In addition to producing melanin to protect epidermal keratinocytes against DNA damage, melanocytes may have important roles in strengthening innate immunity against pathogens. We have developed a functional, pigmented, human full-thickness 3D skin equivalent to determine whether the presence of melanocytes impacts epidermal bacterial growth and regulates the expression of genes involved in the immune response. We introduced primary epidermal melanocytes to construct a 3-cell full-thickness skin equivalent with primary dermal fibroblasts and epidermal keratinocytes. Immunohistochemistry verified the appropriate ratio and spatial organisation of melanocytes. Alpha-MSH induced melanogenesis, confirming an appropriate physiological response. We compared this 3-cell skin equivalent with the 2-cell version without melanocytes in response to inoculation with 3 species of bacteria: Staphylococcus epidermidis, Corynebacterium striatum, and Cutibacterium acnes. There was a significant decrease in the colonisation of bacteria in the skin equivalents containing functional melanocytes. There was increased expression of immune-response genes (S100A9, DEFB4A, IL-4R) following microorganism exposure; however, there were marked differences between the unpigmented and pigmented skin equivalents. This physiologically relevant human 3D-skin equivalent opens up new avenues for studying complex skin pigmentation disorders, melanoma, and UV damage, as well as the rapidly evolving field of the skin microbiome and the balance between commensal and pathogenic species.

epidermal melanocyte hair follicle melanocyte superoxide dismutase TO THE EDITOR Canities or senile hair graying, a universally recognized sign of aging, remains unresolved in terms of physiological causes, although a strong genetic contribution is understood (Gunn et al., 2009Gunn D.A. Rexbye H. Griffiths C.E. et al.Why some women look young for their age.PLoS One. 2009; 4: e8021Crossref PubMed Scopus (124) Google Scholar). As the hair fiber continues to grow long after melanin production ceases, we suggest that melanocytes in the hair follicle may be more sensitive to the impact of chronological aging than are keratinocytes. Moreover, follicular melanocytes also age more markedly than those in the overlying epidermis. The hair follicle provides a unique opportunity to decouple the impact of age on two hair follicular tissue functions: hair formation and hair pigmentation. Previous studies have pointed to a link between cellular aging mechanisms, including oxidative stress and hair graying (Arck et al., 2006Arck P.C. Overall R. Spatz K. et al.Towards a “free radical theory of graying”: melanocyte apoptosis in the aging human hair follicle is an indicator of oxidative stress induced tissue damage.FASEB J. 2006; 20: 1567-1569Crossref PubMed Scopus (155) Google Scholar; Wood et al., 2009Wood J.M. Decker H. Hartmann H. et al.Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair.FASEB J. 2009; 23: 2065-2075Crossref PubMed Scopus (141) Google Scholar). Commo et al., 2004Commo S. Gaillard O. Thibaut S. et al.Absence of TRP-2 in melanogenic melanocytes of human hair.Pigment Cell Res. 2004; 17: 488-497Crossref PubMed Scopus (56) Google Scholar and others (Nishimura et al., 2005Nishimura E.K. Granter S.R. Fisher D.E. Mechanisms of hair graying: incomplete melanocyte stem cell maintenance in the niche.Science. 2005; 307: 720-724Crossref PubMed Scopus (538) Google Scholar) have suggested that repopulation of the early anagen hair bulb with precursors of active melanocytes is also increasingly likely to fail with aging. However, most studies differentiate follicles on the basis of the level of pigmentation, not donor age. We also know that hair graying may be partially reversed in some skin disorders or after certain drug therapies (Reynolds et al., 1998Reynolds A. Murray P.I. Colloby P.S. Darkening of eyelashes in a patient treated with latanoprost.Eye (Lond). 1998; 12: 741-743Crossref PubMed Scopus (18) Google Scholar; Shaffrali et al., 2002Shaffrali F.C. McDonagh A.J. Messenger A.G. Hair darkening in porphyria cutanea tarda.Br J Dermatol. 2002; 146: 325-329Crossref PubMed Scopus (27) Google Scholar). Thus, graying may not necessarily indicate a complete deletion of the melanocyte stem cell population. We wanted to explore the impact of chronological age on melanocyte behavior to further understand the graying process and to identify associated molecular changes. This study provides analysis of race, age, and anatomically matched cultures of adult human epidermal and hair follicle melanocytes (HFMs) (Supplementary Table S1 online), and to our knowledge, this is previously unreported. Cultured HFMs showed at least three distinct sub-populations, including highly pigmented/dendritic bulbar melanocytes, less-differentiated tripolar cells, and an undifferentiated amelanotic bipolar sub-population (Supplementary Figure S1 online). By contrast, epidermal melanocytes (EMs) largely consisted of a homogeneous population of highly dendritic and uniformly weakly pigmented cells (Supplementary Figure S1 online). Unlike EMs, the most active melanocytes of the bulb do not survive when explanted ex vivo. Less-differentiated melanocytes, from elsewhere in the bulb and the outer root sheath, persist in graying, and can be cultured and induced to make melanin (Tobin and Paus, 2001Tobin D.J. Paus R. Graying: gerontobiology of the hair follicle pigmentary unit.Exp Gerontol. 2001; 36: 29-54Crossref PubMed Scopus (224) Google Scholar; Slominski et al., 2004Slominski A. Tobin D.J. Shibahara S. et al.Melanin pigmentation in mammalian skin and its hormonal regulation.Physiol Rev. 2004; 84: 1155-1228Crossref PubMed Scopus (1300) Google Scholar). Download .pdf (.25 MB) Help with pdf files Supplementary Information Proliferation of HFMs and EMs was examined in matched cultures derived from young, middle-aged, and older donors (Supplementary Table S1 online). Both EM and HFM proliferation decreased with age, HFM by 49.7±2.4% and EM by 42.6±5.7%. Tyrosinase expression was reduced with age in EMs by 51.6% and by 77.1% in HFMs (Supplementary Table S2 and S3 online; Supplementary Figure 1 online). In marked contrast, tyrosinase-related protein-1 expression was increased with age in both EMs (+24.3%) and HFMs (+43.6%) (Supplementary Table S2 and S3 online; Figure 1). It was noteworthy that dopachrome tautomerase was greatly reduced in EMs obtained from older donors, but the expression of the native form of this important melanogenic enzyme was elevated by 67.2% with age in HFMs (Supplementary Table S2 and S3 online), although the glycosylated form was much less affected. Recent data from our laboratory have shown that dopachrome tautomerase levels may be associated with protection against oxidative stress in EMs (Gledhill et al., unpublished). Dopachrome tautomerase may also provide such protection from quinone metabolites (Michard et al., 2008Michard Q. Commo S. Rocchetti J. et al.TRP-2 expression protects HEK cells from dopamine- and hydroquinone-induced toxicity.Free Radic Biol Med. 2008; 45: 1002-1010Crossref PubMed Scopus (16) Google Scholar), highlighting an additional non-pigmentary role for this enzyme in maintaining cellular redox status. The precise relationship between dopachrome tautomerase and eumelanogenic HFMs remains unresolved, as this relationship seems to vary between follicles from different body sites (Commo et al., 2004Commo S. Gaillard O. Thibaut S. et al.Absence of TRP-2 in melanogenic melanocytes of human hair.Pigment Cell Res. 2004; 17: 488-497Crossref PubMed Scopus (56) Google Scholar; Thibaut et al., 2009Thibaut S. De Becker E. Caisey L. et al.Human eyelash characterization.Br J Dermatol. 2009; 162: 304-310Crossref PubMed Scopus (35) Google Scholar). Recent data have suggested that HFMs and EMs may be regulated independently; even by shared signaling pathways (Van Raamsdonk et al., 2009Van Raamsdonk C.D. Barsh G.S. Wakamatsu K. et al.Independent regulation of hair and skin color by two G protein-coupled pathways.Pigment Cell Melanoma Res. 2009; 22: 819-826Crossref PubMed Scopus (37) Google Scholar). As accumulation of oxidative stress is a leading cellular aging mechanism, and melanin synthesis itself is an oxidative process (Pawelek and Lerner, 1978Pawelek J.M. Lerner A.B. 5,6-Dihydroxyindole is a melanin precursor showing potent cytotoxicity.Nature. 1978; 276: 626-628Crossref PubMed Scopus (154) Google Scholar), we examined the relative expression of key anti-oxidant enzymes (such as catalase, superoxide dismutase (SOD)-1, and SOD-2) in fully matched HFMs and EMs obtained from donors of different ages. SOD-1 and SOD-2 protein expressions were similar in HFMs and EMs and were not changed with age (data not shown). However, by contrast, catalase expression was markedly reduced in HFMs derived from older donors as seen by immunocytochemical examination (Figure 2a1, a2 vs. b1, b2) and western blotting (68.8% reduction with age; Figure 2c; Supplementary Table S4 online), and catalase activity was also reduced with age (42.0% Figure 2e). Catalase expression was also reduced in EMs with age (51.6% Figure 2c; Supplementary Table S4 online). Lowered catalase protein expression/activity with elevated H2O2 production, due to superoxide dismutation by SOD, may lead to an accumulation of H2O2 in cells. This is likely to have cytotoxic implications; such a scenario has been implicated in vitiligo (Wood et al., 2008Wood J.M. Gibbons N.C. Chavan B. et al.Computer simulation of heterogeneous single nucleotide polymorphisms in the catalase gene indicates structural changes in the enzyme active site, NADPH-binding and tetramerization domains: a genetic predisposition for an altered catalase in patients with vitiligo?.Exp Dermatol. 2008; 17: 366-371Crossref PubMed Scopus (20) Google Scholar). Although we did not measure H2O2 levels in our EMs and HFMs, we have subsequently shown that addition of H2O2 reveals an “aged” HFM phenotype that is refractory to stress responses (Kauser et al., manuscript in preparation). Keratinocytes surrounding melanocytes in the epidermis may well be a source of H2O2 that is uncharged and freely diffusible (Pelle et al., 2005Pelle E. Mammone T. Maes D. et al.Keratinocytes act as a source of reactive oxygen species by transferring hydrogen peroxide to melanocytes.J Invest Dermatol. 2005; 124: 793-797Crossref PubMed Scopus (71) Google Scholar), and there is evidence of H2O2 damage to proteins in graying hair follicles (Wood et al., 2009Wood J.M. Decker H. Hartmann H. et al.Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair.FASEB J. 2009; 23: 2065-2075Crossref PubMed Scopus (141) Google Scholar). Thus, the generalized loss of catalase in the hair follicle could contribute to an “aged” melanocyte phenotype, heightened by the greater age-related susceptibility of HFMs compared with their epidermal counterparts. Such oxidative damage may also, as suggested by others (Wood et al., 2009Wood J.M. Decker H. Hartmann H. et al.Senile hair graying: H2O2-mediated oxidative stress affects human hair color by blunting methionine sulfoxide repair.FASEB J. 2009; 23: 2065-2075Crossref PubMed Scopus (141) Google Scholar), cause damage to other systems in the hair follicle and affect the hair fiber itself. It may be expected that higher expression/activity of catalase is required by melanocytes with higher pigmentation levels (also in pigmented vs. the gray/white anagen hair bulbs); however, it is also the case that hair fiber production, by very high metabolic and mitotic activity in white/gray hair bulbs and associated reactive oxygen species production, does not seem to be impaired by the reduced catalase expression/activity in these follicles. In summary, we have demonstrated that we can successfully transfer an aged HFM phenotype from ex vivo skin to in vitro cell culture, and we now propose that catalase expression/activity is an important effector in the responses of melanocytes to aging. Our data lend support to the hypothesis that the susceptibility of HFMs to oxidative stress over time may be a major factor in the loss of hair pigment, the reduction in overall numbers of melanocytes per follicle, and ultimately for the increase in number of “white” hairs. This research was supported by Unilever R&D, UK. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

Proopiomelanocortin (POMC) can be processed to ACTH and melanocortin peptides. However, processing is incomplete in some tissues, leading to POMC precursor release from cells. This study examined POMC processing in human skin and the effect of POMC on the melanocortin-1 receptor (MC-1R) and melanocyte regulation. POMC was secreted by both human epidermal keratinocytes (from 5 healthy donors) and matched epidermal melanocytes in culture. Much lower levels of alpha-MSH were secreted and only by the keratinocytes. Neither cell type released ACTH. Cell extracts contained significantly more ACTH than POMC, and alpha-MSH was detected only in keratinocytes. Nevertheless, the POMC processing components, prohormone convertases 1, 2 and regulatory protein 7B2, were detected in melanocytes and keratinocytes. In contrast, hair follicle melanocytes secreted both POMC and alpha-MSH, and this was enhanced in response to corticotrophin-releasing hormone (CRH) acting primarily through the CRH receptor 1. In cells stably transfected with the MC-1R, POMC stimulated cAMP, albeit with a lower potency than ACTH, alpha-MSH, and beta-MSH. POMC also increased melanogenesis and dendricity in human pigment cells. This release of POMC from skin cells and its functional activity at the MC-1R highlight the importance of POMC processing as a key regulatory event in the skin.

Cotricotropin- releasing hormone ( CRH) and related peptides are produced in skin that is dependent on species and anatomical location. Local peptide production is regulated by ultraviolet radiation ( UVR), glucocorticoids and phase of the hair cycle. The skin also expresses the corresponding receptors ( CRH- R1 and CRH- R2), with CRH- R1 being the major receptor in humans. CRH- R1 is expressed in epidermal and dermal compartments, and CRH- R2 predominantly in dermal structures. The gene coding for CRH- R1 generates multiple isoforms through a process modulated by UVR, cyclic adenosine monophosphate ( cAMP) and phorbol 12- myristate 13-acetate. The phenotypic effects of CRH in human skin cells are largely mediated by CRH- R1alpha through increases in concentrations of cAMP, inositol triphosphate ( IP3), or Ca2+ with subsequent activation of protein kinases A ( PKA) and C ( PKC) dependent pathways. CRH also modulates the activity of nuclear factor of kappa light polypeptide gene enhancer in B- cells ( NF- kappaB), activator protein 1 ( AP-1) and cAMP responsive element binding protein ( CREB). The cellular functions affected by CRH depend on cell type and nutritional status and include modulation of differentiation program( s), proliferation, viability and immune activity. The accumulated evidence indicates that cutaneous CRH is also a component of a local structure organized similarly to the hypothalamo- pituitary- adrenal axis.

Human skin is a local source of corticotropin-releasing hormone (CRH) and expresses CRH and CRH receptors (CRH-R) at mRNA and protein levels. Epidermal melanocytes respond to CRH by induction of cAMP with up-regulation of pro-opiomelanocortin gene expression and subsequent production of adrenocorticotropin hormone. However, the role of CRH/CRH-R in melanocyte biology is complicated by the significant heterogeneity of cutaneous melanocyte subpopulations, from continuously active and UV-responsive melanocytes in epidermis to UV nonresponsive, hair growth cycle-coupled melanogenesis in hair follicles. In the present study we report that normal human scalp hair follicle melanocytes express CRH at the mRNA level. Furthermore, CRH, urocortin and CRH-R 1 and 2 were differentially expressed in follicular melanocytes, fibroblasts, and keratinocytes depending on anatomic location and differentiation status in situ and in vitro. Stimulation of follicular melanocytes with CRH and CRH peptides, modified for selectivity for CRH-R1 and/or CRH-R2, variably induced cell melanogenesis, dendricity, and proliferation. CRH-peptides also stimulated the expression and activity of Tyrosinase, and expression of Tyrosinase-related protein-1 and-2. However, a modified urocortin peptide highly selective for CRH-R2 down-regulated melanocyte differentiation phenotype. This study indicates that CRH peptides can differentially influence hair follicle melanocyte behavior not only via CRH-R1 signaling but also by complex cross-talk between CRH-R1 and CRH-R2.

The presence of albumin in the human epidermis has been reported more than a decade ago, but until now, it was assumed that this protein is synthesized in the liver and transported to the avascular skin. To our knowledge, transcription of albumin in the human epidermis was never considered. In this report, we present for the first time evidence for autocrine synthesis of albumin in the human epidermis in keratinocytes in situ and in vitro. Using double immunofluorescence labelling, we identified that albumin colocalized together with its transcription factor PCD/DCoH/HNF-1alpha in suprabasal keratinocytes in human full-thickness skin sections and in keratinocytes cultured in serum-free medium. Moreover, albumin and HNF-1alpha protein expression was confirmed by Western blotting in undifferentiated and differentiated keratinocytes as well as in human epidermal suction blister roof extracts. Reverse-transcriptase polymerase chain reaction analysis from human epidermal keratinocytes and epidermal suction blister roofs revealed the transcription of albumin. Using in vivo fluorescence excitation spectroscopy at the surface of human skin, we confirmed albumin as a major constituent yielding a lambda(max) at 295 nm, which was assigned to the single tryptophan 214 fluorophore in this protein. This in vivo result is in agreement with albumin concentrations of 10(-3) M, underlining the importance of this protein in epidermal homeostasis.

The proopiomelanocortin (POMC) gene and protein are expressed principally in the pituitary and brain (e.g., hypothalamus). The POMC gene, protein, and derived peptides, however, can also be detected in the skin. It appears that POMC can also be processed in the skin, a tissue that also expresses the prohormone convertases PC1 and PC2 and 7B2 protein. All POMC peptides may be produced in the skin epidermis, dermis, and adnexa by epithelial cells, melanocytes, and mesenchymal cells (e.g., immune cells, fibroblasts, and endothelial cells), and can also be released from cutaneous sensory nerve endings (cf. Slominski et al, 2004).

The proopiomelanocortin (POMC)-derived peptides, ACTH and alpha-MSH, are the principal mediators of human skin pigmentation via their action at the melanocortin-1 receptor (MC-1R). Recent data have demonstrated the existence of a functionally active beta-endorphin/mu-opiate receptor system in both epidermal and hair follicle melanocytes, whereby beta-endorphin can regulate melanogenesis, dendricity, and proliferation in these cells. However, a role for ACTH and alpha-MSH in the regulation of the human follicular pigmentary unit has not been determined. This study was designed to examine the involvement of ACTH and the alpha-MSH/MC-1R system in human follicular melanocyte biology. To address this question we employed RT-PCR and immunohisto/cytochemistry, and a functional role for these POMC peptides was assessed in follicular melanocyte cultures. Human scalp hair follicle melanocytes synthesized and processed POMC. ACTH and alpha-MSH in association with their processing enzymes and MC-1R are expressed in human follicular melanocytes at the message level in vitro and at the protein level both in situ and in vitro. The expression of the POMC/MC-1R receptor system was confined only to subpopulations of poorly and moderately differentiated melanocytes. In addition, functional studies revealed that ACTH and alpha-MSH are able to promote follicular melanocyte differentiation by up-regulating melanogenesis, dendricity, and proliferation in less differentiated melanocyte subpopulations. Thus, these findings suggest a role for these POMC peptides in regulating human hair follicle melanocyte differentiation.

The human skin holds the full machinery for pro-opiomelanocortin processing. The alpha-melanocyte-stimulating hormone (alpha-MSH)/melanocortin-1-receptor cascade has been implicated as a major player via the cAMP signal in the control of melanogenesis. Only very recently the beta-endorphin/mu-opiate receptor signal has been added to the list of regulators of melanocyte dendricity and melanin formation. In this context it was reported that (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) can act as an allosteric inhibitor of tyrosinase, the key enzyme in melanogenesis, and this inhibition is reversible by both alpha- and beta-MSH. It was also shown earlier that 7BH(4), the isomer of 6BH(4), is twice as active in this inhibition reaction. However, as yet it is not known whether 7BH(4) is indeed present in loco in the melanosome. We here provide evidence that this isomer is present in this organelle in a concentration range up to 50 x 10(-6) M. Determination of beta-MSH in melanosomal extracts yielded 10 pg/mg protein. Moreover, we demonstrate reactivation of the 7BH(4)/tyrosinase inhibitor complex by beta-MSH, whereas alpha-MSH failed to do so. Furthermore, we show intra-melanosomal l-dopa formation from dopachrome by 7BH(4) in a concentration range up to 134 x 10(-6) M. Based on these results, we propose a new receptor-independent mechanism in the control of tyrosinase/melanogenesis by beta-MSH and the pterin 7BH(4).

We are currently experiencing a spectacular surge in our knowledge of skin function both at the organ and organismal levels, much of this due to a flurry of cutaneous neuroendocrinologic data, that positions the skin as a major sensor of the periphery. As our body's largest organ, the skin incorporates all major support systems including blood, muscle and innervation as well as its role in immuno-competence, psycho-emotion, ultraviolet radiation sensing, endocrine function, etc. It is integral for maintenance of mammalian homeostasis and utilizes locally-produced melanocortins to neutralize noxious stimuli. In particular, the cutaneous pigmentary system is an important stress response element of the skin's sensing apparatus; where stimuli involving corticotrophin-releasing hormone (CRH) and proopiomelanocortin (POMC) peptides help regulate pigmentation in the hair follicle and the epidermis. These pigmentary units are organized into symmetrical functional pigmentary units composed of corticotropin-releasing hormone, and the melanocortin POW peptides melanocyte stimulating hormone, adrenocorticotropic hormone and also the opiate beta-endorphin., These new findings have led to the concept of "self-similarity" of melanocortin systems based on their expression both at the local (skin) and systemic (CNS) levels, where the only major apparent difference appears to be one of scale. This review explores this concept and describes how the components of the CRH/POMC systems may help regulate the human hair follicle pigmentary unit. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Corticotropin‐releasing hormone (CRH) is the most proximal element of the hypothalamic‐pituitary‐adrenal axis (HPA) and is the chief regulator of pituitary POMC gene expression and the subsequent production and secretion of POMC peptides. Previously, our laboratories documented cutaneous expression of CRH, urocortin and functional CRH receptors (CRH‐Rs), suggesting their role in skin physiology and pathology. Human skin predominately expressed CRH‐R1 with CRH‐R2 being expressed primarily in the adnexal structures. While CRH‐R activity has been implicated in the regulation of epidermal cell function, a role for these receptors in human hair biology has not yet been demonstrated. This study was designed to investigate the effects of modified CRH peptides (D‐Glu20)‐CRH, (D‐Pro5)‐CRH and (D‐Pro4)‐urocortin with respective selectivity for CRH‐R1 and CRH‐R2 on behaviour of cultured hair follicle melanocytes (HFMs) derived from scalp of seven normal individuals. HFMs were stimulated with these peptides (10−7−10−10 m) for 72 h. (D‐Glu20)‐CRH (10−8 m) and (D‐Pro5)‐CRH (10−9 and 10−10 m) markedly increased cell dendricity, melanogenesis and proliferation (P < 0.01) compared with unstimulated levels. While (D‐Pro4)‐urocortin failed to stimulate cell dendricity, this peptide did stimulate melanogenesis (10−8 m) (P < 0.01) and exhibited a biphasic proliferative response; stimulating pigment cell division at 10−7 and 10−8 m (P < 0.01) but inhibiting proliferation at 10−9 and 10−10 m (P < 0.01). Here, we demonstrate the existence of functionally active CRH‐Rs in cultured human scalp HFM and show that signalling via these receptors modulates follicular melanocyte dendricity, melanogenesis and proliferation. Thus, activation of CRH‐Rs may have a pivotal role in the regulation of follicular melanocyte homeostasis.

Mesenchymal cells are involved in reciprocal mesenchymal–epithelial interactions during development and growth of skin and its appendages. Fibroblasts exhibit topographic differentiation and so constitute a highly diverse family of cells with distinct and characteristic traits. This heterogeneity is also seen in the skin where hair growth inductive fibroblasts called follicular papilla (FP) fibroblasts are distinct from fibroblasts of the connective tissue sheath (CTS), and both, in turn, are distinct from peripheral interfollicular dermal fibroblasts (DFs). POMC peptides and their cognate receptors are expressed variably by several skin cells types, including fibroblasts. However, it is not clear how the POMC system is regulated in different skin fibroblasts populations. We characterized the expression of the POMC peptide family and their receptors and pro‐hormone convertases (PCs) in human haired scalp during the hair growth cycle and in matched sets of DF, FP and CTS fibroblasts cultured from normal adult male scalp. Expression of POMC peptides, PC 2 and µ‐OR in FP fibroblasts was highest during anagen. By contrast, β‐end, 7B2 and PC1 were broadly undetectable during anagen but expression levels increased considerably during the apoptosis‐driven catagen phase. Matched sets of cultured FP and DF fibroblasts showed similar protein expression levels of α‐MSH, ACTH, PC2, 7B2, µ‐OR and MC1‐R. However, FP cells expressed higher levels of PC1 and β‐end peptide and higher µ‐OR mRNA levels than DF cells. Thus, follicular and interfollicular fibroblasts represent heterogeneous subpopulations that are likely to respond variably to POMC peptides.