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Originally published In Press as doi:10.1074/jbc.M507595200 on November 4, 2005

J. Biol. Chem., Vol. 281, Issue 1, 121-128, January 6, 2006
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The Guanine Nucleotide Exchange Factor CNrasGEF Regulates Melanogenesis and Cell Survival in Melanoma Cells*{boxs}

Eva M. Amsen1, Nam Pham2, Youngshil Pak2, and Daniela Rotin3

From the Program in Cell Biology, The Hospital for Sick Children, and the Biochemistry Department, University of Toronto, Toronto, Ontario M5G 1X8, Canada

Received for publication, July 13, 2005 , and in revised form, October 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
cAMP-dependent Ras activation has been demonstrated in numerous cell types, particularly of neuronal (including melanoma cells) and endocrine origin, but the Ras activator involved has not been identified. In B16 melanoma cells, cAMP activates the Ras/Erk pathway, leading initially to stimulation but subsequently to long term (>24-h) inhibition of melanogenesis (dendrite extension and melanin production). Here we identify CNrasGEF as the Ras guanine nucleotide exchange factor (GEF) involved. We demonstrate that CNrasGEF is expressed endogenously in B16 melanoma cells and that cAMP-mediated activation of Ras and Erk1/2 in these cells can be augmented by CNrasGEF overexpression and reduced by its knockdown by RNA interference. Moreover, we show that CNrasGEF participates in the regulation of melanogenesis. Knockdown of CNrasGEF leads to increased dendrite extension and melanin production observed ~50 h after forskolin/isobutylmethylxanthine treatment, suggesting that CNrasGEF inhibits melanogenesis in the long term. Independently, we find that overexpression of CNrasGEF leads to apoptosis, whereas its knockdown by RNAi enhances cell proliferation, independent of cAMP. Collectively, these results suggest that CNrasGEF regulates melanogenesis but that it also has a distinct role in regulating cell proliferation/apoptosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
cAMP exhibits differential mitogenic effects in different cell types (1). In several endocrine, neuronal, or Schwann cells and in some 3T3 cells, cAMP promotes cell proliferation, often by activating Ras. For example, in thyroid cells, cAMP-stimulated mitogenesis following thyroid stimulating hormone binding to its receptor is mediated via Ras activation, independent of protein kinase A (2). Moreover, cAMP-dependent activation of Erk in NIH-3T3 cells was recently demonstrated to be carried out independently of Rap1-Epac and instead was proposed to be mediated via Ras activation (3). The cAMP-dependent Ras activator involved in these cases has not been identified. Recently, a pathway for cAMP-dependent Ras/Erk activation (independent of protein kinase A or Rap1) has been proposed in melanoma cells (4).

Melanocytes are specialized epidermal cells of neurocrest origin that synthesize melanin and are responsible for skin pigmentation and protection from UV radiation (5, 6). Melanoma cells are transformed melanocytes that give rise to a very aggressive skin cancer, melanoma. B16 mouse melanoma cells, particularly B16-F10, have been characterized extensively with regard to their tumorigenic and metastatic potential (e.g. Refs. 7–9) and the signaling pathways responsible for melanin production (5). Melanin synthesis takes place in intracellular organelles called melanosomes. Upon stimulation by UV radiation, several factors are released, including {alpha}-melanocyte-stimulating hormone (10), a strong melanogenic factor that acts by binding to the melanocortin receptor, MC1R. MC1R is a G protein-coupled receptor coupled to G{alpha}s, causing elevation of intracellular cAMP upon ligand binding. cAMP is a critical component of melanogenesis, which comprises both melanin synthesis and movement of melanosomes to newly formed dendrites at the cell periphery, from where melanin can be released and distributed. The melanogenic effects of {alpha}-melanocyte-stimulating hormone can be mimicked by elevation of intracellular cAMP (e.g. with forskolin and IBMX)4 (5). The rate-limiting enzyme in melanin synthesis is tyrosinase (11).

Earlier work has investigated the role of cAMP in the signaling pathways regulating melanogenesis. Activation of protein kinase A and cAMP-response element-binding protein by elevated cAMP levels leads to production of the microphthalmia transcription factor (12, 13), in turn causing activation of transcription of the tyrosinase gene promoter (14, 15), thus promoting melanogenesis. However, cAMP-dependent and protein kinase A-independent pathways have also been linked to the regulation of melanogenesis, including the phosphatidylinositol 3-kinase and Ras/Erk pathways (4). Ballotti and colleagues (4, 5, 16, 17) have demonstrated that contrary to many other cells types, cAMP activates Ras (but not Rap1) in B16 melanoma cells, leading to activation of B-Raf, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), and Erk. Ras/Erk activation in these cells has two effects: a short term stimulatory effect, whereby Erk phosphorylates microphthalmia transcription factor (on Ser73), leading to increased transcriptional activity of the tyrosinase promoter (14, 17, 18), and a long term (>24-h) effect, which results in inhibition of melanogenesis. The latter effect was unraveled by demonstrating sustained inhibition (observed after 48 h) of melanogenesis with a specific mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor (PD98059) or overexpression of constitutively active Ras or mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (16). This is believed to be caused by phosphorylation of microphthalmia transcription factor (also on Ser73), resulting in its ubiquitination and proteasomal degradation, leading to attenuation of melanogenesis (5, 19).

Based on the critical role played by cAMP-mediated Ras activation in melanogenesis, Busca et al. (4) have proposed the existence of a cAMP-dependent Ras activator (guanine nucleotide exchange factor; GEF) in melanoma cells. We and others have previously identified a guanine nucleotide exchange factor, called CNrasGEF (also known as PDZ-GEF1, nRapGEF, RA-GEF, and RAPGEF2) (20–23). Our previous work showed that when expressed heterologously in HEK293T cells, CNrasGEF can activate Ras in response to elevation of cAMP levels (independent of protein kinase A), achieved by treatment with 8-Br-cAMP or forskolin plus IBMX (20) or by agonist (isoproterenol) stimulation of co-expressed b1 adrenergic receptor, a G protein-coupled receptor that activates G{alpha}s and leads to elevation of intracellular cAMP levels (24). CNrasGEF can also activate Rap1, independent of cAMP (20–23). We thereforeinvestigatedwhetherCNrasGEFcouldparticipateinthecAMP-dependent Ras activation in melanoma cells. Our work here shows that CNrasGEF is highly expressed in B16 melanoma cells, it can activate Ras and Erk1/2 in these cells upon cAMP elevation, and it plays an important role in regulating melanogenesis. We also show that CNrasGEF regulates cell survival of B16 cells, an effect not dependent on cAMP.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs and Vectors—FLAG-tagged WT-CNrasGEF and FLAG-tagged {Delta}CDC25-CNrasGEF (lacking the catalytic Ras/Rap1-activating domain) were expressed from a pCMV5 vector as described previously (20). Constructs expressing GFP-tagged WT- and {Delta}CDC25-CNrasGEF were generated by cloning the respective cDNA sequences from the pCMV5 constructs into pEGFP-c3 (WT) or pEGFP-c2 ({Delta}CDC25-CNrasGEF). A 64-bp oligonucleotide was inserted into the multiple cloning sites of pSuper-EGFP, a modified pEGFP-c1 vector (Clontech) that incorporates the promoter from pSuper H1 RNA flanked by multiple cloning sites (kindly provided by Dr. C. C. Hui, The Hospital for Sick Children). The 64-bp oligonucleotide was designed to form a small hairpin RNA targeting the following sequence unique to the N-terminal region of mouse CNrasGEF (mKIAA0313): AAACTGCACCTCACTGACAGC. As a control, a scrambled version of the above oligonucleotide was designed, containing the following sequence (not recognizing any coding regions in the mouse genome): AAGAACCTGTCAGCCACCTAC. The oligonucleotide strands were purchased from Sigma Genosys and were annealed and phosphorylated before cloning into pSuper-EGFP.

pCGT vectors containing T7-tagged V12-H-Ras or N17-H-Ras were kindly provided by Dr. Dafna Bar-Sagi (State University of New York, Stony Brook, NY).

Cell Culture and Transfections—B16-F10 murine melanoma cells (hereafter called B16 cells), purchased from ATCC, were cultured in Dulbecco's modified Eagle's medium containing 10% FBS and 100 units of penicillin, 100 µg of streptomycin. Cells were maintained at 37 °C and 5% CO2. Cells were transfected using Lipofectamine according to the manufacturer's (Invitrogen) instructions.

Analysis of Cell Proliferation and Morphology—Cells were transfected with pEGFPc1, pSuper-EGFP-CNrasGEF-RNAi (hereafter called GFP-CNrasGEF-RNAi or CNrasGEF-RNAi for short), pEGFPc3-WT-CNrasGEF (called WT-CNrasGEF), or pEGFPc2-{Delta}CDC25-CNrasGEF (called {Delta}CDC25-CNrasGEF) and sorted by fluorescence-activated cell sorting (FACS) the next day to obtain a homogeneously transfected population, since transfection efficiencies ranged from ~30 to 80%. The GFP-positive cells were reseeded at low density onto 6-well plates in medium containing 2% FBS and penicillin/streptomycin, since complete serum depletion did not allow the cells to survive for more than 24 h. After attaching overnight, cells were stimulated (or not) with 20 µM forskolin and 20 µM IBMX (Sigma) for periods of up to 72 h.

Cells were photographed using a Leica DM IRE2 microscope with Openlab software at different sections of the wells, at 0, 8, 24, 48, and 72 h after stimulation, returning to the same cells at every time point. Cells with and without dendrites were counted, where "dendrites" were cell extensions that appeared over time. The percentage of cells with dendrites was calculated, and the 95% confidence interval for proportions of cells with dendrites (, where p represents proportion and n is total number of cells) was determined as described (25) and shown as error bars where appropriate (see Fig. 3).

To construct growth curves, the total cell number/mm2 was plotted against growth time. The 95% confidence interval for counted cells (, where n = total number of cells) was determined, corrected for cells/mm2, and shown as error bars where applicable (25). The growth rate was determined by normalizing the number of cells/mm2 to the number obtained at the first measured time point, calculating the 2log value, and plotting against time.

Short term dendrite formation was analyzed as follows. B16 cells were transfected as described above. Transfected cells were recovered for 15 h in Dulbecco's modified Eagle's medium with 10% FBS, penicillin/streptomycin, and with or without 20 µM forskolin/IBMX. Cells were analyzed using a Leica DM IRE2 microscope with Openlab software by counting GFP-fluorescent cells with and without dendrites, as described above.

Apoptosis Analysis—Cells were transfected and sorted as described above and reseeded onto 4-well polystyrene vessel glass slides (BD Falcon). After 20–24 h, cells were fixed in 1% paraformaldehyde in phosphate-buffered saline (pH 7.4) and permeabilized using ethanol/acetic acid (2:1). Fixed cells were stained for DNA damage using the ApopTag Red kit (Chemicon) according to the manufacturer's protocol, and mounted using Vectashield with 4',6-diamidino-2-phenylindole (Vector Laboratories). Cells were visualized and photographed using a Leica DM IRE2 microscope with Openlab software. Cells stained for apoptosis were counted and presented as percentage of total cells. The 95% confidence interval for proportions , where p represents proportion, and n is the total number of cells) was determined as described (25).

Determination of Melanin Production—B16 cells were grown to 50% confluence and transfected (or not) with CNrasGEF-RNAi or the scrambled control as described above. The next day, cells were reseeded 1:2 into new dishes, and the medium was replaced with Dulbecco's modified Eagle's medium containing 2% FBS, penicillin/streptomycin, and with or without 20 µM forskolin/100 µM IBMX. After 54 h of stimulation, cells were collected by trypsinization and divided into two equal batches. Half of the collected cells were lysed for protein analysis as described below. The other half was used to isolate melanin, according to a previously described protocol with slight modifications (26). Briefly, cells were washed twice in phosphate-buffered saline, resuspended in 200 µl of double-distilled H2O and 1 ml of ethanol/ether (1:1), and incubated at room temperature for up to 1 h to remove impurities. Subsequently, the pellet was resuspended in 0.5 ml of 1 N NaOH, 10% Me2SO, and melanin was dissolved for 15–30 min at 70 °C. Melanin absorption was measured with a Beckman Coulter DU640B spectrophotometer at 470 nm.

To determine the effect of Ras on melanin synthesis, cells were transfected with V12-Ras or N17-Ras and collected 48 h after stimulation with forskolin/IBMX. Melanin was isolated as described above.

Western Blot Analysis—For protein analysis in growth, morphology, and melanin production experiments, cells were washed once with phosphate-buffered saline and lysed in lysis buffer (150 mM NaCl, 50 mM Hepes, 1% Triton X, 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 1 mM phenylmethylsulfonyl fluoride). Total protein levels were determined using the Bio-Rad protein assay. An antibody generated against the carboxyl terminus of human CNrasGEF (20) was used to detect mouse CNrasGEF in all experiments. {beta}-Actin (antibody from Sigma) was used as a loading control and to ensure that knockdown (where applicable) was not affecting all proteins in the cell nonspecifically. Relative CNrasGEF levels were quantified as protein band intensities using FluorChem densitometry software and normalized to {beta}-actin levels.

Ras Activation Assay—B16 cells were transfected, serum-starved overnight, and then subjected to treatments with 500 µM 8-Br-cAMP (Sigma) for 15 min. Cells were lysed with lysis buffer (25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 25 mM NaF, 10 mM MgCl2, 1 mM EDTA, 1 mM NaVO4, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 250 µM phenylmethylsulfonyl fluoride), and the level of Ras-GTP in the lysates was determined using an activation-specific probe, as described (27). Briefly, to determine the levels of active Ras (Ras-GTP) in cells, agarose-bound glutathione S-transferase fusion protein of the Ras-binding domain of Raf-1 (Upstate%20Biotechnology">Upstate Biotechnology Inc.) was used to precipitate Ras-GTP from cell lysates, and the amount of Ras-GTP was determined by immunoblotting with anti-H/N-Ras antibodies (Quality Biotech), since our unpublished work5 demonstrated endogenous expression of N- and H-Ras (but not K-Ras) in B16 cells. Relative Ras and CNrasGEF levels were quantified as protein band intensities using FluorChem densitometry software and normalized to total Ras levels.

Analysis of Erk1/2 Activation—B16 cells were transfected with GFP-CNrasGEF-RNAi, GFP-scrambled RNAi (control), and GFP-WT-CNrasGEF or GFP-{Delta}CDC25-CNrasGEF and FACS-sorted the next day. Sorted cells were reseeded in 6-well dishes and grown in Dulbecco's modified Eagle's medium containing 20% FBS for 24 h before serum starvation overnight. To study the effect of cAMP elevation, cells were stimulated (or not) with 20 µM forskolin/100 µM IBMX for 15 min and lysed on ice in a buffer with phosphatase inhibitors (100 mM NaCl, 50 mM Hepes, pH 7.4, 1% Triton X-100, 1 mM NaVO4, 5 mM EDTA, 50 mM NaF, 1 mM NaPPi, 10 mM p-nitrophenyl phosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Western blot was carried out on these lysates using antibodies against CNrasGEF and phosphorylated Erk1/2 (Promega). Total Erk1/2 levels were determined after stripping the blot and reprobing with an antibody against Erk1/2 (Promega).

cAMP Production Assay—B16 cells were cultured as described above, and grown overnight in medium with different serum concentrations (0, 2, or 10% FBS). Cells were stimulated (or not) with 20 µM forskolin/IBMX for 1 h or 500 µM 8-Br-cAMP for 15 min. Intracellular cAMP levels were measured using the cAMP Biotrak enzyme immunoassay system (Amersham Biosciences) according to the manufacturer's protocol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
CNrasGEF Is Expressed Endogenously in B16 Melanoma Cells and Is Able to Activate Ras and Erk1/2 in These Cells—To study the role of CNrasGEF in B16 cells, we first tested the level of its endogenous expression in these cells. As seen in Fig. 1, CNrasGEF is highly expressed in B16 (F10) melanoma cells relative to other cell lines analyzed. We have previously shown that overexpressed CNrasGEF in HEK293T cells activates Ras upon cAMP elevation (20, 24). Here we tested whether it can also activate Ras in B16 cells in response to cAMP. Fig. 2, A and C, demonstrates that treatment of B16 cells with the membrane-permeant analogue of cAMP, 8-Br-cAMP, leads to Ras activation, in agreement with previous studies of Busca et al. (4). Overexpression of CNrasGEF in these cells causes an increase in Ras activation, which is further augmented by treatment with 8-Br-cAMP (Fig. 2A). Thus, CNrasGEF can enhance Ras activation in B16 melanoma cells.



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  		FIGURE 1.
Endogenous expression of CNrasGEF in B16 melanoma cells. Cell lysate (50 µg of protein/lane) from HEK293T (293T), NG108-15 (NG108), or B16-F10 (B16) cells, as well as HEK293T cells overexpressing CNrasGEF, were immunoblotted with anti-CNrasGEF antibodies (right) or preimmune serum (left). The rightmost lane (HEK293T-transfected) represents a lower exposure time than the rest of the autoradiogram.

 
To further analyze the requirement of CNrasGEF for Ras activation in B16 cells, we knocked down CNrasGEF in these cells by RNAi. To achieve knockdown, we utilized a modified pEGFPc1 vector with an incorporated pSuper H1 RNA promoter and multiple cloning sites, where we introduced the CNrasGEF-RNAi hairpin oligonucleotide (see "Experimental Procedures"). This vector (called GFP-CNrasGEF-RNAi, or CNrasGEF-RNAi for short) thus expresses EGFP from a separate (cytomegalovirus) promoter than the RNAi. As seen in Fig. 2, overexpression of the CNrasGEF-RNAi construct in B16 cells resulted in a substantial reduction in the amount of CNrasGEF (but not of total Ras), suggesting that our knockdown strategy is effective. Some (variable) nonspecific knockdown due to overexpression of GFP alone or the scrambled control was also seen (Fig. 2, B and C, top), but it was not as pronounced as the knockdown by the specific CNrasGEF-RNAi. On average, specific knockdown by RNAi accounted for a ~35% difference in CNrasGEF levels compared with the scrambled control. Transfection efficiency of both knockdown construct and scrambled control was ~40–50%. We then tested the effect of knockdown of CNrasGEF on cAMP-stimulated Ras activation in these cells. FACS to isolate only the GFP-expressing cells could not be performed for these experiments, because the yield of sorted cells is too low for biochemical assays measuring Ras activation. Nevertheless, Fig. 2, A and C, demonstrates that reduction of CNrasGEF levels with RNAi resulted in a corresponding reduction in cAMP-dependent Ras activation. When normalized to levels of total Ras and actin, activated Ras levels after RNAi and stimulation with 8-Br-cAMP were ~50% lower than levels in untransfected cells (n = 7) and ~25% lower than in cells transfected with the scrambled control (n = 2).

To examine the effect of CNrasGEF on the activation of Erk1/2, we transfected cells with GFP-tagged constructs expressing wild type CNrasGEF, the inactive {Delta}CDC25 mutant, CNrasGEF-RNAi, or the scrambled control. Cells were FACS-sorted and reseeded the day after transfection in order to measure the levels of activated Erk1/2 in transfected cells only. Fig. 2D (top) shows that, consistent with its activation of Ras, CNrasGEF mediates the cAMP-dependent activation of Erk1/2. In response to forskolin/IBMX, the levels of phosphorylated Erk1/2 increase in untransfected cells. Overexpression of WT-CNrasGEF (but not the catalytically inactive {Delta}CDC25 mutant) further augments the cAMP-dependent increase of phosphorylated Erk1/2. Knockdown of CNrasGEF with RNAi (with ~30–80% efficiency relative to scrambled controls) reveals a reduction of ~10–40% in levels of phosphorylated Erks (normalized to total Erk) (Fig. 2D, bottom), with higher levels of knockdown resulting in greater reduction of phospho-Erk levels. The reduction was stronger with Erk1 (top band) than Erk2.



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  		FIGURE 2.
CNrasGEF leads to cAMP-dependent Ras and Erk1/2 activation in B16 melanoma cells. A–C, activation of Ras by CNrasGEF. B16 cells were transfected (or not) with wild type FLAG-tagged CNrasGEF (Flag-CNrasGEF), CNrasGEF-RNAi, or a scrambled sequence used as a control for the RNAi (B). Cells were then treated (or not) with the cAMP analogue, 8-Br-cAMP, and activated Ras (Ras-GTP) was precipitated from lysates with the glutathione S-transferase fusion protein of the Ras-binding domain of Raf-1. The lower panels in A and the top and bottom panels in C represent controls for total Ras, amount of transfected CNrasGEF (immunoblotted with anti-FLAG antibodies), and amount of endogenous CNrasGEF, depicting knockdown of CNrasGEF with RNAi and some nonspecific knockdown with the scrambled control. There was some variability in the extent of knockdown by the scrambled control (compare B and C, top), but it was consistently smaller than the CNrasGEF-RNAi. The knockdown of CNrasGEF in B and C depicted is from cells treated with 8-Br-cAMP, but a similar effect was seen with untreated cells. Results shown in A–C are representative of seven experiments. Cells were not FACS-sorted for analysis of Ras activation. D, activation of Erk1/2 by CNrasGEF. Cells were transfected (or not) with GFP-WT-CNrasGEF, GFP-{Delta}CDC25-CNrasGEF, or GFP-tagged CNrasGEF-RNAi or a scrambled control. The population of transfected cells was enriched by FACS sorting. Sorted cells were treated (or not) with forskolin/IBMX. Active Erks were detected using Western blot with an antibody against phosphorylated Erk1/2, and levels were normalized to levels for total Erk1/2, detected after stripping and reprobing of the blot. The levels of endogenous or ectopic CNrasGEF were determined using an antibody against CNrasGEF. The top and bottom panels of Fig. 2D are representative of three experiments. Tfxn, transfection.

 
Taken together, these results suggest that CNrasGEF is able to activate Ras and Erk1/2 in B16 cells in response to cAMP stimulation and that its knockdown by RNAi reduces this activation.

CNrasGEF Inhibits the Sustained (Long Term) Dendrite Extension in B16 Melanoma Cells—Melanoma cells, including B16 cells, are derived from the neurocrest and extend dendrites, which are needed for pigment distribution. Dendrite extension and melanin production are the hallmarks of melanogenesis and are regulated by cAMP (5). The net stimulatory effect of cAMP on dendrite extensions is the combined result of several cAMP-dependent pathways: those stimulating dendrite extension and those inhibiting it. Upon prolonged cAMP stimulation, the Ras/ERK pathway has been shown to inhibit dendrite extension. To analyze the role of CNrasGEF in dendrite formation/extension, we transfected B16 cells with GFP alone or GFP-tagged CNrasGEF-RNAi, WT-CNrasGEF, or the catalytically inactive {Delta}CDC25-CNrasGEF, in which the CDC25 domain has been deleted and which cannot bind Ras. For these experiments, transfected cells were FACS-sorted to isolate only the cells that took up the corresponding (GFP-expressing) plasmids. Fig. 3A demonstrates the effectiveness of knockdown of CNrasGEF by overexpression of GFP-CNrasGEF-RNAi and FACS. Following FACS, cells were seeded and grown for 15 h in medium with low (2%) serum prior to the addition of forskolin plus IBMX, to stimulate cAMP production (Table 1). Dendrite extension was analyzed at 0 and 48 h poststimulation with forskolin/IBMX or in low serum medium alone at corresponding time points.



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  		FIGURE 3.
CNrasGEF inhibits long term dendrite formation in B16 melanoma cells. A, upper panel, knockdown of endogenous CNrasGEF following transfection with GFP-CNrasGEF-RNAi vector and FACS. The GFP-vector alone was used as a control. Knockdown in this example was ~70% after quantification. The lower panel depicts the same blot reprobed with {beta}-actin to verify equal protein loading and shows that the RNAi targeted CNrasGEF and did not knock down the expression of all cellular proteins nonspecifically. B, morphology of B16 melanoma cells after a 48-h treatment with forskolin/IBMX (e–h) or in the absence of forskolin/IBMX at the same time point (a–d). Cells were transfected with the GFP-vector alone (GFP) (a and e), CNrasGEF-RNAi (b and f), wild type CNrasGEF (WT-CNrasGEF) (c and g), or CNrasGEF lacking its catalytic CDC25 domain ({Delta}CDC25-CNrasGEF)(d and h), all GFP-tagged. Cells were FACS-sorted and reseeded as described above. The arrows depict examples of dendrites. C and D, quantification of cells with dendrites after 2 days in the absence (C) or presence (D) of forskolin/IBMX. The percentage of all unstimulated cells that show dendrite extension over time was analyzed as described under "Experimental Procedures." Only experiments in which knockdown exceeded 50% were included. The error bars represent 95% confidence intervals. Data are a summary of 2–5 independent experiments. Numbers in the bars indicate the total number of cells counted per condition. The asterisks indicate p values from {chi}2 tests (*, p < 0.05; ***, p < 0.0001).

 


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  		TABLE 1
cAMP (or its analogue) levels in B16 melanoma cells

Cells were grown in Dulbecco's modified Eagle's medium with the indicated amount of serum (FBS) and treatments. cAMP levels were measured as described under "Experimental Procedures."

 
Data for dendrite extension of control cells and cells with knocked down CNrasGEF were collected only from experiments in which knockdown exceeded 50%. Fig. 3B depicts the morphology of B16 melanoma cells after a 48-h treatment with forskolin/IBMX (bottom) or in low serum medium at the same time point. Extensive dendrite formation is observed in the stimulated cells expressing GFP, CNrasGEF-RNAi, or {Delta}CDC25-CNrasGEF, but not in cells overexpressing WT-CNrasGEF. The number of cells with and without dendrites under every condition were counted and represented as the percentage of cells with dendrites in Fig. 3, C and D. As seen in Fig. 3D, the percentage of cells that extended dendrites after a 48-h stimulation with forskolin/IBMX was increased in the CNrasGEF-knocked down cells relative to cells expressing GFP alone or cells overexpressing the catalytically inactive {Delta}CDC25-CNrasGEF mutant. In contrast, there was a strong reduction in the proportion of cells extending dendrites in cells overexpressing WT-CNrasGEF. Similar results (although more modest) were seen at 24 h, but not at an early time point (8 h) (not shown), after forskolin/IBMX stimulation, suggesting that the effect of CNrasGEF on inhibition of dendrite extension is a late effect. Although the effect of knocking down CNrasGEF on dendricity appears modest, it is both statistically significant ({chi}2 test p values <0.0001) and highly reproducible and may reflect the fact that the RNAi approach did not produce a complete knockdown. Moreover, in experiments where RNAi did not produce a visible knockdown, no increase in dendricity was observed. Dendrite extension in all transfected cells grown in the presence of 2% serum but in the absence of forskolin/IBMX treatment was quite low (Fig. 3C) and correlates with lack of elevation of intracellular cAMP levels (Table 1). Collectively, these results suggest that CNrasGEF is involved in the cAMP-dependent, sustained (long term) inhibition of melanogenesis. This inhibitory effect was not related to the proapoptotic effect of CNrasGEF (see below), because dendrite formation was measured only in living, nonapoptotic cells.

In addition to inhibiting melanogenesis in the long run, Ras has also been proposed to stimulate melanogenesis early (<15 h) after its activation by cAMP (17). Our results suggest that the early stimulatory effect is also regulated by CNrasGEF, since overexpression of CNrasGEF increased early dendrite formation, an effect requiring the presence of its CDC25 (catalytic) domain. However, cAMP stimulation of CNrasGEF did not further augment this short term dendrite formation (supplemental Fig. 1).

CNrasGEF Inhibits Melanin Production—To directly test melanin production, B16 melanoma cells transfected as above were treated with forskolin plus IBMX for 54 h and harvested (FACS sorting was not possible due to insufficient yield of sorted cells for spectrophotometrical detection of melanin produced). Melanin levels were analyzed at 470 nm, as described (26), and normalized for total protein concentration. As shown in Fig. 4A, melanin production in untransfected cells increased upon prolonged stimulation with forskolin/IBMX. Melanin levels were increased even further in cells in which CNrasGEF was knocked down with RNAi (Fig. 4A, inset). Use of a scrambled control revealed partial decrease in CNrasGEF levels (inset) and, accordingly, resulted in intermediate amounts of melanin produced. (The average percentage knockdown by CNrasGEF-RNAi was 57% relative to untransfected cells and 33% relative to the scrambled control). These data suggest that CNrasGEF inhibits long term melanin production in B16 melanoma cells. This effect was only seen in the presence of forskolin/IBMX, since melanin levels were similar in all unstimulated cells regardless of CNrasGEF levels. Similar results were obtained at 72 h poststimulation with forskolin/IBMX (not shown). These results are in agreement with the above data (Fig. 3) describing dendrite extension and again demonstrate the sustained inhibition of melanogenesis by CNrasGEF. This inhibition can also be mimicked by overexpression of a constitutively active V12-Ras but not by a dominant negative N17-Ras (Fig. 4B).

The effect of overexpression of wild type CNrasGEF on melanin synthesis could not be analyzed, since the apoptotic effect of CNrasGEF overexpression began at an earlier stage than the time point at which melanin production could be measured (see below).

CNrasGEF Inhibits Proliferation of B16 Cells and Promotes Their Apoptosis—In the course of our experiments, we noticed extensive cell death in cells overexpressing WT-CNrasGEF. To quantify the effect, we transfected B16 cells with GFP alone, WT-CNrasGEF, CNrasGEF-RNAi, or the catalytically inactive {Delta}CDC25-CNrasGEF (all GFP-tagged), FACS-sorted them, and analyzed the effect of CNrasGEF on cell proliferation. As shown in Fig. 5A, the rate of B16 cell proliferation was slowest in cells overexpressing WT-CNrasGEF relative to those overexpressing GFP alone, {Delta}CDC25-CNrasGEF, or those in which CNrasGEF was knocked down by RNAi; In fact, the latter exhibited the fastest proliferation rate, suggesting that CNrasGEF provides inhibitory signals for cell proliferation of B16 melanoma cells.



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  		FIGURE 4.
Sustained (long term) melanin production in B16 melanoma cells is stimulated by knockdown of CNrasGEF. A, B16 cells were transfected (or not) with CNrasGEF-RNAi or a scrambled control and stimulated (or not) with forskolin/IBMX 24 h after transfection (equal to time 0). At 54 h poststimulation, cells were harvested, and the amount of melanin produced per mg of protein was analyzed, as described under "Experimental Procedures." The average percentage knockdown by CNrasGEF-RNAi was 57% versus untransfected cells and 33% versus the scrambled control. The error bars represent average ± S.E. of two separate experiments, where protein levels were measured in duplicate, and melanin absorption was determined at two different concentrations. The asterisks indicate p values from {chi}2 tests (*, p < 0.05; **, p < 0.01). For CNrasGEF-RNAi, p < 0.01 relative to both untransfected cells and scrambled control. B, B16 cells were transfected (or not) with V12-Ras (constitutively active) or N17-Ras (dominant negative) and stimulated (or not) with forskolin/IBMX. Melanin levels were analyzed at 48 h poststimulation. The error bars represent average ± S.E. of three independent experiments. The asterisks indicate p values relative to untransfected cells, as described in A.

 
We subsequently analyzed whether the retarded proliferation of the B16 melanoma cells overexpressing WT-CNrasGEF was due to apoptosis, using terminal dUTP nick-end labeling assays. Fig. 5B demonstrates that cells overexpressing WT-CNrasGEF had a ~4–5-fold increase in the number of apoptotic cells relative to control cells (transfected with the GFP vector alone), and a more than 2-fold increase in apoptotic cells relative to those overexpressing the catalytically inactive {Delta}CDC25-CNrasGEF construct. Both proliferation rate and apoptosis were not affected by cAMP (data not shown). These results, therefore, show that B16 cells are exquisitely sensitive to the amounts of CNrasGEF they express and that overexpression of this GEF leads to enhanced apoptosis.



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  		FIGURE 5.
CNrasGEF attenuates proliferation and promotes apoptosis in B16 melanoma cells. A, growth curves of B16 melanoma cells transfected with GFP control, WT-CNrasGEF, CNrasGEF-RNAi, or {Delta}CDC25-CNrasGEF. Data represent cells/mm2± 95% confidence interval (n = 2500–5000 cells/transfection at 87 h after seeding) from two (knockdown) to five (overexpression) experiments. Only experiments in which knockdown exceeded 50% were included. Inset, logarithmic growth curve (see "Experimental Procedures"). Although data shown are of cells stimulated with forskolin/IBMX, identical results were obtained with unstimulated cells (not shown). B, terminal dUTP nick-end labeling assays performed on B16 melanoma cells overexpressing GFP control, WT-CNrasGEF, CNrasGEF-RNAi, or {Delta}CDC25-CNrasGEF (all GFP-tagged), revealing extensive apoptosis in cells overexpressing the wild type CNrasGEF at 24 h after sorting and reseeding (48 h after transfection). Data are the percentage of apoptotic cells ± 95% confidence interval (n = 450–850 cells counted per transfection), taken from three independent experiments.

 
Overall, our data suggest that in B16 cells, CNrasGEF plays critical roles in regulating cell proliferation and cAMP-dependent differentiation (melanogenesis).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The process of melanogenesis, manifested as dendrite extension and melanin production/secretion, heavily relies on the elevation of cAMP and is tightly regulated by numerous factors. An important one is cAMP-dependent Ras activation that leads to Erk activation, a process that is not mediated by protein kinase A or Rap1 (4). Our work presented here suggests that CNrasGEF could be the (or an) exchange factor that mediates this cAMP-dependent Ras activation. In support of this notion, we demonstrate here that cAMP-dependent Ras and Erk1/2 activation in B16 melanoma cells (first documented in Ref. 4) can be augmented by overexpression of CNrasGEF or decreased by knockdown of this GEF (Fig. 2).

Prolonged activation of Ras and Erk in B16 melanoma cells inhibits the cAMP-dependent formation of dendrites and synthesis of melanin (16). This raises the possibility that Ras activation via CNrasGEF in these cells could be involved in this process. Our data clearly demonstrate that alterations in the levels of expression of CNrasGEF (by overexpression or knockdown) affect B16 dendricity and melanin production in response to cAMP (Figs. 3 and 4), whereby CNrasGEF inhibits the long term dendrite extension and melanin synthesis. We propose that this negative regulatory effect is the result of Ras activation by CNrasGEF. We confirmed that Ras indeed inhibits melanin synthesis by using constitutively active and dominant negative Ras mutants (Fig. 4B). These data support previous findings (16), and offer additional evidence that CNrasGEF indeed has a role in the Ras pathway in B16 cells.

As well as inhibiting long term melanogenesis, CNrasGEF is also capable of stimulating early (<15 h) dendrite formation in B16 cells, an effect previously proposed to be Ras-dependent (17). Our work shows, however, that this effect was not cAMP-dependent, although it required catalytically active CNrasGEF. The pathway(s) involved in this function of CNrasGEF is currently unknown.

In addition to revealing a regulatory role for CNrasGEF in melanogenesis, we also observed very striking effects of CNrasGEF on cell survival of B16 cells (Fig. 5), where this GEF appeared to promote apoptosis, independent of cAMP. Interestingly, we also found extensive cell death in another cell line of neuronal origin, the rat glial/mouse neuroblastoma hybrid cell line NG108-15, when transfected with WT-CNrasGEF (data not shown). This apoptotic effect may not be universal, however, since HEK293T cells do not seem to be adversely affected by overexpression of CNrasGEF (20). In both B16 and NG108–15 cells, the proapoptotic effects of CNrasGEF were not dependent on cAMP but were dependent on its intact catalytic activity, since little apoptosis was seen upon overexpression of {Delta}CDC25-CNrasGEF. Although the mechanisms involved are currently not known, it is possible that Rap1 activation via CNrasGEF plays a role in regulating cell survival/apoptosis. Rap1 is expressed in B16 melanoma cells (4) and has been shown to be involved in regulating cell survival/apoptosis in some cells, such as thyroid cells (e.g. see Ref. 29), where thyroid-stimulating hormone-mediated Akt phosphorylation is augmented by Rap1 activation (30) and hepatocytes (31). However, in the latter cases, cAMP appears to be involved, whereas in our studies, the survival rate was similar in the presence or absence of cAMP stimulation, and previous work from several groups, including ours, demonstrated that CNrasGEF-mediated activation of Rap1 was not dependent on cAMP (20, 22, 23). This suggests that other pathways, not yet known, may play a role in the CNrasGEF-dependent proapoptotic effects. Similar to other cell types, numerous pro- and antiapoptotic factors participate in the regulation of melanocyte survival (e.g. see Refs. 32–34), and it remains to be shown which of these participate in the proapoptotic effect(s) of CNrasGEF.

The findings described here have some broad implications. First, cAMP-dependent Ras and Erk activation (independent of protein kinase A and Epac/Rap1) has been proposed in several cellular systems in addition to melanoma cells, particularly in neuronal and endocrine cells (3, 29, 35), where a cAMP-dependent Ras GEF has been sought after for quite some time. We propose that CNrasGEF could fulfill this role at least in some of these cells. Second, melanogenesis itself, which includes melanosome exocytosis, provides an example of a specialized case of lysosomal movement/exocytosis, where defects in such a process lead to a list of genetic disorders often associated with albinism (28). The involvement of CNrasGEF in regulating melanogenesis may suggest that CNrasGEF could be involved in some disorders in which aberrant signaling would result in defective melanosome exocytosis. Future work is required to test these hypotheses.


   FOOTNOTES
 
* This work was supported in part by the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society and by the Canadian Institute of Health Research (CIHR). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 Supported through a studentship, fully or in part, by the Ontario Student Opportunity Trust Fund-Hospital for Sick Children Foundation Student Scholarship Program. Back

2 Supported by a fellowship from the CIHR. Back

3 A CIHR Investigator and now holder of a Canada Research Chair (Tier I) award. To whom correspondence should be addressed: Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5098; Fax: 416-813-5771; E-mail: drotin{at}sickkids.ca.

4 The abbreviations used are: IBMX, isobutylmethylxanthine; FACS, fluorescence-activated cell sorting; GEF, guanine nucleotide exchange factor; 8-Br-cAMP, 8-bromocyclic AMP; GFP, green fluorescent protein; FBS, fetal bovine serum; RNAi, RNA interference; WT, wild type. Back

5 E. M. Amsen, N. Pham, Y. Pak, and D. Rotin, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Dr. C. C. Hui for the pSuper-EGFP vector and Dr. Dafna Bar-Sagi for Ras constructs.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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Y. Pak, W. K. Glowacka, M. C. Bruce, N. Pham, and D. Rotin
Transport of LAPTM5 to lysosomes requires association with the ubiquitin ligase Nedd4, but not LAPTM5 ubiquitination
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