Interactions between Growth Hormone, Insulin-Like Growth Factor I, and Basic Fibroblast Growth Factor in Melanocyte Growth
[b]Abstract] Melanocytes, highly differentiated neural crest-derived cells, are located in the basal layer of the epidermis, where they play a role in protecting against UV damage in the skin. Previous studies suggest that both growth hormone (GH) and the insulin-like growth factor I (GH/IGF-I) system may be important for melanocyte growth and function. We have therefore characterized the role of the GH/IGF system in melanocyte growth in vitro and its interaction with the local growth factor basic fibroblast growth factor (bFGF). Analysis of the effects of GH, IGF-I, and bFGF and combinations of these growth factors on melanocyte growth in vitro revealed that 1) GH stimulates the growth of melanocytes when combined with IGF-I, des(1–3)IGF-I [an analog of IGF-I that has a reduced binding affinity for IGF-binding proteins (IGFBPs)], or bFGF, either separately or in combination; 2) in contrast to the lack of effect of GH or bFGF alone, both IGF-I and des(1–3)IGF-I enhance melanocyte growth in a dose-dependent manner; and 3) IGF-I is more efficacious in eliciting a growth response at low concentrations compared to des(1–3)IGF-I. Using Western ligand blotting, affinity cross-linking, immunoprecipitation, RIA, and Northern analysis, we show that cultured human melanocytes synthesize and secrete minimal amounts of IGFBP. IGFBP-4 is the major IGFBP produced by these cells when cultured in complete growth medium or in the presence of either IGF-I or des(1–3)IGF-I alone. 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 play a role in enhancing the modulation of IGF action.
[b]Intro] MELANOCYTES are specialized neural crest-derived cells situated in the basal layer of the epidermis. They play a protective role against UV damage in the skin by producing melanin in response to UV stimulation, which is passed, via their dendrites, onto adjacent keratinocytes. The relationship between melanocytes and keratinocytes is thus a crucial feature of the epidermis and is known to involve numerous growth factors, including basic fibroblast growth factor (bFGF) (1). Insulin-like growth factor I (IGF-I), a potent mitogen for keratinocytes, may also be involved in this relationship. An essential role for the IGF system in normal epidermal development is indicated by the finding that IGF-I receptor (IGF-IR) knockout mice have an extremely thin epidermis (2). IGF-binding proteins (IGFBPs), modulators of IGF action, have also been identified in skin (3, 4, 5), cultured keratinocytes (6, 7), and dermal fibroblasts (8). Furthermore, the IGFBPs have been shown to regulate IGF-I action on keratinocytes (9) and fibroblasts in vitro (10) and show cell-specific regulation by several growth factors present in skin (10, 11).
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.
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. 1aGo, 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. 1aGo). 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. 1aGo; P < 0.05). Neither GH nor bFGF alone significantly enhanced melanocyte growth (Fig. 1Go, c and d).
GH and bFGF in combination stimulate melanocyte growth and potentiate the effects of IGF-I and des(1, 2, 3)IGF-I
Melanocyte growth was also assessed in response to combinations of the following growth factors]
IGFBP-4, in low abundance, is the primary IGFBP in CM
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. 1aGo), 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).
[b]
Discuss]* We have investigated the effects of IGF-I, GH, bFGF, and combinations of these growth factors on the growth of normal human melanocytes in vitro. Whereas GH is ineffective alone, melanocyte growth is enhanced when GH is combined with either IGF-I, des(1, 2, 3)IGF-I (an analog of IGF-I that has a reduced binding affinity for IGFBPs), or the local growth factor bFGF, either separately or in combination. We have also shown that IGF-I and des(1, 2, 3)IGF-I both enhance melanocyte growth in a dose-dependent manner, with IGF-I being more potent, further suggesting a role for IGF-I in melanocyte function. Using WLB, affinity cross-linking, immunoprecipitation, RIA, and Northern analysis, we have shown that cultured human melanocytes produce minimal amounts of IGFBP. IGFBP-4 was identified as the major IGFBP secreted by these cells when cultured in complete growth medium or in the presence of either IGF-I or des(1, 2, 3)IGF-I alone.
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.
GH and IGF-I and -II were first identified by their endocrine activity. Specifically, IGF-I was found to mediate the linear growth-promoting actions of GH. It is now evident that these two growth factor systems also exert widespread activity throughout the body and that their actions are not always interconnected. The literature highlights the importance of the GH and IGF systems in normal skin homeostasis, including dermal/epidermal cross-talk. GH activity, sometimes mediated via IGF-I, is primarily evident in the dermis, particularly affecting collagen synthesis. In contrast, IGF action is an important feature of the dermal and epidermal compartments, predominantly enhancing cell proliferation, survival, and migration. The locally expressed IGF binding proteins play significant and complex roles, primarily via modulation of IGF actions. Disturbances in GH and IGF signaling pathways are implicated in the pathophysiology of several skin perturbations, particularly those exhibiting epidermal hyperplasia (e.g., psoriasis, carcinomas). Additionally, many studies emphasize the potential use of both growth factors in the treatment of skin wounds; for example, burn patients. This overview concerns the role and mechanisms of action of the GH and IGF systems in skin and maintenance of epidermal integrity in both health and disease.
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