Inhibition of PAX3 by TGF-b
Modulates Melanocyte Viability
Guang Yang,2Yitang Li,2Emi K. Nishimura,3Hong Xin,1Anyu Zhou,2Yinshi Guo,1,4Liang Dong,1Mitchell F. Denning,1
Brian J. Nickoloff,1and Rutao Cui1,*
1Department of Pathology, Oncology Institute, Cardinal Bernardin Cancer Center, Stritch School of Medicine, Loyola University Chicago,
2160 South First Avenue, Maywood, IL 60153-5385, USA
2Department of Medical Oncology, Dana-Farber Cancer Institute, Children’s Hospital Boston, Harvard Medical School, 44 Binney Street,
Boston, MA 02115, USA
3Department of Stem Cell Medicine, Cancer Research Institute, Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan
4Present address: Renji Hospital, Shanghai JiaoTong University School of Medicine, Shanghai 200127, China
The protein encoded by paired-box homeotic gene 3
ciated transcription factor (Mitf) in the melanocyte
lineage. Here, we show that PAX3 expression in skin
is directly inhibited by TGF-b/Smads. UV irradiation
of TGF-b/Smads upregulates PAX3 in melanocytes,
which is associated with a UV-induced melanogenic
response and consequent pigmentation. Further-
more, the TGF-b-PAX3 signaling pathway interacts
with the p53-POMC/MSH-MC1R signaling pathway,
of p53-POMC/MSH-MC1R signaling is required for
the UV-induced melanogenic response because
PAX3 functions in synergy with SOX10 in a cAMP-re-
the transcription of Mitf. This study will provide a rich
foundation for further research on skin cancer pre-
vention by enabling us to identify targeted small mol-
ecules in the signaling pathways of the UV-induced
melanogenic response that are highly likely to induce
naturally protective pigmentation.
Tanning is an acquired pigmentation process involving the syn-
dendrites and distribute melanin to surrounding keratinocytes
(Jablonski and Chaplin, 2000). The crucial role of skin pigmenta-
tion seems to be to absorb ultraviolet (UV) radiation, serving as
a natural sun screen. UV radiation induces melanin production
in melanocytes, and melanin acts as a physical barrier that scat-
ters incident UV irradiation, thereby reducing penetration of UV
irradiation through the epidermis. The UV sensor/effector for
skin pigmentation occurs through p53 activation in keratino-
cytes, with its key mechanistic role being the transcriptional
activation of proopiomelanocortin/melanocyte-stimulating hor-
mone (POMC/MSH) (Cui et al., 2007), the ligand of the melano-
cortin-1 receptor (MC1R). Molecular and genetic data suggest
that MC1R has a crucial role in tanning and pigmentation in hu-
man and mouse. MC1R binding with MSH activates the cAMP
pathway. Microphthalmia-associated transcription factor (Mitf)
lanocytes (Abdel-Malek et al., 1999; Bertolotto et al., 1998; Price
et al., 1998). Mitf is transactivated by PAX3 and SOX10 proteins
directly (Potterf etal., 2000)and regulates thetranscriptionof the
major pigment enzyme genes, including tyrosinase (Bentley
et al., 1994), tyrosinase-related protein 1 (Tyrp1), and dopa-
chrome tautomerase (DCT) (Yasumoto et al., 1997).
scription factors (containing a paired-box domain and a paired-
type homeodomain), is essential for the melanocyte lineage in
both developing and adult mice (Scholl et al., 2001). Although
PAX3null mice dieshortly afterbirth, micewith anaturally occur-
ring PAX3 loss-of-function mutation (Splotch, Sp/+) have severe
belly due to defective neural crest-derived melanocyte develop-
ment (Epstein et al., 1993). PAX3 mutations in humans produce
type I and type III Waardenburg syndrome (Read and Newton,
nocyte deficiencies in the skin and inner ear. Collectively, these
studies indicate important roles for PAX3 both in differentiation
pathways such as pigmentation and in the proliferation and/or
survival of developing melanocytes. However, the network of
genes that regulates PAX3 expression or activity in melanocytes
has not yet been elucidated.
TGF-b superfamily members (activin, bone morphogenic
proteins, TGF-bs, and decapentaplegic) control many funda-
mental aspects of cellular behavior, including proliferation,
migration, adhesion, differentiation, and/or survival, primarily
by local paracrine extracellular signaling molecules (ten Dijke
and Hill, 2004). Several independent groups have observed
the growth-inhibiting effects of TGF-b in primary melanocytes,
but not in melanoma cells (Herlyn et al., 1990; Myatt et al.,
2000; Rodeck et al., 1994). The central elements of TGF-b signal
554 Molecular Cell 32, 554–563, November 21, 2008 ª2008 Elsevier Inc.
transduction include multimeric serine/threonine kinase recep-
tor complexes on the cell surface, which activate Smad proteins
that accumulate in the nucleus and control transcription of TGF-
b/smads’ target genes (Siegel and Massague, 2003). UV irradi-
ation downregulates the expression of TGF-b/Smads directly in
human skin in vivo (Gambichler et al., 2007; Quan et al., 2004)
and/or blocks cellular responsiveness to TGF-b by downregu-
lating the TGF-b II receptor/Smads signaling (Quan et al.,
2001). TGF-b also decreases melanin synthesis in melanocytes
(Martinez-Esparza et al., 1999). Collectively, these observations
led us to examine the possibility that TGF-b might also partici-
pate in regulating the pigmentation response to UV irradiation.
We postulated that crosstalk between keratinocytes and mela-
nocytes contributes to the pigmentation response of skin to UV
Here, we confirm and extend observations that the expression
and secretion of TGF-b are repressed by UV irradiation in kerati-
nocytes. We found that PAX3 transcription in melanocytes is
suppressed by TGF-b/Smad directly. Furthermore, we demon-
strate that upregulation of PAX3 in melanocytes results from
the loss of TGF- b from keratinocytes. In addition, we found
that activation of the p53-POMC/MSH-MC1R signaling pathway
is required in melanogenesis. PAX3 functions in synergy with
SOX10 in a cAMP-response element binding protein/cAMP-
response element (CREB/CRE)-dependent manner to regulate
the transcription of Mitf. Finally, overexpression of PAX3 protein
was frequently observed in melanomas from sun-exposed skin
compared with non-sun-exposed skin.
TGF-b Level Was Reduced by UV Irradiation
protein level after UV irradiation of skin in vivo (Choi et al., 2007;
Gambichler et al., 2007; Quan et al., 2001). To further explore the
modulation of TGF-b in skin after UV irradiation, we initially stud-
ied cultured human primary keratinocytes before and after expo-
sure toUVB light (100J/m2).This dose has beendescribed in our
erythema dose (Diffey et al., 1997), which is commonly used as
a measure of sunlight exposure. We found that UV markedly
repressed expression of TGF-b protein by 6 hr in keratinocytes
(Figure 1A). This result is consistent with previous findings using
human skin in vivo (Gambichler et al., 2007; Quan et al., 2004).
Furthermore, we found that UV-induced repression of TGF-b oc-
curred at the transcriptional level (Figure 5). Having established
that UV irradiation represses protein expression of TGF-b in
keratinocytes, we next investigated whether UV irradiation im-
pairs TGF-b secretion by keratinocytes. An ELISA assay of the
corresponding culture medium demonstrated >75% repression
of TGF-b secretion by keratinocytes at 24 hr after UV irradiation
TGF-b Inhibits Melanocyte Proliferation
in fibroblasts (Elliott and Blobe, 2005; Siegel and Massague,
Figure 1. PAX3 Is Repressed by TGF-b in HPM
(A) TGF-b is repressed by UV irradiation. Human primary
keratinocytes (HPKs) were irradiated with UV as de-
scribed. Protein was collected at time 0 and at different
time points after stimulation. TGF-b protein levels, which
were analyzed by western blot, are shown along with ac-
tin, which served as loading control.
(B) HPKs were irradiated with UV as described. TGF-b
levels were measured in culture medium. Secretion of
TGF-b was repressed by UV irradiation.
(C) Human primary melanocytes (HPMs) were stimulated
with different doses of TGF-b as indicated. RNA and pro-
tein were collected at 24 hr after TGF-b stimulation. The
left panels represent PAX3 RNA levels as measured by
quantitative RT-PCR and normalized to GAPDH. Results
are expressed as the mean of the experiment done in trip-
licate ± the standard error of the mean (SEM). Repression
is calculated relative to PAX3 levels in vehicle (DMSO)-
treated cells. Protein levels of PAX3 and its downstream
gene, Mitf, which were analyzed by western blot, are
shown on the right along with actin, which served as load-
(D) HPMs were stimulated with TGF-b. RNA and protein
were collected at time 0 and at different time points after
stimulation. The left panels represent PAX3 RNA levels
as measured by quantitative RT-PCR and normalized to
GAPDH. Results are expressed as the mean of the exper-
iment done in triplicate ± SEM. Induction is calculated rel-
ative to PAX3 levels in vehicle (DMSO)-treated cells. Pro-
tein levels of PAX3 and its downstream gene, Mitf, which
were analyzed by western blot, are shown on the right
along with actin, which served as loading control.
The Role of TGF-b in Melanogenesis
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Lang, D., Lu, M.M., Huang, L., Engleka, K.A., Zhang, M., Chu, E.Y., Lipner, S.,
Skoultchi, A., Millar, S.E., and Epstein, J.A. (2005). Pax3 functions at a nodal
point in melanocyte stem cell differentiation. Nature 433, 884–887.
Li, D., Turi, T.G., Schuck, A., Freedberg, I.M., Khitrov, G., and Blumenberg, M.
(2001). Rays and arrays: the transcriptional program in the response of human
epidermal keratinocytes to UVB illumination. FASEB J. 15, 2533–2535.
Lin, J.Y., and Fisher, D.E. (2007). Melanocyte biology and skin pigmentation.
Nature 445, 843–850.
Liu, G., Ding, W., Liu, X., and Mulder, K.M. (2006). c-Fos is required for
TGFbeta1 production and the associated paracrine migratory effects of
human colon carcinoma cells. Mol. Carcinog. 45, 582–593.
Martin, M., Vozenin, M.C., Gault, N., Crechet, F., Pfarr, C.M., and Lefaix, J.L.
(1997). Coactivation of AP-1 activity and TGF-beta1 gene expression in the
stress response of normal skin cells to ionizing radiation. Oncogene 15,
Martinez-Esparza, M., Solano, F., and Garcia-Borron, J.C. (1999). Indepen-
dent regulation of tyrosinase by the hypopigmenting cytokines TGF beta1
and TNF alpha and the melanogenic hormone alpha-MSH in B16 mouse
melanocytes. Cell Mol. Biol. (Noisy-le-grand) 45, 991–1000.
Martinez-Esparza, M., Ferrer, C., Castells, M.T., Garcia-Borron, J.C., and
Zuasti, A. (2001). Transforming growth factor beta1 mediates hypopigmenta-
tion of B16 mouse melanoma cells by inhibition of melanin formation and
melanosome maturation. Int. J. Biochem. Cell Biol. 33, 971–983.
Mayanil, C.S., Pool, A., Nakazaki, H., Reddy, A.C., Mania-Farnell, B., Yun, B.,
George, D., McLone, D.G., and Bremer, E.G. (2006). Regulation of murine
TGFbeta2 by Pax3 during early embryonic development. J. Biol. Chem. 281,
Medrano, E.E. (2003). Repression of TGF-beta signaling by the oncogenic
protein SKI in human melanomas: consequences for proliferation, survival,
and metastasis. Oncogene 22, 3123–3129.
Milewski, R.C., Chi, N.C., Li, J., Brown, C., Lu, M.M., and Epstein, J.A. (2004).
Identification of minimal enhancer elements sufficient for Pax3 expression in
neural crest and implication of Tead2 as a regulator of Pax3. Development
Box genes are frequently expressed in cancer and often required for cancer
cell survival. Oncogene 22, 7989–7997.
Myatt, N., Aristodemou, P., Neale, M.H., Foss, A.J., Hungerford, J.L., Bhatta-
charya, S., and A Cree, I. (2000). Abnormalities of the transforming growth
factor-beta pathway in ocular melanoma. J. Pathol. 192, 511–518.
Passeron, T., Valencia, J.C., Bertolotto, C., Hoashi, T., Le Pape, E., Takahashi,
K., Ballotti, R., and Hearing, V.J. (2007). SOX9 is a key player in ultraviolet
B-induced melanocyte differentiation and pigmentation. Proc. Natl. Acad.
Sci. USA 104, 13984–13989.
Factor-binding element in the human c-myc promoter involved in transcrip-
tional regulation by transforming growth factor beta 1 and by the retinoblas-
toma gene product. Proc. Natl. Acad. Sci. USA 88, 10227–10231.
Potterf, S.B., Furumura, M., Dunn, K.J., Arnheiter, H., and Pavan, W.J. (2000).
Transcription factor hierarchy in Waardenburg syndrome: regulation of MITF
expression by SOX10 and PAX3. Hum. Genet. 107, 1–6.
Landis, M.W., and Fisher, D.E. (1998). alpha-Melanocyte-stimulating hormone
signaling regulates expression of microphthalmia, a gene deficient in Waar-
denburg syndrome. J. Biol. Chem. 273, 33042–33047.
Pruitt, S.C., Bussman, A., Maslov, A.Y., Natoli, T.A., and Heinaman, R. (2004).
Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro and in vivo.
Gene Expr. Patterns 4, 671–685.
Quan, T., He, T., Voorhees, J.J., and Fisher, G.J. (2001). Ultraviolet irradiation
ing its type-II receptor and inducing Smad7. J. Biol. Chem. 276, 26349–26356.
Quan, T., He, T., Kang, S., Voorhees, J.J., and Fisher, G.J. (2004). Solar
ultraviolet irradiation reduces collagen in photoaged human skin by blocking
transforming growth factor-beta type II receptor/Smad signaling. Am. J.
Pathol. 165, 741–751.
Read, A.P., and Newton, V.E. (1997). Waardenburg syndrome. J. Med. Genet.
Reed, J.A., Lin, Q., Chen, D., Mian, I.S., and Medrano, E.E. (2005). SKI path-
ways inducing progression of human melanoma. Cancer Metastasis Rev.
Rees, J.L. (2004). The genetics of sun sensitivity in humans. Am. J. Hum.
Genet. 75, 739–751.
Rodeck, U., Bossler, A., Graeven, U., Fox, F.E., Nowell, P.C., Knabbe, C., and
Kari, C. (1994). Transforming growth factor beta production and responsive-
ness in normal human melanocytes and melanoma cells. Cancer Res. 54,
Scholl, F.A., Kamarashev, J., Murmann, O.V., Geertsen, R., Dummer, R., and
Schafer, B.W. (2001). PAX3 is expressed in human melanomas and contrib-
utes to tumor cell survival. Cancer Res. 61, 823–826.
Seoane, J., Le, H.V., Shen, L., Anderson, S.A., and Massague, J. (2004). Inte-
gration of Smad and forkhead pathways in the control of neuroepithelial and
glioblastoma cell proliferation. Cell 117, 211–223.
Siegel, P.M., and Massague, J. (2003). Cytostatic and apoptotic actions of
TGF-beta in homeostasis and cancer. Nat. Rev. Cancer 3, 807–821.
Smit, D.J., Smith, A.G., Parsons, P.G., Muscat, G.E., and Sturm, R.A. (2000).
Domains of Brn-2 that mediate homodimerization and interaction with general
and melanocytic transcription factors. Eur. J. Biochem. 267, 6413–6422.
Tassabehji, M., Read, A.P., Newton, V.E., Harris, R., Balling, R., Gruss, P., and
Strachan, T. (1992). Waardenburg’s syndrome patients have mutations in the
human homologue of the Pax-3 paired box gene. Nature 355, 635–636.
Trends Biochem. Sci. 29, 265–273.
Verastegui, C., Bille, K., Ortonne, J.P., and Ballotti, R. (2000). Regulation of the
microphthalmia-associated transcription factor gene by the Waardenburg
syndrome type 4 gene, SOX10. J. Biol. Chem. 275, 30757–30760.
Wang, J., Ouyang, W., Li, J., Wei, L., Ma, Q., Zhang, Z., Tong, Q., He, J., and
Huang, C. (2005). Loss of tumor suppressor p53 decreases PTEN expression
and enhances signaling pathways leading to activation of activator protein 1
and nuclear factor kappaB induced by UV radiation. Cancer Res. 65,
Weigert, C., Sauer, U., Brodbeck, K., Pfeiffer, A., Haring, H.U., and Schleicher,
E.D. (2000). AP-1 proteins mediate hyperglycemia-induced activation of the
human TGF-beta1 promoter in mesangial cells. J. Am. Soc. Nephrol. 11,
Yasumoto, K., Yokoyama, K., Takahashi, K., Tomita, Y., and Shibahara, S.
(1997). Functional analysis of microphthalmia-associated transcription factor
in pigment cell-specific transcription of the human tyrosinase family genes.
J. Biol. Chem. 272, 503–509.
Zhu, B.K., and Pruitt, S.C. (2005). Determination of transcription factors and
their possible roles in the regulation of Pax3 gene expression in the mouse
B16 F1 melanoma cell line. Melanoma Res. 15, 363–373.
The Role of TGF-b in Melanogenesis
Molecular Cell 32, 554–563, November 21, 2008 ª2008 Elsevier Inc. 563