yao YG, Bu ZY, Zheng M. Expression of VEGFR-2 on HaCaT cells is regulated by VEGF and plays an active role in mediating VEGF induced effects. Biochem Biophys Res Commun 2006;349:31–8. [22] Man XY, Yang XH, Cai SQ, Yao YG, Zheng M. Immunolocalization and expression of vascular endothelial growth factor receptors (VEGFRs) and neuropilins (NRPs) on keratinocytes in human epidermis. Mol Med 2006;12:127–36. [23] Redondo P, Jimenez E, Perez A, Garcia-Focillas. N-acetylcysteine downregulates vascular endothelial growth factor production by human keratinocytes in vitro. Arch Dermatol Res 2000;292:621–8. [24] Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol 1988;106:761–71. [25] Mehta VB, Zhou Y, Radulescu A, Besner GE. HB-EGF stimulates eNOS expression and nitric oxide production and promotes eNOS dependent angiogenesis. Growth Factors 2008;26:301–15. [26] Kuwabara M, Kakinuma Y, Ando M, Katare RG, Yamasaki F, Doi Y, et al. Nitric oxide stimulates vascular endothelial growth factor production in cardiomyocytes involved in angiogenesis. J Physiol Sci 2006;56:95–101. [27] Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, et al. The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol 1999;9:845–8.
[28] Dimmeler S, Dernbach E, Zeiher AM. Phosphorylation of the endothelial nitric oxide synthase at ser-1177 is required for VEGF-induced endothelial cell migration. FEBS Lett 2000;477:258–62. [29] Krischel V, Bruch-Gerharz D, Suschek C, Kro¨ncke KD, Ruzixka T, Kolb-Bachofen V. Biphasic effect of exogenous nitric oxide on proliferation and differentiation in skin derived keratinocytes but not fibroblasts. J Invest Dermatol 1998;111:286–91. [30] Nakai K, Fujii S, Yamamoto A, Igarashi J, Kubota Y, Kosaka H. Effects of high glucose on NO synthesis in human keratinocyte cell line (HaCaT). J Dermatol Sci 2003;31:211–8. [31] Nakai K, Kadiiska MB, Jiang JJ, Stadler K, Mason RP. Free radical production requires both inducible nitric oxide synthase and xanthine oxidase in LPStreated skin. Proc Natl Acad Sci USA 2006;103:4616–21. [32] Chen JX, Meyrick B. Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium. Lab Invest 2004;84:182–90. [33] Detmar M, Yeo KT, Nagy JA, Van de Water L, Brown LF, Berse B, et al. Keratinocyte-derived vascular permeability factor (vascular endothelial growth factor) is a potent mitogen for dermal microvascular endothelial cells. J Invest Dermatol 1995;105:44–50. [34] Kim HJ, Kim TY. Regulation of vascular endothelial growth factor expression by insulin-like growth factor-II in human keratinocytes, differential involvement of mitogen-activated protein kinases and feedback inhibition of protein kinase C. Br J Dermatol 2005;152:418–25. [35] Kim MS, Kim YK, Eun HC, Cho KH, Chung JH. All-trans retinoic acid antagonizes UV-induced VEGF production and angiogenesis via the inhibition of ERK activation in human skin keratinocytes. J Invest Dermatol 2006;126:2697– 706. [36] Dance M, Montagner A, Yart A, Masri B, Audigier Y, Perret B, et al. The adaptor protein Gab1 couples the stimulation of vascular endothelial growth factor receptor-2 to the activation of phosphoinositide 3-kinase. J Biol Chem 2006;281:23285–9. [37] Grummer MA, Sullivan JA, Magness RR, Bird IM. VEGF acts through novel, pregnancy-enhanced receptor signaling pathways to stimulate eNOS activity in uterine artery endothelial cells. Biochem J 2009;417:501–11.
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HB-EGF-induced VEGF production and eNOS activation depend on both PI3 kinase and MAP kinase in HaCaT cells Kozo Nakai a,*, Kozo Yoneda a, Tetsuya Moriue a, Junske Igarashi b, Hiroaki Kosaka b, Yasuo Kubota a a b
Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan Department of Cardiovascular Physiology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 March 2009 Received in revised form 21 May 2009 Accepted 2 June 2009
Background: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of growth factors that have been implicated in skin patho-physiology. Although endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) appear to be involved in mitogenesis and chemotaxis in epidermal keratinocytes, the activation of eNOS and VEGF production induced by HB-EGF and its signaling mechanism remains undefined. Objective: We examined possible signal transduction pathways by which HB-EGF leads to eNOS activation and VEGF production in human epidermal keratinocyte cell line (HaCaT cells). Methods: The phosphorylation of epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK; p42/p44 MAPK), Akt and eNOS were examined by Western blotting analysis. VEGF production was determined by enzyme-linked immunosorbent assay. Various inhibitors were utilized to investigate the signaling mechanisms of eNOS activation and VEGF production. Results: HB-EGF-induced phosphorylation of EGFR with maximum phosphorylation at 1 h. HB-EGFinduced phosphorylation of p42/p44 MAPK in a few minutes. It activated Akt with maximum phosphorylation at 1 h and eNOS with maximum phosphorylation at 3 h. The HB-EGF-induced eNOS activation was significantly blocked by the p42/p44 MAPK inhibitor U0126 and the phosphatidylinositol 3-kinase (P13K) inhibitor LY294002. HB-EGF increased VEGF production. The HB-EGF-induced VEGF production was blocked by U0126 and LY294002. Finally, the HB-EGF-induced activation of Akt and eNOS was suppressed by VEGF competitive antagonist, CBO-P11. Conclusion: These results demonstrate that HB-EGF-induced eNOS activation depends on p42/p44 MAPK, PI3K/Akt pathways and endogenous VEGF in HaCaT cells. ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Keywords: HB-EGF VEGF eNOS HaCaT cell
1. Introduction The study of proliferation and migration of epidermal keratinocytes are focused in the pathogenesis of cutaneous wounds and various skin diseases such as psoriasis. Several mitogens have been implicated in these processes, one of which is heparin-binding epidermal growth factor-like growth factor (HBEGF). Studies have shown that HB-EGF is overexpressed in psoriatic lesional skin and that it is a potent mitogenic and chemotactic factor for epidermal keratinocytes [1,2], and participates in the epidermal response to cutaneous wounds [3]. The biological actions of HB-EGF are mediated through some members of the epidermal growth factor receptor (EGFR) superfamily, also known as erbB1, and erbB4 [4]. On activation, these receptors undergo homo- or heterodimerization, followed by activation of intrinsic tyrosine kinase activity, leading to a myriad of signaling
* Corresponding author. Tel.: +81 87 891 2162; fax: +81 87 891 2163. E-mail address: [email protected] (K. Nakai).
events. To this end, a variety of cellular responses occur, such as cell proliferation, migration, and differentiation. The mitogen-activated protein kinases (MAPKs) are a welldocumented family of serine/threonine kinases that include p42/ p44 MAPK, p38 MAPK and c-Jun N-terminal kinase. p42/p44 MAPK are the most characterized, and mediate proliferative and chemotactic responses in various cells, including epidermal keratinocytes [5]. Phosphoinositide 30 -OH kinase (PI3K) is a lipid kinase that phosphorylates the 3-hydroxyl of the head group of phosphatidylinositol, thereby activating several downstream protein kinases, such as Akt [6,7]. Akt is also involved in cell growth by eliciting cell survival/antiapoptotic effects [8,9]. Although the direct effects of MAPK and PI3K on cell growth and migration have been well reported, these kinases also influence endothelial nitric oxide synthase (eNOS) activation [10] and vascular endothelial growth factor (VEGF) production [11], which might partly relate to cell growth and migration, especially in vascular endothelial and smooth muscle cells. It has also been reported that eNOS is a key regulator of the proliferation of epidermal keratinocytes [12]. There is strong evidence supporting
0923-1811/$36.00 ß 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2009.06.002
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PI3K activation of Akt, which in turn is responsible for regulating the phosphorylation and activation of eNOS [13–15]. However, it is becoming increasingly clear that Akt is not the only protein kinase that can phosphorylate eNOS at the Ser1177 residue. Other protein kinases, including protein kinase A, protein kinase G, and AMP kinase have also been shown to phosphorylate eNOS at the Ser1177 residue [16–18]. Thus, it may be possible that eNOS at the Ser1177 residue can be phosphorylated by different protein kinases depending upon cell types and each given stimuli. The derangement of VEGF production by epidermal keratinocytes concerns various skin diseases including carcinogenesis and psoriasis [19], as well as wound healing [20]. In addition, the expression of VEGF receptor (VEGFR) and the proliferation and migration of epidermal keratinocytes by VEGF have been reported [21,22]. EGFR stimulation by EGF has been shown to increase VEGF production [23]. However, there are no reports on whether or not HB-EGF increases VEGF production, and the related signaling mechanisms have not been defined in human epidermal keratinocytes. In the present study, by using human epidermal keratinocyte cell line, HaCaT cells, we examined the signaling mechanisms required for HB-EGF-induced eNOS activation and
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VEGF production that are most likely related to various cellular functions of epidermal keratinocytes. 2. Materials and methods 2.1. Reagents Dulbecco’s modified Eagle’s medium (DMEM), HB-EGF and LNAME were obtained from Sigma (St. Louis, MO, USA). AG1478, LY294002, U0126 and VEGF competitive antagonist (CBO-P11) were purchased from Calbiochem-Novabiochem (San Diego, CA, USA). 4,5-Diaminofluorescein diacetate (DAF-2/DA) was purchased from Daiichi Pure Chemicals Co., Tokyo, Japan. eNOS antibody and activated EGFR antibody were obtained from BD Transduction Laboratories (Lexington, KY, USA). The antibodies against EGFR, p42/p44, phospho-p42/p44, Akt, phospho-AktSer473 and phospho-eNOSSer1177 were purchased from Cell Signaling Technology (Danvers, MA, USA). 2.2. Cell culture HaCaT cells (a generous gift from Professor N.E. Fusenig, German Cancer Research Center, Germany) were cultured in
Fig. 1. HB-EGF increased NO production and promoted cell proliferation in HaCaT cells. (a) After HB-EGF (50 ng/ml) treatment for 5 h, HaCaT cells were stained with DAF-2DA for 15–30 min. The intensity of fluorescence was measured by a fluorescence microplate reader. (b) HaCaT cells were treated with HB-EGF (50 ng/ ml) for 12 h. Cell number was assessed using Cell Counting Kit-8. The results of relative levels of OD from pooled data. Values represent means S.E. (n = 6). **P < 0.005; ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HBEGF-treated group.
Fig. 2. EGFR activation, eNOS phosphorylation and related signaling pathways in HB-EGF-treated HaCaT cells. HaCaT cells were treated with HB-EGF (50 ng/ml) and lysed at specific time points for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (c) EGFR antibody or activated EGFR antibody, (d) p42/p44 antibody or phospho-p42/p44 antibody, (e) Akt antibody or phospho-AktSer473 antibody, and (f) eNOS antibody or phospho-eNOSSer1177 antibody. Similar results were obtained for more than three independent experiments. (a, b) eNOS expression in skin and HaCaT cells, and the HB-EGF-induced eNOS phosphorylation in HaCaT cells. Human umbilical vein endothelial cells and bovine aortic endothelial cells were used as positive control.
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DMEM with 10% heat-inactivated fetal calf serum (FCS) under 37 8C in a humidified atmosphere of 5% CO2 and 95% air [24]. On the treatment day, the medium was replaced with DMEM containing 1% FCS. Cells were about 50% confluent and treated with or without HB-EGF and inhibitors at the same time. 2.3. Measurement of intracellular NO production Production of intracellular NO was determined using DAF-2/ DA. DAF-2/DA is a cell-permeable compound that is converted to DAF-2 by intracellular esterases in the presence of NO. A triazole derivative of DAF-2 emits light; the intensity of this light is proportional to the amount of NO present. The fluorescence was detected using a fluorescence microplate reader, Fluoroskan Ascent FL1 (Thermo Fisher Scientific, Waltham, MA, USA), at excitation and emission wavelengths of 485 nm and 538 nm.
2.4. Cell proliferation assay Cell proliferation was assayed with Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), in which WST-8 (2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) is converted to formazan by cellular dehydrogenase. The absorbance of formazan, which is proportional to cell number, was determined at 450 nm. 2.5. Measurement of VEGF secretion Cell-free supernatants were collected and stored at 70 8C until required for assay. Secreted VEGF levels in the culture media of HaCaT cells were determined by sandwich ELISA by using a commercially available ELISA human VEGF kit (Quantikine Human VEGF Immunoassay, R&D Systems, Minneapolis, MN, USA). According to the manufacture’s protocol, absorption of avidin-
Fig. 3. AG1478 inhibited the HB-EGF-induced activation of EGFR, p42/p44 MAPK, Akt and eNOS. HaCaT cells were treated with HB-EGF (50 ng/ml) plus AG1478 (10 mM) and lysed for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (a) EGFR antibody or activated EGFR antibody, (b) p42/p44 antibody or phospho-p42/p44 antibody, (c) Akt antibody or phospho-AktSer473 antibody, and (d) eNOS antibody or phospho-eNOSSer1177 antibody. (Upper) Shown are the results from representative data. (Lower) The results of densitometric analysis from pooled data. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
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horseradish peroxidase color reaction was measured at 405 nm and VEGF concentrations were determined using a standard graph prepared using serial dilutions of human recombinant VEGF as a standard.
densitometry analysis using Image J software (National Institutes of Health, Bethesda, MD, USA).
2.6. Western blotting analysis
Data were given as mean S.E. A statistical analysis was performed by an analysis of variance followed by the Fisher’s protected least significant difference. P-values <0.05 were considered statistically significant.
Cells were washed with PBS. Then, lithium dodecyl sulfate sample buffer was added. An equal amount of protein was separated on 7% Tris–Acetate gel (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane. The membrane was then probed with antibodies against EGFR, activated EGFR, p42/ p44, phospho-p42/p44, Akt, phospho-AktSer473, eNOS and phospho-eNOSSer1177. Membrane-bound primary antibodies were visualized by using appropriate secondary antibodies conjugated with horseradish peroxidase and chemiluminescent substrate (Pierce, Rockford, IL, USA). The images were subjected to
2.7. Statistical analysis
3. Results 3.1. HB-EGF increased NO production and promoted cell proliferation in HaCaT cells First, we examined whether HB-EGF increases NO production in HaCaT cells. By using DAF-2/DA, we have confirmed that NO
Fig. 4. The PI3K pathway and MAPK pathway involve HB-EGF-induced eNOS activations in HaCaT cells. HaCaT cells were treated with HB-EGF (50 ng/ml) plus U0126 (10 mM) or LY294002 (10 mM) and lysed for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (a) p42/p44 antibody or phospho-p42/p44 antibody, (b) Akt antibody or phospho-AktSer473 antibody, and (c) eNOS antibody or phospho-eNOSSer1177 antibody. (Upper) Shown are the results from representative data. (Lower) The results of densitometric analysis from pooled data. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
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production is slightly, but significantly, increased in HB-EGFtreated HaCaT cells at 5 h (Fig. 1a). The increase in NO production induced by HB-EGF was suppressed by L-NAME (1 mM), a NOS inhibitor. As HB-EGF increases NO production in human vascular endothelial cells and promotes angiogenesis [25], we investigated whether NO is necessary for HB-EGFinduced proliferation of human epidermal keratinocyte cell line. As shown in Fig. 1b, HB-EGF (50 ng/ml) increased proliferation of HaCaT cells at 12 h. L-NAME (1 mM) suppressed the HB-EGFinduced proliferation of HaCaT cells. These data suggest that NO production is involved in HB-EGF-induced cell proliferation in HaCaT cells. L-NAME did not inhibit the base line increase in HaCaT cell proliferation. 3.2. Time course dependent activation of EGFR, eNOS and related signaling pathways in HB-EGF-treated HaCaT cells HB-EGF elicits a variety of cellular responses that are initiated by binding to EGFR. Following ligand binding, EGFR is activated and, in turn, phosphorylates and activates several other endogenous proteins including MAPK and PI3K/Akt in keratinocytes. Subsequently, eNOS may be phosphorylated and activated. We have confirmed the eNOS expression in human skin and HaCaT cells (Fig. 2a), and the HB-EGF-induced eNOS phosphorylation in HaCaT cells (Fig. 2b). To gain an insight into the order and mechanisms of the activation of these proteins, we have investigated the time course dependant phosphorylation of these proteins in HaCaT cells. As previously demonstrated, the activation of EGFR increased rapidly 5 min after stimulation and reached a maximum at 30–60 min (Fig. 2c). The phosphorylation of p42/p44 increased rapidly and reached its peak 5 min after stimulation, and gradually decreased (Fig. 2d). Interestingly, the phosphorylation of Akt was slightly increased at 5 min, but strongly increased and reached its peak at 60 min (Fig. 2e). Similarly, the phosphorylation of eNOS was slightly increased at 5 min, but strongly increased and reached its peak at 180 min (Fig. 2f). Thus, transit activation of p42/p44 MAPK, PI3K/Akt and eNOS occurred at 5 min. However, PI3K/Akt and eNOS were activated strongly at 60 min and 180 min, respectively. We decided to investigate the phosphorylation of EGFR at 60 min, p42/p44 at 60 min, Akt at 60 min and eNOS at 180 min. To examine whether the HB-EGF-induced phosphorylation of p42/p44, PI3K/Akt and eNOS was EGFR dependent, we used an inhibitor of EGFR kinase, AG1478 and confirmed its effects on HBEGF-induced phosphorylation of the aforementioned kinases. The maximum phosphorylations of these proteins were all inhibited by AG1478 (10 mM, Fig. 3).
Akt pathway, in HB-EGF-induced activation of eNOS in HaCaT cells. 3.4. HB-EGF-induced VEGF production does not require NO in HaCaT cells EGF increases VEGF production in normal human keratinocytes [23]. As HB-EGF increases NO production in HUVEC [25], and NO stimulates VEGF production in cardiomyocytes [26], there is a possibility that HB-EGF-induced NO could increase VEGF production in HaCaT cells. Thus, we examined secreted VEGF levels in the culture media of HaCaT cells by ELISA method. As shown in Fig. 5a, HB-EGF increased VEGF production in HaCaT cells. However, L-NAME did not affect the HB-EGF-induced VEGF production in HaCaT cells. To determine the signaling pathways underlying VEGF production in response to HB-EGF, AG1478, LY294002 and U0126 were administered (Fig. 5b). HB-EGFinduced VEGF production was completely inhibited by AG1478 (10 mM) or U0126 (10 mM). LY294002 (10 mM) slightly, but significantly, suppressed the HB-EGF-induced VEGF production. These results clearly illustrate that the activation of both PI3K and p42/p44 MAPK are involved in HB-EGF-induced VEGF production in HaCaT cells, but p42/p44 MAPK is involved, more than PI3K.
3.3. The PI3K pathway and MAPK pathway involve HB-EGF-induced eNOS activations in HaCaT cells To determine the mechanisms underlying eNOS activation in response to HB-EGF, we attempted to identify the role of PI3K by using LY294002, a PI3K inhibitor. As p42/p44 MAPK pathway appeared to be activated first following the activation of EGFR, we decided to investigate the effects of U0126, a p42/p44 inhibitor. U0126 (10 mM) suppressed HB-EGF-induced phosphorylation of p42/p44 (Fig. 4a), but it did not suppress phosphorylation of Akt (Fig. 4b). LY294002 (10 mM) did not suppress HB-EGF-induced phosphorylation of p42/p44 (Fig. 4a), but it did suppress phosphorylation of Akt (Fig. 4b). Interestingly, both U0126 and LY294002 inhibited phosphorylation of eNOS (Fig. 4c). These results suggest that the activation of both PI3K/Akt pathway and p42/p44 MAPK pathway are involved, and p42/p44 MAPK pathway did not require PI3K/
Fig. 5. HB-EGF-induced VEGF production does not require NO, but depends on both PI3K pathway and MAPK pathway in HaCaT cells. HaCaT cells were treated with (a) HB-EGF (5–50 ng/ml) plus L-NAME (1 mM), and (b) HB-EGF (50 ng/ml) plus AG1478 (10 mM), U0126 (10 mM) or LY294002 (10 mM). Secreted VEGF levels in the culture media at 5 h of HaCaT cells were determined by sandwich ELISA by using a commercially available ELISA human VEGF kit. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
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Fig. 6. VEGF is required for the HB-EGF-induced activation of eNOS in HaCaT cells. HaCaT cells were treated with HB-EGF (50 ng/ml) plus VEGF inhibitor (20 mM) and lysed for immunoblot analyses. Samples were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with (a) EGFR antibody or activated EGFR antibody, (b) p42/p44 antibody or phospho-p42/p44 antibody, (c) Akt antibody or phospho-AktSer473 antibody, and (d) eNOS antibody or phosphoeNOSSer1177 antibody. (Upper) Shown are the results from representative data. (Lower) The results of densitometric analysis from pooled data. Values represent the mean S.E. derived from more than three independent experiments. ***P < 0.0005 versus untreated cells. ##P < 0.005; ###P < 0.005 versus HB-EGF-treated group.
3.5. VEGF is required for the HB-EGF-induced activation of eNOS in HaCaT cells After confirming that HB-EGF increased VEGF production, we investigated whether HB-EGF-induced VEGF is involved in the activation of eNOS in HaCaT cells, since VEGFR is expressed in HaCaT cells [21]. In addition, VEGF phosphorylates the eNOS via Akt pathway in endothelial cells [27,28]. The HB-EGF-induced phosphorylations of Akt and eNOS were significantly suppressed by the VEGF competitive antagonist, CBO-P11 (20 mM) in HaCaT cells. However, the phosphorylation of p42/p44 MAPK was not suppressed (Fig. 6).
4. Discussion In this study, we demonstrated the HB-EGF-induced activation of p42/p44 MAPK, PI3K/Akt and eNOS through the EGFR in HaCaT cells. These activated signaling pathways were completely blocked by an EGFR kinase inhibitor, AG1478. Our data demonstrated that HB-EGF promoted the VEGF production in HaCaT cells. We further showed that MAPK inhibitor and PI3K inhibitor blocked the HBEGF-induced eNOS activation and VEGF production in HaCaT cells. Finally, VEGF inhibitor prevented the HB-EGF-induced Akt and eNOS activation without affecting the activity of EGFR in HaCaT cells.
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Fig. 7. Schematic representation of HB-EGF-induced VEGF production and eNOS activation in HaCaT cells. HB-EGF first activates EGFR, and subsequently activates p42/p44 MAPK and PI3K pathway. There may be a PI3K/Akt/eNOS activation pathway. The activation of p42/p44 MAPK can directly lead to the eNOS activation. The activation of both p42/p44 MAPK pathway and PI3K pathway leads to an increase in VEGF production. Then, VEGF activates eNOS via VEGFR/PI3K/Akt pathway.
In our study, it is suggested that NO is involved in the cell proliferation of HB-EGF-treated HaCaT cells. However, various factors must be involved in HB-EGF-induced proliferation of HaCaT cell. The reason why L-NAME did not inhibit the base line increase in the cell proliferation may be because cell proliferation did not totally depend on NO. As shown in Fig. 1a, the slight levels of NO production were observed in untreated HaCaT cells. Presumably, this NO production may involve the effects of internal EGF family. However, the levels of this constitutive NO production may be too low to affect cell proliferation. Thus, it is suggested that L-NAME inhibited the NO production, but did not inhibit the base line increase in the cell proliferation of HaCaT cells. The action mechanism of NO to increase the proliferation remains controversial, since NO is a signaling mediator with many diverse and often opposing biological activities. In addition, endogenous NO
and exogenous NO have different roles. Low levels of exogenous NO increase keratinocyte proliferation in vitro while higher doses inhibit proliferation [29]. Endogenous NO is synthesized by three isoforms of NOS. There are two types of NOS: constitutive NOS and inducible NOS. Constitutive NOS includes neuronal NOS (nNOS) and endothelial NOS (eNOS). All of these NOS are expressed in human epidermal keratinocytes [30]. Inducible NOS is mostly induced during cytokine-triggered inflammatory processes in skin [31]. Although the physiological and pathological roles of nNOS and eNOS in skin are still unclear, eNOS deficiency is known to impair epithelial proliferation in mice [12]. Over the years, much insight has been gained into the signaling pathways involved in the activation of eNOS, i.e. phosphorylation of eNOS at the Ser1177 residue. HB-EGF is reported to activate eNOS and NO production via a PI3K-dependent pathway through its interaction with EGFR in
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human umbilical vein endothelial cells [24]. The regulation of the phosphorylation of eNOS by PI3K activation of Akt is the most well known pathway [13–15]. However, it is important to emphasize that Akt is unlikely to be the only protein kinase that modulates eNOS phosphorylation. It is known that activation of p42/p44 MAPK also regulates eNOS phosphorylation and eNOS activity. Inhibition of p42/p44 MAPK activation has been shown to suppress estrogen-induced eNOS activation in ovine pulmonary endothelial cells [18]. Hypoxia increases eNOS phosphorylation partially via p42/p44 MAPK pathway in porcine coronary artery endothelium [32]. Our study demonstrated that HB-EGF-induced EGFR activation resulted in an early activation of p42/p44 MAPK, and inhibition of this pathway reduced eNOS phosphorylation in HaCaT cells. Interestingly, the treatment with HB-EGF also activated PI3K/Akt pathway, and inhibition of this pathway had reduced eNOS phosphorylation in HaCaT cells. Although it is difficult to determine whether both pathways are involved independently or synergistically, to our knowledge, this is the first report demonstrating the involvement of both p42/p44 MAPK and PI3K in HB-EGF-induced phosphorylation of eNOS in HaCaT cells. There are many reports about the derangement of VEGF production by epidermal keratinocytes, since VEGF may relate to various skin diseases, including psoriasis [19], as well as wound healing [20]. Previous studies have shown that EGF can increase VEGF production in human epidermal keratinocytes [23,33]. Nacetylcysteine downregulates the VEGF production, suggesting the involvement of p42/p44 MAPK. In this study, we found that HBEGF increases VEGF production in HaCaT cells. According to previous reports, it is demonstrated that insulin-like growth factor-II induces VEGF gene expression via MAPK pathway and PI3K pathways [34], and that PI3K/Akt pathway rather than p42/ p44 MAPK pathway is involved in UV-induced VEGF production in HaCaT cells [35]. We thus investigated whether or not p42/p44 MAPK and PI3K/Akt pathways are involved in HB-EGF-induced VEGF production in HaCaT cells. Our results showed that HB-EGFinduced VEGF production was suppressed by U0126, a specific p42/p44 MAPK inhibitor, and slightly suppressed by LY294002, a specific PI3K inhibitor. Thus, p42/p44 MAPK pathway is mostly involved, but PI3K/Akt pathway is also involved in HB-EGFstimulated VEGF production in HaCaT cells. Interestingly, HaCaT cells have been reported to express VEGFR. Among three major VEGFR (VEGFR-1, VEGFR-2 and VEGFR-3), VEGFR-1 and VEGFR-2 are primarily expressed in human epidermal keratinocytes [22]. VEGFR-2 stimulation through PI3K activation is well known [36]. Although VEGF activates p42/p44 MAPK in uterine artery endothelial cells, this is not necessary for eNOS activation since U0126 blocks p42/p44 MAPK phosphorylation but not eNOS activation [37]. In HaCaT cells, our results showed that U0126 suppressed the HB-EGFinduced phosphorylations of p42/p44 MAPK and eNOS. In addition, VEGF inhibitor suppressed the HB-EGF-induced phosphorylations of Akt and eNOS without affecting the phosphorylation of p42/p44 MAPK. Thus, we speculate that HB-EGF activated eNOS via p42/p44 MAPK pathway, and that the HB-EGF-induced VEGF also activated PI3K/Akt and eNOS via VEGFR in HaCaT cells. However, the exact nature of this discrepancy may require further study. To summarize our results is difficult, since a considerable number of multiple signaling pathways are involved in HB-EGFinduced eNOS activation and VEGF production in HaCaT cells. However, we suggest a possible mechanism for HB-EGF-induced VEGF production and eNOS activation in HaCaT cells (Fig. 7, Scheme). HB-EGF first activates EGFR, and subsequently activates p42/p44 MAPK and PI3K pathway. There may be a PI3K/Akt/eNOS activation pathway. The activation of p42/p44
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MAPK can directly lead to the eNOS activation, since U0126 did not suppress the Akt activation. The activation of both p42/p44 MAPK pathway and PI3K pathway leads to an increase in VEGF production, because both U0126 and LY294002 suppressed the increase in VEGF production. VEGF inhibitor suppressed the activation of Akt but not p42/p44MAPK. At least a few hours may be needed for HB-EGF-induced VEGF production in HaCaT cells. By viewing the strong activation of eNOS at 180 min, this activation might depend on VEGFR/PI3K/Akt pathway in HaCaT cells. In conclusion, we have demonstrated the HB-EGF-induced VEGF production and eNOS activation, and suggested possible signaling pathways in HaCaT cells. Acknowledgements This work was supported by grants-in-aid for scientific research to K. Nakai from the Ministry of Education, Science, Sports and Culture of Japan. We thank Ms. Fumiko Nishiyama for technical help and Mr. Ian Willey for editorial help in the preparation of this manuscript.
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