Terminal differentiation and senescence in the human melanocyte: repression of tyrosine-phosphorylation of the extracellular signal-regulated kinase 2 selectively defines the two phenotypes.

Abstract

Melanocytes are pigmented cells distributed in humans in several organs like the epidermis, the leptomeninges, the eye, and the inner ear. Epidermal melanocytes, whether derived from adult or neonatal skin, proliferate well in a medium supplemented with phorbol esters and other mitogens before they undergo senescence. Potent cAMP inducers like cholera toxin are also growth promoters for neonatal melanocytes but only transient growth stimulators for cells derived from adults. We used this cellular system to delineate biochemical pathways involved in proliferation and in terminal differentiation. Here we show that after a period of 4-8 wk of sustained proliferation in the presence of cholera toxin, the adult melanocytes became round, flat, and enlarged. These changes were associated with terminal growth and preceded by a five-to sixfold increase in cAMP levels and an 8-to 10-fold increase in melanin content. The simultaneous addition of phorbol esters and cholera toxin did not prevent cells from reaching terminal differentiation. Identified targets for phorbol esters are protein kinase C (PKC) and the mitogen-activated kinases (MAPKs), also called extracellular signal-regulated kinases (ERKs). PKC was found to be similarly regulated in proliferating and in terminally differentiated melanocytes. Proliferating melanocytes in early or late passage showed identical activation of the kinase ERK2. This kinase was rapidly phosphorylated upon phorbol 12-myristate 13-acetate (PMA) addition and specifically accumulated in the nucleus of the cells, whereas in unstimulated cells it had a perinuclear distribution. In contrast, senescent and terminally differentiated cells were unable to phosphorylate tyrosine residues of the ERK2 gene product in spite of presenting normal amounts of ERK2 protein. In addition, ERK2 did not show the nuclear accumulation observed in proliferating melanocytes after PMA activation and remained localized in the perinuclear area. These results demonstrate that senescent and terminally differentiated melanocytes share a common block in a critical pathway thought to integrate multiple intracellular signals transmitted by various second messengers and specifically prevent the continuation of the signal transduction cascade initiated by PMA activation of PKC. (Lorton and Nordlund, 1991;Tassabehji et al., 1992;Urabe et al., 1993). When humans reach their fourth or fifth decade of life, the number of melanocytes in the epidermis, hair bulbs, eyes, and mucus membranes and the number of oral and cutaneous nevi begin to decrease (Nordlund, 1986). At the same time, there is a significant increase in the incidence of skin cancers. It is not known if the decrease in melanocyte numbers as humans age is because of premature senescence, terminal differentiation, or both phenomena. Possibly one result of the loss of melanocytes is a permissive environment for the development of malignancies. The possibility to grow human melanocytes in vitro to study the cellular and molecular mechanisms involved in melanocyte mig...

Significance of Nuclear Relocalization of ERK1/2 in Reactivation of c-fos Transcription and DNA Synthesis in Senescent Fibroblasts

“…In addition, similar observations were reported in senescent and terminally differentiated melanocytes (24). In this case, however, ERK2 was retained in the cytoplasm only because it was not phosphorylated and thus inactive.…”

Immortalization of human embryonic fibroblasts by overexpression of c-myc and simian virus 40 large T antigen

“…This phenomenon has been reported for various animal cells including human cells (Hayflick and Moorhead, 1961;Rheinwald and Green, 1975;Rohme, 1981;Tassin et al, 1979;Thornton et al, 1983;Medrano et al, 1994). Maximum population doubling numbers to get for animal embryonic fibroblast before reaching the replicative senescence is reported to be proportional to the maximum life span of donor animal (Martin et al, 1970;Schneider and Mitsui, 1976;Rhome, 1981).…”

Human melanocyte senescence and melanoma susceptibility genes

“…These range from 8 to 90 pd for neonatal melanocytes, and from 2.4 to 40 pd for adult melanocytes (Eisinger and Marko, 1982;Gilchrest et al, 1984;Bennett et al, 1985;Graeven and Herlyn, 1992;Medrano et al, 1994;Haddad et al, 1998). One factor affecting the lifespan appears to be the different mitogenic supplements used.…”

“…CT is one of a set of supplements that increase cellular cAMP signaling; others include melanocyte-stimulating hormone (MSH), theophylline, dibutyryl cAMP and isobutyl methylxanthine (IBMX). Comparisons between the two specific media led to the conclusion that lifespans of neonatal human melanocytes were shorter in a medium containing CT and IBMX (13-15 pd) than in the same basic medium without cAMP agonists but containing TPA (40-60 pd) (Medrano et al, 1994;Bandyopadhyay et al, 2001). The cAMP agonists appeared more important than the TPA in producing this difference, since neonatal melanocytes grown with TPA as well as CT and dibutyryl cAMP were reported to grow for only about 12 pd (Halaban et al, 2000).…”

“…Pathways include both stimulation of transcription of specialized melanocytic proteins (for reviews, see Busca`and Ballotti, 2000;Goding, 2000) and post-translational activation of the key melanosomal enzyme tyrosinase (Wong and Pawelek, 1975). Human melanocytes approaching senescence show further increases in pigmentation and expression of some melanocytic proteins, even where already grown with cAMP agonists (Gilchrest et al, 1984;Medrano et al, 1994;Bennett et al, 1985;reviewed by Bennett and Medrano, 2002). A similar increase in melanin content is seen in senescent mouse melanocytes, but not in Ink4a-Arf null mouse melanocytes at or after the same time in culture, indicating that it requires this locus; moreover, increased pigmentation of these cells was seen on restoring p16 without Arf (Sviderskaya et al, 2002).…”