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Original Studies |
Centre for Hormone Research, Royal Children’s Hospital, Parkville, Victoria 3052, Australia
Address all correspondence and requests for reprints to: Stephanie R. Edmondson, Centre for Hormone Research, Royal Children’s Hospital, Flemington Road, Parkville, Victoria 3052, Australia. E-mail: edmondss{at}cryptic.rch.unimelb.edu.au
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Abstract |
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Introduction |
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The IGF-I system appears to play a role in melanocyte growth and function. Melanocytes, both in vitro and in vivo, express IGF-I messenger ribonucleic acids (mRNAs) and IGF-I receptor (IGF-IR) (12, 13, 14) and in vitro grow in response to IGF-I (15). IGF-I also plays an important role in the growth of some melanomas (16). Recent reports have noted enhanced growth of melanocytic lesions of patients treated with recombinant human GH (17, 18). The presence of GH receptor (GHR) mRNA in cultured melanocytes suggests that GH may have a direct action on these cells (12). Alternatively, locally synthesized or circulating IGFs may mediate the effects of GH on melanocytes.
In the present study, we have characterized the role of GH/IGF system in melanocyte growth in vitro, including its interaction with another key local growth factor, bFGF. We have further examined the potential role of melanocyte-derived IGFBPs in modulating IGF action.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanocyte growth medium [MGM-2; which is melanocyte basal medium (MBM) and includes 20 µg/mL bovine pituitary extract, 5 µg/mL insulin, 1 ng/mL bFGF, 0.5 µg/mL hydrocortisone, 10 ng/mL phorbol 12-myristate 13-acetate, and 10 ng/mL GA], MBM, and two primary melanocyte cell cultures were obtained from Clonetics (San Diego, CA). Hanks’ Balanced Salt Solution, gentamicin (10 mg/mL), geneticin (G418), and 0.025% trypsin-1 mmol/L ethylenediamine tetraacetate (EDTA) were all obtained from Life Technologies (Grand Island, NY). Dispase was purchased from Collaborative Biomedical Products (Bedford, MA). FCS, DMEM, and phosphate-buffered saline (calcium and magnesium free; PBS) were obtained from Trace (Melbourne, Australia). Tissue culture flasks and 24-well plates were obtained from Nunc (Copenhagen, Denmark). RIA grade BSA, protein A-Sepharose, and L-DOPA (3,4-dihydroxyphenylalanine) were purchased from Sigma Chemical Co. (St. Louis, MO). Nitrocellulose filters were purchased from Schleicher & Schuell, Inc. (Dassel, Germany). [125I]IGF-I (2000 Ci/mmol), 14C-labeled protein molecular mass (14–22 kDa) markers, [32P]dCTP (3000 Ci/mmol), [32P]ATP (3000 Ci/mmol), and Hybond-N nylon membranes were obtained from Amersham (Aylesbury, UK). [125I]IGF-II (1500 Ci/mmol) was a gift from Dr. L. A. Bach (Department of Medicine, Austin and Repatriation Medical Centre, Melbourne, Australia). The complementary DNA (cDNA) for human IGFBP-4 was provided by Dr. S. Shimasaki (Whittier Institute, La Jolla, CA) (19). Recombinant human IGF-I was a gift from Dr. A. Sköttner (Pharmacia-Upjohn, Stockholm, Sweden). Recombinant human bFGF and the random priming kit were obtained from Boehringer Mannheim (Sydney, Australia). Des(1, 2, 3)IGF-I was purchased from Gropep (Adelaide, Australia). 2,3-Bis(2-methoxy-4-nitro-5-sulfonyl)-5[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) was obtained from Scima R (Victoria, Australia). Disuccinimidyl suberate was obtained from Pierce Chemical Co. (Rockford, IL).
Isolation of melanocytes
Melanocytes were purchased from Clonetics (newborn foreskin-derived) or were isolated from foreskins (collected after obtaining ethical approval from the Department of Surgery, Royal Children’s Hospital, Melbourne, Australia). Foreskins (newborn; 11 months old; and 8, 9, and 11 yr old) were collected in keratinocyte serum-free medium containing gentamicin (5 µg/mL) and were stored at 4 C overnight. After transfer to 70% ethanol for 10 min, foreskins were rinsed in PBS, cut into 5 x 5-mm pieces, immersed in Hanks’ Balanced Salt Solution containing dispase at 25 caseinolytic units/mL, and incubated at 4 C for 18 h. The epidermal layer was removed and placed in 2 mL 0.025% trypsin/1 mmol/L EDTA at 37 C for 20–30 min. During this period cells were dispersed by intermittent vigorous pipetting. Trypsin was inactivated by the addition of 0.5 mL FCS, then the cell suspension was filtered through sterile gauze and pelleted at room temperature (1000 rpm) for 10 min. Cells were resuspended in MGM-2 and plated into two 75-cm2 flasks. Flasks were incubated at 37 C in 5% CO2 overnight. Medium was changed every 3 days, during which time the majority of the keratinocytes and occasional fibroblasts had died, and melanocytes remained adhered to the flask. Differential trypsinization was used to completely remove contaminating keratinocytes and fibroblasts. Specifically, cells were incubated in trypsin-EDTA at 4 C for 5 min, and loosely adhered melanocytes were removed, leaving fibroblasts and keratinocytes. The melanocytes were pelleted and replated into a fresh 75-cm2 flask. The following day the melanocytes were assessed for contamination with fibroblasts and keratinocytes. On rare occasions keratinocytes and fibroblasts were still present. For complete removal of fibroblasts, geneticin (G418; 100 µg/mL) was added to the cultures according to the method of Halaban and Alfano (20). Cells were grown in the presence of G418 for only 1 week. In the primary melanocyte cell cultures used for these studies all contaminating keratinocytes had died by passage 3. If keratinocytes were present in melanocyte cultures at passage 3, the cells were discarded.
Identification of melanocytes
Melanocytes isolated in this laboratory were positively identified following a modified method described by Hu et al. (21). Primary melanocytes and human skin fibroblasts (negative control) were plated into 24-well tissue culture plates with their appropriate growth medium (fibroblasts were grown in DMEM-10% FCS). When confluent, medium was removed, and cells were washed in PBS and fixed in 5% formalin for 4 C at 30 min. Cells were rinsed with ultrapure water (Continental Water Systems, Australia) and then incubated with 0.1% L-DOPA in PBS at 37 C for 3.5 h. L-DOPA was then removed, and the cells were fixed in 10% formalin for 60 min at 22 C. Formalin was removed, and cells were air-dried. Positive identification of melanocytes was seen as brown staining when cells were microscopically assessed; fibroblasts did not stain brown.
Melanocyte growth assays
Primary melanocytes (n = 3; passages 6–8) were plated at 1 x 104 cells/200 µL·well on 96-well poly-D-lysine-coated plates (Collaborative Biomedical Products, Bedford, MA). After overnight incubation, MGM-2 was removed and replaced with experimental medium. Specifically, cells were exposed in triplicate to MGM-2, MBM-0.1% BSA, or increasing doses of growth factors diluted in MBM-0.1% BSA [IGF-I, 0–100 ng/mL; des(1, 2, 3)IGF-I, 0–100 ng/mL; GH, 0–100 ng/mL; bFGF, 0–50 ng/mL]. To assess the effect of combinations of growth factors, only the highest concentration of each was used; IGF-I, des(1, 2, 3)IGF-I, GH at 100 ng/mL, or bFGF at 50 ng/mL. Assays were carried out for a period of 15 days, with medium (130 µL of the 200-µL volume) replaced every 3 days. At the end of the experimental period cell number was measured using an XTT colorimetric assay (22).
XTT colorimetric assay
XTT was dissolved in prewarmed (60 C) PBS at 1 mg/mL. Phenazine methosulfate (PMS; 100 mmol/L) was prepared and stored in the dark at -20 C. Immediately before use, PMS was added to XTT (1 mg/mL), so that the final concentration was 125 µmol/L. Fresh XTT-PMS (40 µL) mix was added to 200 µL medium in each well of the 96-well plate. Cells were incubated at 37 C in 5% CO2 for 30, 60, 90, and 120 min, and absorbency (cell number) was measured at 450 nm.
Statistical analysis of melanocyte growth assay
Experiments were conducted on cells obtained from three primary cell cultures. For statistical analysis of melanocyte growth in response to increasing doses of each growth factor alone, triplicate optical density values (at 450 nm; cell number) were averaged, and data were expressed as the percent increase in absorbance compared to that of cells treated with MBM-0.1% BSA. Data for each dose-response curve were statistically analyzed by one-way ANOVA, using Dunnett’s multiple comparison test. Dose responses for IGF-I and des(1, 2, 3)IGF-I at comparable concentrations were compared using unpaired t test. Data for melanocyte growth in response to combinations of growth factors were expressed as the percent increase in absorbance compared to cells treated with MBM-0.1% BSA and were statistically analyzed by unpaired t test. Statistical analyses were determined using Prism GraphPad Software (GraphPad Software, Inc., San Diego, CA).
Identification of secreted IGFBPs by Western ligand blot (WLB)
Melanocyte-conditioned medium (1 mL of 15 mL total; CM) was concentrated by ethanol precipitation, and samples were electrophoresed as previously described (7) and according to the method of Laemmli (23). Protein samples were run on a 12% SDS-polyacrylamide gel with 1 mL unconditioned melanocyte medium (negative control), 5 µL human serum (positive control), and 14C-labeled mol wt markers (Amersham). Separated proteins were electrophoretically transferred to nitrocellulose filters and probed with [125I]IGF-II (1 x 106 cpm). Filters were exposed to x-ray film (up to 4 weeks; X-Omat AR, Eastman Kodak Co., Rochester, NY) or a phosphorimaging screen (Storm, Molecular Dynamics, Inc., Sunnyvale, CA). X-Ray autoradiographs were scanned using a Bio-Rad model GS-670 Imaging densitometer (Bio-Rad Laboratories, North Ryde, Australia). IGFBP secretion by cultured melanocytes (n = 7) was initially assessed using complete growth medium (MGM-2). Specifically, samples were assessed for period (days) of conditioning, confluence of cells, passage number, and donor age.
Identification of secreted IGFBPs by [125I]IGF-I binding and cross-linking analysis
Melanocyte CM (2 mL) was precipitated as described previously and resuspended in 220 µL 10 mmol/L Tris-HCl (pH 7.4). Fifty-five microliters of these samples were incubated in a final volume of 60 µL with [125I]IGF-II (50,000 cpm) in the presence or absence of 60 ng IGF-I (competitor) in duplicate for 2 h at 37 C with rotation. Controls included [125I]IGF-II (50,000 cpm) in the absence or presence of 60 ng IGF-I. Samples were then incubated on ice for 10 min followed by the addition of disuccinimidyl suberate to a final concentration of 1 mmol/L and a further incubation for 15 min on ice. The cross-linking reaction was quenched by the addition of 5 µL ice-cold 16 x quenching buffer (80 mmol/L Tris-HCl (pH 7.4) and 80 mmol/L EDTA (pH 8.0)]. Samples were analyzed by SDS-PAGE (as described for WLB above), then dried under vacuum at 80 C for 2 h and autoradiographed.
Identification of IGFBPs by immunoprecipitation
For further identification of melanocyte-derived IGFBPs, CM was immunoprecipitated with antisera specific for human IGFBP-3 (human anti-IGFBP-3 antiserum, donated by Prof. Robert Baxter, Kolling Institute, Sydney, Australia) and human IGFBP-4 (anti-IGFBP-4 antiserum, Hop, donated by Dr. Steven Chernausek, Children’s Hospital Medical Center, OH). Specifically, 2 mL melanocyte CM were preabsorbed with 320 µL of a 20% suspension of protein A-Sepharose [suspended in Tris-buffered saline, i.e. 25 mmol/L Tris-HCl (pH 7.4) and 0.9% (wt/vol) NaCl containing 0.1% BSA) by gentle agitation for 2 h at 4 C. The protein A-Sepharose was pelleted, and the CM was transferred to a new tube. Antiserum (1:100) and 320 µL of a fresh 20% suspension of protein A-Sepharose were then added to the CM, and samples were incubated overnight with gentle agitation for 2 h at 4 C. Fibroblast CM (500 µL) was used as a positive control. Samples were then centrifuged (5000 rpm at 4 C), and the protein A-Sepharose pellet was washed three times in Tris-buffered saline (1 mL; described above) before dissolving in Laemmli sample buffer, boiled for 5 min, run on SDS-PAGE, and ligand blotted as described previously.
Identification of IGFBPs by RIA
Melanocyte CM was subjected to RIA for IGFBP-1, -2, -3, and -6. These assays were performed by Ms. Sara Holman in Prof. Robert Baxter’s laboratory (Kolling Institute, Royal North Shore Hospital, Australia) using previously described assays (24, 25, 26, 27).
Northern blot analysis
Total RNA from melanocytes at passages 5–8 grown in MGM-2 was isolated by the guanidium thiocyanate method (28) and quantified by spectrophotometer at 260 nm. Total RNA (30 µg) was run in a 1.2% agarose-formaldehyde gel as described by Sambrook et al. (29). Samples were transferred to Hybond-N nylon membrane and probed as previously described (11) using [32P]dCTP-labeled human IGFBP-4 cDNA. Membranes were exposed to x-ray film (X-Omat, Eastman Kodak Co., Rochester, NY) for 3–12 days.
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Results |
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Melanocyte growth was assessed in response to increasing
concentrations of IGF-I, des(1, 2, 3)IGF-I, GH, or bFGF. As shown in Fig. 1a
, IGF-I stimulated melanocyte growth in a dose-dependent manner.
IGF-I at 1, 10, and 100 ng/mL induced growth responses of 38 ±
11%, 45 ± 11%, and 47 ± 16%, respectively, compared to
basal medium (P < 0.05). Des(1, 2, 3)IGF-I, an analog of
IGF-I that has minimal affinity for IGFBPs (30), also induced growth
responses at 10 and 100 ng/mL of 26 ± 15% and 48 ± 16%,
respectively, over basal medium (P < 0.05; Fig. 1a). The growth responses to
des(1, 2, 3)IGF-I at 0.1 and 1.0 ng/mL were significantly less than those
for IGF-I at the same concentrations (Fig. 1a; P <
0.05). Neither GH nor bFGF alone significantly enhanced melanocyte
growth (Fig. 1, c and d).
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Melanocyte growth was also assessed in response to combinations of
the following growth factors: IGF-I (100 ng/mL), des(1, 2, 3)IGF-I (100
ng/mL), GH (100 ng/mL), and bFGF (50 ng/mL). As shown in Fig. 2, GH or
bFGF in combination with IGF-I or des(1, 2, 3)IGF-I enhanced melanocyte
growth 2- to 4-fold compared to any of the growth factors alone
(P < 0.05). The addition of GH to IGF-I and
des(1, 2, 3)IGF-I induced growth responses of 143 ± 36% and
201 ± 56%, respectively. bFGF in combination with IGF-I and
des(1, 2, 3)IGF-I induced growth responses of 303 ± 150% and
302 ± 119%, respectively. Although GH or bFGF alone did not
stimulate melanocyte growth (Figs. 1
, c and d, and
2) GH and bFGF in combination were able
to induce a significant growth response (198 ± 90%) compared to
either growth factor alone (P < 0.05). This
GH/bFGF-stimulated growth response was not significantly different from
that seen with GH or bFGF combined with either IGF-I or des(1, 2, 3)IGF-I
or a combination of both GH and bFGF with either IGF-I or
des(1, 2, 3)IGF-I. Complete MGM-2 induced melanocyte growth significantly
greater than that induced by all growth factors alone or in combination
(P < 0.05).
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Des(1, 2, 3)IGF-I (at 10 and 100 ng/mL) was no more effective than
IGF-I in stimulating melanocyte growth and was less effective at lower
concentrations (0.1 and 1.0 ng/mL; Fig. 1a), raising questions about a
role for IGFBPs in IGF action on melanocytes. We, therefore, assessed
the secretion of IGFBPs by melanocytes grown in complete growth medium
(MGM-2).
CM was analyzed for the presence of IGFBPs by WLB as described in
Materials and Methods. A major band was seen at 24 kDa, and
inconsistent bands were seen at approximately 38 and 42 kDa (Fig. 3). Results were obtained after 4 weeks
of autoradiographic exposure, indicating that IGFBP abundance in
melanocyte CM was extremely low. The results shown in Fig. 3 are
typical for all CM analyzed. The IGFBP profile was not altered by age
of donor (newborn to 11 yr), passage number (passages 6–8), or cell
density. The IGFBP profile was maintained when melanocytes were grown
in the presence of IGF-I (100 ng/mL in MBM-0.1% BSA) or des(1, 2, 3)IGF-I
(100 ng/mL in MBM-0.1%BSA) alone (data not shown).
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). A specific band at
approximately 31 kDa (lanes 1 and 2) was identified that was completely
abolished with unlabeled IGF-I (lanes 3 and 4). This 31-kDa band is
consistent with IGFBP-4 (24 kDa) cross-linked with IGF-I (7 kDa).
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shows results for a typical
immunoprecipitation of melanocyte CM followed by WLB. As shown in Fig. 5, the 24-kDa protein was consistently immunoprecipitated from
melanocyte CM using anti-IGFBP-4 antiserum (lane 5). Anti-IGFBP-3
antiserum did not precipitate any protein from the same melanocyte CM
(lane 3), but was able to precipitate IGFBP-3 from human fibroblast CM
(lane 7).
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IGFBP-4 mRNA is detected in cultured human melanocytes
To further confirm that melanocytes synthesize IGFBP-4, total RNA
(30 µg) from melanocytes grown to complete confluence in MGM-2 was
subjected to Northern analysis. As shown in Fig. 6, the predicted 2.4-kb IGFBP-4 mRNA band
(31) was clearly identified in melanocyte RNA (lanes 1 and 2) and was
also detected in fibroblast total RNA used as a positive control (lane
3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The response of melanocytes to GH in combination with bFGF or IGF-I suggests that GH plays a role in melanocyte growth in vitro, consistent with earlier findings of GHR mRNA detected in cultured human melanocytes (12). Our data show that these GHRs are responsive in vitro. If our data reflect the in vivo situation, then the observed increase in the size of melanocytic naevi in patients treated with human GH (17, 18) may be due to the direct action of GH on melanocytes.
As GH was unable to elicit a growth response when added alone, our findings suggest that GH action on melanocyte growth does not simply involve local production of IGF-I. The mechanisms involved in GH/IGF-I/bFGF synergy are unknown, but might involve up-regulation of GHRs or other postreceptor processes.
bFGF is an important melanocyte growth factor produced by neighboring keratinocytes (32) and dermal fibroblasts (33), but not by melanocytes (32). Under our experimental conditions, bFGF was only able to induce a melanocyte growth response when IGF-I or GH was also present in basal medium. Our finding that bFGF alone did not produce a dose-dependent growth response is perhaps not surprising. Previous studies assessing the proliferative effect of bFGF on cultured melanocytes have always included additional growth factors or phorbol esters in the experimental medium (32, 34). For example, Pittelkow and Shipley (15) demonstrated a synergistic effect of insulin and bFGF, in the presence of phorbol 12-myristate 13-acetate, on melanocyte proliferation. Furthermore, bFGF-induced proliferation of neuronal precursor cells was dependent on the presence of IGF in the medium (35), and this may also be the case with melanocytes. Binding of bFGF to its receptor is exquisitely regulated by specific protoeglycans found in extracellular matrix (36). IGF-I can regulate turnover in articular cartilage explants (37) and proteoglycan synthesis in a chondrocyte cell line (38). It is therefore possible that cultured melanocytes produce extracellular matrix components, such as proteoglycans, in response to IGF-I or GH, which, in turn, augment the bFGF response. It is also possible that intracellular signaling events downstream of receptor activation contribute to synergistic growth stimulation by IGF-I/GH, IGF-I/bFGF, GH/bFGF, or a combination of all three growth factors.
In contrast to GH, IGF-I alone was able to stimulate melanocyte growth in a dose-dependent manner. This is in contrast to a report by Herlyn et al. (39) in which IGF-I alone at 10 ng/mL was unable to sustain melanocyte growth, but is supportive of data showing that 5 µg/mL insulin (probably acting via the IGF-IR) can enhance melanocyte growth (40). The differences in IGF-I responses among all studies may relate to the different formulations of experimental medium.
IGF-I activity can be augmented or inhibited by a family of at least six IGFBPs that are produced and regulated in a cell-specific manner (41). Our melanocyte growth studies revealed that des(1, 2, 3)IGF-I (which has minimal binding to IGFBPs) was not more effective than IGF-I, suggesting that IGFBPs may play a minor role, if any, in the IGF-I response in vitro. This hypothesis is supported by our analysis of conditioned medium, which clearly showed that melanocytes secrete very low levels of IGFBPs, essentially IGFBP-4. Alternatively, the apparent reduced efficacy of des(1, 2, 3)IGF-I compared with that of IGF-I at low concentrations may suggest that IGFBP-4 augments IGF-I growth stimulation in these cells. This observation would be consistent with recent studies in which IGFBP-4 enhanced IGF-I-stimulated survival of rat neuronal cells (42) and IGFBP-4 knockout mice, which are born smaller than their wild-type counterparts (43). Whereas IGFBP-4 is usually inhibitory, these recent findings and our current results in melanocytes all support the possibility that IGFBP-4 can enhance IGF action in some circumstances.
IGFBP-4 is the major IGFBP produced by a range of neuronal cell lines (44), and our finding that IGFBP-4 is the major IGFBP produced by melanocytes is consistent with their neural crest origin. IGFBP-4 production may thus play an important role in the regulation of IGF action in these cell types. IGF-I is a primary mitogen for a number of malignant melanoma cell lines (45). Studies on melanoma cell lines (46, 47) indicate synthesis of a range of IGFBPs (IGFBP-2, -3, -4, -5, and -6), with regulation by serum or IGF-I. It is thus possible that aberrantly expressed IGFBPs play a more significant role in regulating the IGF-I response of melanoma cells.
The role of melanocyte-derived IGFBPs in modulating IGF action in vivo remains uncertain, particularly since adjacent basal keratinocytes produce abundant amounts of IGFBP-3 (3, 5, 7, 11). The juxtaposition of melanocytes with basal keratinocytes suggests that IGFBP-3 may also play a significant role in targeting or inhibiting IGF-I interaction with melanocyte IGF-IR.
In conclusion, these studies provide support for a role for both GH and IGF-I in the growth of human melanocytes in vitro, involving synergy with bFGF. Low levels of melanocyte-derived IGFBP-4 may enhance IGF action in vitro, which may be further modulated in vivo by other locally produced IGFBPs.
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Footnotes |
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Received December 11, 1998.
Revised February 3, 1999.
Accepted February 9, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in serum
and plasma. J Immunol Methods. 159:81–91.[CrossRef][Medline]
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P < 0.05.
