Control of melanocyte
differentiation by a MITF–
PDE4D3 homeostatic circuit
Mehdi Khaled, Carmit Levy, and David E. Fisher1
Department of Dermatology, Cutaneous Biology Research
Center, Massachusetts General Hospital, Harvard Medical
School, Massachusetts 02114, USA
Cyclic AMP (cAMP) is a ubiquitous second messenger
that regulates a variety of biological processes. The mag-
nitude and duration of cAMP expression are regulated by
both production and hydrolysis. Melanocyte-stimulating
hormone (MSH) plays a crucial role in pigment cell differ-
entiation via cAMP-regulated expression of the master
transcription factor MITF. We report the identification
of phosphodiesterase 4D3 as a direct target of the MSH/
cAMP/MITF pathway. This creates a negative feedback
loop that induces refractoriness to chronic stimulation
of the cAMP pathway in melanocytes. This homeostatic
pathway highlights a potent mechanism controlling me-
lanocyte differentiation that may be amenable to phar-
macologic manipulation for skin cancer prevention.
Supplemental material is available at http://www.genesdev.org.
Received April 15, 2010; revised version accepted August 30,
Cyclic AMP (cAMP) is a second messenger that regulates
key processes (such as cell growth, differentiation, and
movement) and specialized actions unique to specific cell
lineages (Houslay et al. 2007). cAMP is typically metab-
olized by phosphodiesterases (PDEs), which control the
magnitude, duration, and subcellular localization of
cAMP (Houslay et al. 2007). In melanocytes the cAMP/
CREB signaling pathway is potently regulated by mela-
nocyte-stimulating hormone (MSH) via the G protein-
coupled receptor MC1R, which in turn transcriptionally
activates expression of MITF (Bertolotto et al. 1998; Price
et al. 1998), the master transcriptional regulator of me-
lanocyte development (Levy et al. 2006). In vitro and in
vivo, the up-regulation of cAMP in melanocytes leads to
increased pigment production and protection of the skin
against the deleterious effects of UV radiation (D’Orazio
et al. 2006). Skin is the most common organ to be affected
by cancer, and cutaneous malignancies most commonly
occur in fair-skinned people with a limited capacity to tan
(Fitzpatrick and Sober 1985; Fitzpatrick 1988; Sturm et al.
2003; Rijken et al. 2004). The role of pigmentation in
modulating human skin cancer risk continues to be in-
completely understood. However, darkly pigmented peo-
ple or those who tan easily (Fitzpatrick phototypes 3–6)
exhibit significantly diminished skin cancer risk relative
to those with light skin who tan poorly (Fitzpatrick
phototypes 1,2) (Lin and Fisher 2007).
Here we report that, in melanocytes, MITF directly
regulates the transcription of the PDE4D3 gene, creating
negative homeostatic control of the cAMP pathway and
lineage differentiation. These findings provide a rationale
for targeting PDE4D3 to modulate MITF expression and
control skin pigmentation.
Results and Discussion
PDE4D expression is MITF-dependent in melanocytes
We used annotated microarray data (http://biogps.gnf.
org/downloads) to search for PDEs that are expressed in
the melanocyte lineage. Among the PDEs known to
degrade cAMP (Conti and Beavo 2007; Omori and Kotera
2007), the following exhibited ‘‘present calls’’ in some or
most of the melanoma lines: PDE1C, PDE3A, PDE3B,
PDE4A, PDE4B, PDE4D, PDE6C, PDE7B, PDE8A, and
PDE10A. When the expression of these PDEs was com-
pared with that of MITF, we observed that two of them,
PDE4D and PDE4B, exhibited fluctuations in expression
that correlated with MITF expression, suggesting that
they could be transcriptionally linked to MITF (Supple-
mental Fig. S1). In this analysis, we used tyrosinase, a
known target of MITF, as a positive control (Bentley et al.
1994; Yasumoto et al. 1994). Interestingly, PDE4C did not
correlate with MITF.
To address this possibility, we first investigated whether
MITF was necessary for PDE4D and PDE4B expression
(Fig. 1A). siRNA-mediated knockdown of MITF in three
different primary human melanocyte cultures signifi-
cantly suppressed expression of PDE4D, but not PDE4B.
This experiment was carried out using primers able to
recognize all isoforms of the PDE4D gene. Expression of
LEF-1, an additional control, was also unaffected. The
knockdown efficiency of MITF was also verified by West-
ern blot (Supplemental Fig. S2).
PDE4D is a complex gene encoding multiple isoforms
(Supplemental Fig. S3) with distinct regulation and tissue
distribution (D’Sa et al. 2002; Richter et al. 2005). To
identify the specific isoforms expressed in primary mela-
nocytes, isoform-specific primers were used and revealed
expression of seven of the nine PDE4Ds known to be
expressed in humans (all except PDE4D4 and PDE4D8)
(Supplemental Fig. S4A). Since MITF is regulated by
signaling pathways using cAMP (Bertolotto et al. 1998;
Price et al. 1998), we examined the effect of forskolin on
the expression of the PDE4D isoforms in melanocytes.
Only PDE4D3 mRNAwas consistently up-regulated upon
forskolin stimulation (Supplemental Fig. S4B). We ob-
served induction of PDE4D3 mRNA levels with a slight
delay (first seen at 6 h), consistent with the possibility of
a mechanistic intermediate between forskolin/cAMP and
PDE4D3 transcription. Slight suppression of PDE4D3
levels at 2 h wasalso observed in fibroblasts (Supplemental
Fig. S5A), suggesting a mechanism unrelated to M-MITF.
Treatment of normal human fibroblasts with forskolin
failed to induce the expression of PDE4D (using common
region primers) or PDE4D3 (Supplemental Fig. S5A), al-
though it did induce phosphorylation of CREB, confirming
[Keywords: cAMP; MITF; PDE4D; skin; pigmentation]
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Articleis online at http://www.genesdev.org/cgi/doi/10.1101/gad.1937710.
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MITF regulates PDE4D3 in melanocytes
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