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Endocrinology, doi:10.1210/en.2007-0949
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Endocrinology Vol. 149, No. 4 1898-1905
Copyright © 2008 by The Endocrine Society

Mechanisms of Adrenocorticotropin-Induced Activation of Extracellularly Regulated Kinase 1/2 Mitogen-Activated Protein Kinase in the Human H295R Adrenal Cell Line

Mandy E. Janes, K. M. Emily Chu, Adrian J. L. Clark and Peter J. King

Centre for Endocrinology, Barts and the London School of Medicine, London EC1M 6BQ, United Kingdom

Address all correspondence and requests for reprints to: Dr. P. J. King, Centre for Endocrinology, John Vane Science Centre, William Harvey Research Institute, Charterhouse Square, London EC1M 6BQ, United Kingdom. E-mail: p.j.king{at}qmul.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
The role of ACTH in stimulating or inhibiting growth of adrenal cells has been a subject of some controversy. Reports that ACTH may stimulate ERK/MAPK in Y1 cells have suggested a role for cAMP in this process. In attempting to extend this work, the ACTH responses in the human H295R cell line have been studied. This cell line makes only a very modest cAMP response to ACTH, yet the ERK1/2 response is highly reproducible and immediate but not prolonged. It is minimally reduced by the protein kinase A inhibitor, H89, but unaffected by protein kinase C and calcium inhibitors. Inhibition of epidermal growth factor receptor or other tyrosine kinase receptor transactivation was without effect, as was inhibition of c-Src activity or c-Src phosphorylation. The most effective inhibitor of this pathway was dansylcadaverine, an inhibitor of receptor internalization. These findings imply that ACTH-induced ERK1/2 activation in H295R cells is dependent on a mechanism distinct from that by which most G protein-coupled receptors activate ERK1/2 but that nevertheless seems to depend on receptor internalization.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
THE PRIMARY ROLE of ACTH is to stimulate steroidogenesis in the adrenal cortex. This is accomplished principally via cAMP-dependent mechanisms, although intracellular calcium influx and possibly other mediators may also contribute to this process (1, 2, 3, 4, 5, 6). However, the influence of ACTH on adrenocortical growth has been an area of some controversy for many years. There is little dispute that, in vivo, hypophysectomy or proopiomelanocortin (POMC) gene deletion or mutation is associated with adrenocortical hypoplasia (7, 8, 9, 10, 11, 12) and that conversely ACTH excess may be associated with adrenal hyperplasia (13, 14, 15). However, the role of POMC-derived peptides other than ACTH that may be cosecreted has revealed that the role of ACTH is more complex than appears at first sight. Important mitogenic roles for pro-{gamma}MSH and an N-terminal POMC peptide have been described (16, 17) and may account for some of the effects ascribed to ACTH or may act synergistically with ACTH. Administration of synthetic ACTH, free of other potential mitogens, argues in favor of a specific ACTH effect in in vivo studies. In mice in which the POMC gene has been deleted, the highly atrophic adrenal glands acquire a virtually normal appearance after 10 d of synthetic ACTH1–24 administration (10). Patients with homozygous defects of the highly specific receptor for ACTH, the melanocortin 2 receptor (MC2R) have hypoplastic adrenal glands with an intact zona glomerulosa but a very atrophic zona fasciculata and reticularis (18), and these observations are confirmed in mice with deletion of the MC2R gene (19). Thus, clinical and animal evidence is highly suggestive of a mitogenic role for ACTH, perhaps in synergy with other peptides.

Attempts to replicate such observations in cultured cells have been problematic. In many primary cell culture studies, ACTH has a growth-inhibitory effect (20, 21) or a biphasic effect with initial growth inhibition followed by later growth stimulation (22). Relatively few stable adrenal cell lines express the MC2R. Of those that do, the mouse adrenal tumor Y1 cell line has been the most studied. It has been suggested that in this cell line, ACTH stimulates MAPK isoforms ERK 1 and ERK2 and cell cycle progression via protein kinase C-dependent mechanisms and inhibits c-Myc expression and hence impairs growth via cAMP-dependent pathways (23). Consistent data have been reported by Le and Schimmer (24) using kin 8 mutants of the Y1 cell line that lack functional protein kinase A. However, the value of this cell line as a model of the physiological mechanisms of ACTH-induced mitogenesis is arguably reduced by the observation that the Ki-ras oncogene is amplified and overexpressed, explaining the high basal levels of phospho-ERK1/2 observed in many studies (23).

In view of the variability of the Y1 cell line for mitogenic studies in our hands, the potential influence of Ki-Ras overexpression and the desire to investigate a model that may be of greater significance to human disease, we studied the H295R cell line. This line, derived originally from a human adrenocortical carcinoma, has proved extremely valuable in a range of studies of adrenal cell biology. Its value for many studies of ACTH action has been limited by the low expression levels of the MC2R and greater resemblance to a glomerulosa cell phenotype (25, 26). Despite this, we found in preliminary studies that there is sufficient expression of the MC2R that a phospho-ERK1/2 response to ACTH can be observed in the absence of a high level of constitutive activity in this cell line without any prior manipulation. This therefore provides a potentially valuable model to begin to dissect out the signaling pathways induced by ACTH in human adrenocortical cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Reagents
Laboratory reagents were supplied by Sigma-Aldrich (Poole, UK) unless otherwise stated. Chemicals were all certified as analytical grade. ACTH (1–39) and [Nle4, D-Phe7] (NDP)-{alpha}MSH was obtained from Bachem (St. Helens, UK). Pertussis toxin, SQ22536, genistein, AG1478, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine (PP2),SU6656, GM6001, calphostin C, and GF109203X were obtained from Calbiochem (Nottingham, UK). Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), tetra(acetoxymethyl)-ester was obtained from AG Scientific (San Diego, CA), and H89 and epidermal growth factor (EGF) were obtained from Sigma. The anti-ERK1/2 and phospho-ERK1/2 antibodies and anti-phospho-Tyr-417 Src antibody were purchased from Cell Signaling Technology (Danvers, MA), and the anti-Src clone GD11 antibody was from Upstate Cell Signaling Solutions (Lake Placid, NY).

Cell culture
H295R cells were a gift from Professor Ian Mason (University of Edinburgh, Edinburgh, UK). Cells were grown in a 1:1 mix of DMEM:F12, 10 ml/liter penicillin/streptomycin, 10% fetal bovine serum (FBS; Life Technologies, Inc., Paisley, UK) and 1x insulin/transferrin/selenium solution (10 µg/ml insulin, 5.5 µg/ml human transferrin, 5 ng/ml sodium selenite, 0.5 mg/ml BSA, and 4.7 µg/ml linoleic acid). Media were replaced every 3 d, and cells were not allowed to reach confluence but were passaged when around 80% confluent. For stimulation experiments, cells were plated into six-well plates and when around 70% confluent were washed once in serum-free medium (SFM) and left subsequently in SFM overnight. The next day, inhibitors were added in SFM to the indicated concentrations for 1 h (unless otherwise stated) before adding a 10x stock of the appropriate stimulant for the times indicated.

Cell proliferation, cAMP, and ERK1/2 assays
Cells cultured in 12-well plates at 30% confluency were washed twice in SFM and left for 48 h in the incubator to induce growth arrest. After this procedure, cells were treated with appropriate reagents for 72 h. Medium was aspirated and 500 µl of SFM added to each well. Proliferation assays were carried out using the CellTiter 96* AQueous proliferation assay (Promega, Madison, WI) based on 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction to a formazan product. Formazan production was measured by absorbance at 490 nm. One hundred microliters of MTS solution were added directly to each well and incubated for 1 h at 37 C. One hundred microliters of each well were pipetted into a 96-well plate and the absorbance read at 490 nm on a kinetic microplate reader (Molecular Devices, Sunnyvale, CA). There were six replicates for each treatment and experiments were performed at least three times.

For measurement of cAMP, cells were plated into 6-well plates and grown to around 90% confluence. Before stimulation, cells were washed and left in SFM for 1 h. Treatments were carried out for 30 min in 1 ml of SFM in the presence of 10–3 M 3-isobutyl-1-methylxanthine. Cells were harvested after transfer of plates to ice. Cells and media were transferred to microcentrifuge tubes, boiled for 5 min, and centrifuged at 10,000 x g for 5 min. The supernatant was transferred to fresh tubes and stored at –20 C until the cAMP assay was performed. cAMP was measured by a competitive protein binding assay (27) on whole-cell extracts.

For ERK1/2 kinase assays, cells were washed once in ice-cold PBS before adding cell lysis buffer [NaCl (150 mM), Tris (pH 7.5) (20 mM), EDTA (1 mM), and 1% Triton X-100] with protease and phosphatase inhibitor cocktails (Sigma); incubated on ice for a further 15 min with intermittent vortexing; and after centrifugation and removal of cell debris, frozen at –80 C until required. Thirty microliters of cell extract samples were heated to 95 C for 5 min in sodium dodecyl sulfate sample buffer, loaded onto 10% polyacrylamide gels (Bio-Rad Laboratories, Hemel Hempstead, UK), and electrophoresed at 120 V in Tris-glycine buffer. Gels were blotted onto polyvinyl difluoride membranes (Amersham, Buckinghamshire, UK), which were subsequently blocked for 1 h in 5% Marvel (Premier Foods, Ltd, St. Albans, UK) and probed with total or phospho-ERK1/2 rabbit antibodies either overnight at 4 C or for 1–2 h at room temperature, washed, and incubated for 1 h at room temperature with peroxidase-labeled antirabbit antibody (Amersham). Blots were developed using the ECLplus chemiluminescence system (Amersham).

To quantify the transcriptional activity of ERK1/2 activated in response to ACTH, a Gal4-Elk-1 luciferase reporter assay system was used (28). This consisted of a Gal4 Elk-1 expression vector encoding the DNA binding domain of Gal4 (residues 1–147) linked to the carboxy-terminal transcription activation domain of Elk-1 (residues 307–428) and a 5xGal4-E1b-luciferase expression vector containing Gal4 binding sites upstream of the luciferase gene. On MAPK activation, Gal4-Elk-1 is phosphorylated allowing transcription of the luciferase reporter. Cells were plated into 6-well plates and transfected with 0.45 µg Gal4 Elk-1, 0.45 µg Gal4-luc, and 0.1 µg Renilla per well using FuGENE 6 transfection reagent (Roche, Stockholm, Sweden) in a ratio of 6 µl FuGENE to 1 µg DNA. After 24 h cells were transferred to SFM and incubated overnight. The next day cells were treated with ACTH or serum. After stimulation, cells were transferred back to SFM and left for a total of 6 h from the beginning of treatment. Cells were harvested and luciferase activity assayed using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
The influence of ACTH on H295R cell proliferation was initially sought using the MTS assay. Figure 1Go shows that exposure to ACTH alone for 72 h did not induce proliferation in contrast to the potent effect of serum. ACTH at various concentrations in the presence of serum appeared to show a trend toward a dose-dependent effect, but this was not different to that obtained with serum alone.


Figure 1
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  		FIG. 1. Influence of ACTH on H295R cell proliferation. Cell proliferation was measured using a formazan dye proliferation assay. ACTH alone ({circ}) was without effect in contrast to serum ({blacksquare}), compared with unstimulated cells ({square}). The effect of combining ACTH at various concentrations with serum is shown (bullet) but is not statistically different from serum alone (means ± SEM, n = 3).

 
H295R cells were stimulated with ACTH (10–7 M) or forskolin (10–5 M) in the presence of 3-isobutyl-1-methylxanthine and harvested for cAMP assay. As seen in Fig. 2AGo, forskolin induces a substantial cAMP response indicating that adenylate cyclase is present and fully functional in these cells. ACTH induces a modest, although significant, response. Ligand binding studies confirm the presence of specific ACTH binding sites.


Figure 2
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  		FIG. 2. Induction of Erk phosphorylation and activation in H295R cells. A, Thirty minutes treatment with ACTH (10–7 M) has a small but significant effect in stimulating cAMP production (***, P < 0.001). Forskolin (Fsk; 10–5 M) in contrast induces a strong cAMP response. Un, Uninduced control. B, ACTH (10–7 M) stimulates ERK1/2 phosphorylation within 5 min, although this effect decays within 30 min. Densitometric estimation of the phospho-ERK1/2 response from three identical experiments is shown below the autoradiograph (means ± SEM, n = 3). C, ACTH dose-response curve 5 min after stimulation. D, NDP-{alpha}-MSH (10–7 M) is without effect in stimulating phospho-ERK1/2, despite the reproducible response to ACTH (10–7 M). E, Gal4/Elk assay shows a small but significant effect of ACTH, which contrasts with the substantial and sustained action of serum (10% FBS) (**, P < 0.01; *, P < 0.05). This ACTH effect is inhibited by the MEK inhibitor, UO126 (10–6 M) (*, P < 0.05).

 
ACTH (10–7 M) induced an immediate ERK1/2 response that had effectively disappeared by 30–60 min (Fig. 2BGo). Dose-response studies suggest an EC50 of approximately 3 x 10–9 M (Fig. 2CGo). RT-PCR analysis demonstrated that H295R cells expressed the MC1R in addition to the MC2R and that because ACTH is an agonist for the MC1R, the possibility arose that it was this receptor that mediated the ERK1/2 response. However, the absence of any ERK1/2 response to NDP-MSH (10–7 M) indicates that the MC2R mediates this action because this receptor is insensitive to NDP-MSH (Fig. 2DGo).

The possibility of a transcriptional response to ERK activation was investigated using the Gal4/ELK assay. This demonstrated a transcriptional response in the first 30 min of stimulation, which was not sustained, in contrast to that induced by serum. This effect was shown to be ERK1/2 dependent because it was blocked by the MAPK/ERK kinase (MEK) inhibitor UO126 (10–6 M) (Fig. 2EGo).

An influence of ACTH on other MAPKs was sought. No phosphorylation of c-Jun N-terminal kinase (JNK) in response to ACTH (10–7 M) could be detected despite readily detectable levels of total JNK in cells. As a positive control, a potent phospho-JNK response to osmotic stress (0.4 M sucrose) was seen. p38 MAPK was not detectable in H295R cells. In addition, no change in AKT phosphorylation was seen in response to ACTH (data not shown).

We next sought to identify a mechanism whereby ACTH stimulated ERK1/2. Although the cAMP response to ACTH was modest, we attempted to mimic this elevation of cAMP using a submaximal dose of forskolin. At 10–6 M forskolin, a comparatively similar cAMP stimulation to that obtained with 10–7 M ACTH was seen (Fig. 3AGo). The protein kinase A inhibitor, H89 (10–5 M), was only partially effective at reducing the forskolin response but ineffective at reducing the ACTH-induced stimulation of ERK1/2 in a 5-min incubation (Fig. 3BGo).


Figure 3
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  		FIG. 3. The role of the cAMP pathway in transducing the ACTH-MAPK signal. A, Forskolin (Fsk) at the lower concentration of 10–6 M is approximately equivalent to ACTH in stimulating cAMP production (30 min treatment) and generates a similar phospho-ERK1/2 response. B, The protein kinase A inhibitor, H89 (10–5 M) reduces the forskolin-induced phosphorylation of ERK1/2, but has no effect on the ACTH-induced phosphorylation of ERK1/2 (5 min incubations with stimulus). A representative blot is shown with densitometry of three experiments normalized to the uninduced control (un; means ± SEM, n = 3).

 
Alternative signaling pathways were explored using the calcium chelator, BAPTA (5 x 10–5 M), the calcium channel blocker, nifedipine (10–5 M) (Fig. 4AGo), and the protein kinase C inhibitors GF109203X (5 x 10–6 M) (Fig. 4BGo) and calphostin C (10–6 M, data not shown), despite inhibiting activation by phorbol-12-myristate-13-acetate (10–7 M). None of these inhibitors influenced ERK1/2 stimulation.


Figure 4
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  		FIG. 4. Effect of calcium and protein kinase C blockade on ACTH-induced MAPK signaling. A, Neither BAPTA (5 x 10–5 M) nor the calcium channel blocker, nifedipine (10–5 M), appears to reduce the stimulation of phospho-ERK production by ACTH. B, The protein kinase C antagonist GF109203X (5 x 10–6 M) is capable of completely blocking the phorbol-12-myristate-13-acetate (PMA; 10–7 M)-induced MAPK response in H295R cells but has no effect on the ACTH induction of MAPK. All inductions were 5-min treatments. For each experiment, representative blots are shown along with densitometry of three experiments normalized to the uninduced control (un; means ± SEM, n = 3).

 
G protein-coupled receptors have been shown in many cases to induce a mitogenic signal by transactivation of a growth factor receptor. However, use of the tyrosine kinase inhibitor, genistein (5 x 10–5 M), or the EGF receptor tyrosine kinase inhibitor, AG1478 (10–6 M), had no influence on ERK1/2 activation by ACTH, despite effectively inhibiting the ERK1/2 induction by EGF (10–8 M) in the same cells (Fig. 5Go, A and B). There is increasing evidence for G protein-independent MAPK signaling mediated by an arrestin acting as a scaffold for c-Src or its related family members. However, immunoblotting cells stimulated with ACTH with an antibody that recognizes phospho-Src shows no evidence of phosphorylation, in contrast to that found after exposure to serum (Fig. 5CGo). Furthermore, two selective Src family inhibitors, PP2 (10–6 M) and SU6656 (10–6 M), had no influence on ERK1/2 activation (Fig. 5DGo).


Figure 5
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  		FIG. 5. Effect of ACTH on tyrosine kinase pathway signaling. A, The tyrosine kinase inhibitor, genistein (5 x 10–5 M) reduces the phospho-ERK1/2 signal generated by EGF (10–8 M) but has minimal effect on the ACTH-induced signal. B, Similarly, the EGF receptor tyrosine kinase inhibitor, AG1478 (10–6 M), fails to inhibit the ACTH signal. C, c-Src can be shown to be phosphorylated in response to serum (10% FBS) but not in response to ACTH. D, Furthermore, the c-Src inhibitors, PP2 (10–6 M) or SU6656 (10–6 M), do not block ACTH-induced MAPK signaling. All inductions were 5-min treatments. Representative blots are shown along with densitometry of three experiments normalized to the uninduced control (un; means ± SEM, n = 3 for A and D, n = 4 for B).

 
Finally, the role of receptor internalization was investigated. In previous ACTH internalization studies, 0.45 M sucrose has proven to be an effective inhibitor of internalization. However, when used in these studies, it appears to have had a potent ERK1/2 stimulation effect independently of ACTH, as did potassium depletion (data not shown). As an alternative, the transglutaminase inhibitor, dansylcadaverine (3 x 10–4 M) was studied. As shown in Fig. 6Go, this significantly suppressed the ERK1/2 response to ACTH but was ineffective at blocking the response to serum. The effectiveness of dansylcadaverine in inhibiting internalization was confirmed using cell surface labeling of the MC2R with EZ-Link Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL) as described (29) (data not shown).


Figure 6
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  		FIG. 6. Inhibition of receptor internalization blocks ACTH-induced MAPK signaling. Dansylcadaverine (3 x 10–4 M) is effective at blocking the phospho-ERK1/2 response to ACTH throughout a 60-min time course but is ineffective at blocking the response to serum (10% FBS) at 5 min. Representative blots are shown along with densitometry of three experiments in which the ACTH and serum responses are quantified at 5 min, normalized to the uninduced control (means ± SEM, n = 4). *, P < 0.05, compared with treatment without inhibitor.

 
Thus, to summarize, ACTH induces a transient ERK1/2 response in the H295R cell. This appears to be mediated via the MC2R, which is expressed in relatively low numbers in these cells (25, 30). NDP-MSH, an agonist for the MC1R, MC3R, MC4R, and MC5R, is inactive in stimulating ERK1/2. cAMP, the generally acknowledged second-messenger pathway for the MC2R (1, 2), produces only a modest response to ACTH in this cell line, despite the presence of an effective adenylate cyclase cAMP-generating system. Distinct differences in the cellular response to MAPK in relation to the duration of its activation have been widely observed. Typically, sustained responses are associated with cellular differentiation (31, 32). MAPK kinetics in response to a particular agonist may relate to the number of receptor sites (32), and it is perhaps not surprising that ACTH only induces a transient response in this cell line. The basis for this distinction seems to lie in the need for stabilization by C-terminal phosphorylation of c-Fos, which occurs only in response to a sustained stimulus (33). This is entirely consistent with our demonstration of a short-term transcriptional response to ACTH in the Gal4/ELK assay, which contrasts with the long-term response found with a 10% serum stimulus. Again, consistent with this model, ACTH alone in contrast to serum does not induce cell proliferation. ACTH in the presence of serum may have a small but statistically insignificant enhancing effect. It has been shown that ACTH can inhibit the mitogenic response of Y1 cells to fibroblast growth factor-2 by activation of a pathway downstream of ERK1/2 involving dephosphorylation of Akt, induction of the cyclin-dependent kinase inhibitor p27Kip1, and degradation of c-Myc (23, 34, 35). It is possible that these are a consequence of the overexpression of Ki-Ras in this cell line because the antimitogenic effects are not observed in the H295R cells. This remains to be determined. Clearly, however, the lack of mitogenic effect of ACTH on the H295R cell line is not consistent with the proposed mitogenic effect of ACTH on the adrenal cortex in vivo, although it has been suggested that these effects are indirect via the induction of other growth factors (7, 36). The effects of ACTH on steroidogenesis have been observed in vitro as well as in vivo, and future studies will examine whether ERK1/2 activation plays a role in this process.

Having established the presence of an ERK1/2 response to ACTH, our attention focused on the mechanism by which this signal was activated. ACTH is only a weak activator of cAMP production in H295R cells. Even a suboptimal dose of forskolin (10–6 M) was more potent than ACTH in this respect, yet it produces a similar ERK1/2 response. This would be highly suggestive of a common mechanism were it not for two pieces of information. First, the protein kinase A inhibitor H89 was an effective if incomplete inhibitor of the forskolin effect but had little or no influence on the ACTH induction of ERK1/2, and second, the adenylate cyclase inhibitor, SQ22536, had no effect on ACTH-induced ERK1/2 signaling (data not shown). We therefore concluded that whereas cAMP production and activation of protein kinase A and possibly exchange proteins activated by cAMP (EPAC) is the probable mechanism of forskolin induction of ERK1/2, ACTH action largely bypasses this route (37), consistent with results seen in protein kinase A-deficient Y1 cells (24, 38).

Alternative pathways for ACTH induction of ERK1/2 were therefore sought. There is evidence from primary cultures of adrenal cells that ACTH can induce calcium influx via opening of plasma membrane calcium channels (4, 5, 6). This might in turn enhance protein kinase C activation and hence the Ras/Raf/MEK pathways. However, these possibilities seem unlikely in view of the inability of a calcium chelator (BAPTA), a calcium channel blocker (nifedipine), and two protein kinase C inhibitors (calphostin C and GF109203X) to influence ACTH-induced ERK1/2 activation.

There has been great interest in the use of surrogate mitogenic signaling mechanisms by various G protein-coupled receptors. For example, the EGF receptor could be activated by a number of agonists acting through their cognate G protein-coupled receptors by a direct transactivation process (39, 40, 41). Other growth factor receptors were also shown to be transactivated in this way (42, 43). Alternatively, EGF receptor ligands could be cleaved from the cell surface by means of activation of a metalloprotease by certain G protein-coupled receptors (44, 45). The EGF ligand could then activate EGF receptor signaling and the Ras/Raf/MEK pathways. In the case of the H295R cell, however, we were unable to demonstrate any influence of either a specific EGF receptor kinase inhibitor (AG1478) or a nonspecific receptor tyrosine kinase inhibitor (genistein). GM6001, a metalloprotease inhibitor, was effective in reducing background levels of phospho-ERK1/2 but does not significantly reduce the ACTH-induced ERK1/2 response (data not shown).

A further pathway of MAPK signal activation by G protein-coupled receptors involves nonreceptor tyrosine kinases such as c-Src or its homologues as intermediates (46, 47, 48). We were unable to demonstrate c-Src phosphorylation in response to ACTH, and neither of the two Src family inhibitors studied, PP2 and SU6656, altered the ERK1/2 response to ACTH.

In the case of several G protein-coupled receptors, a requirement for receptor internalization has been described (49). Arrestin-G protein-coupled receptor complexes appear to have a central role in this phenomenon, acting as adapters with not only clathrin but also a variety of other molecules involved in signal generation, particularly via MAPK pathways (50, 51, 52, 53). We have previously shown that in mouse Y1 cells, the MC2R is internalized relatively inefficiently via clathrin-coated pit mechanisms that may be inhibited with hyperosmolar sucrose (54). The effect of this treatment was tested on H295R cells but had a pronounced nonspecific effect on ERK1/2 induction in the absence of ACTH, presumably as a result of the osmotic stress on the cell. An alternative inhibitor of internalization in the form of dansylcadaverine was tested, and this was found to be an effective inhibitor of ACTH-induced but not serum-induced ERK1/2 activation, implying the involvement of receptor internalization in the ACTH-mediated activation pathway.

In summary, ACTH induces a transient activation of the ERK1/2 pathway in the H295R cell line. This appears to be dependent on receptor internalization, but identification of specific signaling pathways has proved elusive. Pathways that have been shown to be active for many other G protein-coupled receptors, i.e. c-Src, EGF receptor transactivation, protein kinase C, do not appear to be involved in the case of the MC2R. cAMP signaling may provide a partial explanation for the ERK1/2 signal but is apparently independent of protein kinase A and thus may involve exchange proteins activated by cAMP-dependent signals. Thus, it seems likely that another less well-recognized ERK1/2 signaling pathway, possibly dependent on an internalization complex, is involved. G protein-coupled receptor internalization via arrestin-independent, clathrin-dependent mechanisms has begun to emerge (55) and might be responsible for MC2R internalization, which appeared to be G protein receptor kinase dependent but arrestin independent in our earlier studies with the mouse Y1 cell (54). Consistent with these atypical features of the internalization of the MC2R, we recently reported that the H295R cell MC2R interacts with nucleoporin 50 and translocates to the nucleus with a time course similar to that of receptor internalization (21).


   Footnotes
 
This work was funded by a Ph.D. studentship awarded by the Research Advisory Board of the Barts and the London Charity (to M.E.J.).

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online January 3, 2008

Abbreviations: BAPTA, Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid; EGF, epidermal growth factor; FBS, fetal bovine serum; JNK, c-Jun N-terminal kinase; MCR, melanocortin receptor; MEK, MAPK/ERK kinase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; NDP, [Nle4, D-Phe7]; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine; POMC, proopiomelanocortin; SFM, serum-free medium.

Received July 12, 2007.

Accepted for publication December 26, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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J. Parmar, R. E. Key, and W. E. Rainey
Development of an Adrenocorticotropin-Responsive Human Adrenocortical Carcinoma Cell Line
J. Clin. Endocrinol. Metab., November 1, 2008; 93(11): 4542 - 4546.
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