stress augmented ultraviolet-irradiation-induced pigmentation

ORIGINAL ARTICLESee related Commentary on pages iv and v

Stress Augmented Ultraviolet-Irradiation-Induced Pigmentation

Kaori Inoue, Junichi Hosoi, Ritsuro Ideta, Naomi Ohta, Ohji Ifuku, and Toru TsuchiyaShiseido Research Center,Yokohama, Japan

It was reported that adrenocorticotropic hormone stim-ulates melanogenesis in cultured melanocytes. Stress(high population density and restraint stress) induced asigni¢cant increase in adrenocorticotropic hormone lev-els in plasma and skin compared to control. The serumobtained from HR-1�HR/De F1 female mice sub-jected to stress showed signi¢cantly increased tyrosinaseactivity in human melanocytes compared to that fromnonstressed mice. The increase in tyrosinase activitywas inhibited in the presence of 10 nM corticostatin, anadrenocorticotropic hormone inhibitor. The aim of thisstudy was to examine whether adrenocorticotropic hor-mone released into the circulation under stressful con-ditions is associated with the regulation of ultraviolet-induced pigmentation. Mice divided into three groupswere housed for 22 d under the following conditions:¢ve mice per cage (control); 10 mice per cage (high po-pulation density); restraint stress 4 h per d. The animals

were exposed to ultraviolet-B irradiation (72 mJ percm2, thrice per wk). After ultraviolet-B irradiation, de-layed tanning was marked in stressed mice.The numberof dihydroxyphenylalanine-positive melanocytes alsosigni¢cantly increased in stressed animals. Pretreatmentwith 100 lg of corticostatin inhibited the augmentationof the stress-induced pigmentary response and the in-crease in dihydroxyphenylalanine-positive melanocytesafter ultraviolet irradiation. Adrenocorticotropic hor-mone released by stress may activate tyrosinase inmelanocytes, resulting in the augmentation of ultravio-let-induced pigmentation. These results suggest thatadrenocorticotropic hormone is at least partly responsi-ble for the sensitivity of the pigmentary response afterultraviolet irradiation under stressful conditions. Keywords: ACTH/melanocyte/pigmentation/stress/UVB. J InvestDermatol 121:165 ^171, 2003

Physiologic stress results in activation of the hypothala-mic�pituitary�adrenal (HPA) axis function and thesympathetic nervous system. The secretion of cortico-tropin-releasing hormone (CRH) from the hypothala-mus increases the synthesis of proopiomelanocortin

(POMC) in the pituitary lobe. POMC is the precursor for adre-nocorticotropic hormone (ACTH) and melanocyte stimulatinghormone (MSH), which have numerous physical actions includ-ing acting as regulators of immune response and melanogenesis.ACTH is secreted into the circulation and then induces the secre-tion of corticoids from the adrenal cortex. The localization ofPOMC-derived peptides is not restricted to cells of the pituitarygland and the central nervous system, however (DeBold et al,1988). For example, ultraviolet (UV) irradiation, a major environ-mental stressor, directly activates the synthesis of POMCmRNA, and POMC-derived peptides, namely ACTH and a-MSH, are synthesized in skin (Slominski et al, 1993; Schauer et al,1994; Chakraborty et al, 1996; Wintzen and Gilchrest, 1996).Furthermore UVB regulates both the expression of CRH geneand the production of CRH peptide in skin cells (Slominskiet al, 1996a, 2001), and glucocorticoid receptors are expressed inthe skin (Karstila et al, 1994; Serres et al, 1996). In rodent skin,

UVB and the topical application of b-MSH act synergisticallyto promote melanogenesis (Bolognia et al, 1989; Slominski andPawelek, 1998; Chakraborty et al, 1999). These ¢ndings stronglysuggest the importance of a localized HPA axis in the skin(Slominski andWortsman, 2000; Slominski et al, 2000).On the other hand, there are a number of reports concerning

the participation of circulatory POMC-derived peptides in thepigmentary response (Holtzmann et al, 1982; 1983; Ghanem et al,1989). The plasma level of a-MSH is a¡ected by seasonal sun ex-posure (Altmeyer et al, 1986). The number of melanocytes in theshielded ear of mice increased after exposure of the other ear toUVB irradiation (Rosdahl, 1979). Pears et al (1992) reported thatskin hyperpigmentation due to hypersecretion of a-MSH fromthe pituitary source was most marked in the light-exposed areasof a patient. Elevations of circulating a-MSH and ACTH levelsare assumed to be responsible for the hyperpigmentation. It is de-batable whether both ACTH and MSH act as paracrine factorsfrom skin or as endocrine factors from pituitary gland to stimu-late melanogenesis.A large number of hormones, neuropeptides, and cytokines

produced by the brain and other tissues are known to be releasedinto circulation as a result of stress (Tidgren et al, 1991; Nukinaet al, 1998). Psychologic stress in£uences cutaneous functions bychanging the level of neuropeptides and hormones (Singh et al,1999). The epidermal cell proliferating activity assessed by prolif-erating cell nuclear antigen was decreased by immobilizationstress in Syrian hamsters (Tsuchiya and Horii, 1996). Stress sup-pressed the contact hypersensitivity response (Hosoi et al, 1998)and delayed barrier recovery (Denda et al, 1998). Kaminaga andShinomiya (1997) reported that the skin color parameter changedand the number of dihydroxyphenylalanine (DOPA) positive

Reprint requests to: Kaori Inoue Ph.D., Shiseido Research Center, 2-12-1, Fukuura, Kanazawa-ku, Yokohama 236-8643, Japan; Email: [email protected]: a-MSH, a-melanocyte stimulating hormone; CRH, cor-

ticotropin-releasing hormone; HPA axis, hypothalamic�pituitary�adrenalaxis; POMC, proopiomelanocortin.

Manuscript received August 22, 2002; revised January 29, 2003; acceptedfor publication February 12, 2003

0022-202X/03/$15.00 . Copyright r 2003 by The Society for Investigative Dermatology, Inc.

165

melanocytes increased remarkably under stressful conditions inbrown guinea pigs. The aim of this study was to examinewhether or not the elevation of hormone levels caused bypsychologic stress under normal but not pathologic conditionsin£uences UV-induced melanogenesis. The F1 hairless mousedevelops pigmented spots on the dorsal skin following repeatedexposure to UVB irradiation (Furuya et al, 1998; Naganuma et al,2001). The F1 hairless mouse of Hos: HR-1�HR/De is a usefulmodel for studies of UVB-induced pigmentation. This studyprovides further evidence for the hormonal e¡ects of stress onUV-irradiation-induced pigmentation.

MATERIALS AND METHODS

Animals Female F1 hairless mice (6^9 wk old) of HR-1�HR/Dedeveloped by Shiseido Research Center were used throughout the study.This strain was derived by crossing Hos:HR-1 (Hoshino, Japan) withHR/De. Animals were housed in plastic cages (17� 25 cm, ¢ve mice percage) maintained at 22721C with a 12:12 (light:dark) photoperiod andprovided with food and water ad libitum. All experiments were performedin accordance with the National Institutes of Health Guide for the Careand Use of Laboratory Animals and were approved by the AnimalResearch Committee of the Shiseido Research Center.

UV irradiation UVB irradiation was supplied by a bank of 10 ToshibaFL 20 SE sunlamps (Toshiba Electric, Tokyo, Japan). These tubes emitwavelengths between 280 nm and 340 nm, with a peak at 304 nm.Irradiance at the dorsal surface of the mice measured by UV Radiometer(Topcon,Tokyo, Japan) was 0.3 mWper cm2. The animals were exposed torepeated UVB irradiation (72 mJ per cm2, thrice per wk; 2�3 wk; 1minimal erythemal dose¼ 36^72 mJ per cm2). During irradiation, themice were placed in one cage but were partitioned into eightcompartments, at a distance from the £uorescent bulbs of 85 cm.

Experimental design

Experiment 1.The e¡ect of stress on hormone levels Two types of stress, a socialstress, namely high population density, and restraint stress, were imposedon mice. To determine the e¡ect of stress on skin or plasma hormone le-vels, mice were divided into three groups. Five (group 1) or 10 (group 2)mice were housed per cage for 4 d (control and 10 per cage). Mice (group3) were restrained daily for 6 h for the ¢rst 3 d, and for 2 h before bloodand skin samples were collected for ACTH assay on the fourth day. Thesegroups were not exposed to UV irradiation. The serum obtained fromstressed animals was also used for the in vitro tyrosinase assay with humanmelanocytes.

Experiment 2.The e¡ect of stress on UV-induced pigmentation Mice divided intothree groups were housed for 22 d, i.e., ¢ve mice per cage (control); 10 miceper cage; restraint stress (4 h per d, thrice per wk), which consisted of reg-ular housing (¢ve mice per cage) and restraint in a 50 ml ventilated conicaltube for 4 h.The animals were exposed to repeated UVB irradiation (72 mJper cm2 thrice per wk). All mice including the other two groups were de-prived of food and water during this 4 h period. Trunk blood and dorsalskin were collected immediately after decapitation for determination ofACTH levels or DOPA stain on day 22.

Experiment 3.The e¡ect of corticostatin (ACTH inhibitor) on UV-induced pigmen-tation under stressful conditions All groups were irradiated with UVB irradia-tion (72 mJ per cm2 thrice per wk). Prior to the irradiation groups 2 and 3were subjected to restraint stress (4 h per d, thrice per wk). Five hours be-fore irradiation 100 ml of vehicle (saline) alone (groups 1 and 3) and 100 mgof corticostatin (Bachem Feinchemikalien, Switzerland) (group 2) was in-jected intraperitoneally. Corticostatic (anti-ACTH) peptides do not act viathe cAMP cell signaling system but rather via inhibition of the binding ofACTH to its receptor, most probably through direct competition with the14^18 sequence of ACTH for receptor binding (Solomon et al, 1991; Zhuand Solomon, 1992). Five mice were in each group.

Experiment 4.The e¡ect of stress on skin color Mice divided into three groupswere housed for 20 d, i.e., ¢ve mice per cage (control); 10 mice per cage;restraint stress (4 h per d, thrice per wk). These groups were not exposedto UV irradiation. Dorsal skin samples were collected for DOPA stainingon day 20.

Measurement of pigmentary response Before every UV irradiation,the dorsal skin color parameter was measured using a Mexameter MX-16(Courage þ Khazaka Electronic, Germany), based on the absorptionprinciple (Edwards and Heggie, 1992). The melanin index is representedby two wavelengths (568 and 660 nm) chosen to achieve di¡erent rates ofabsorption by the melanin pigments.

Dopa staining DOPA staining was performed according to the originalmethod (Staricco and Pinkus, 1957). At the end of experiments, skinspecimens taken from the back of mice were incubated in 2 N sodiumbromide at 371C for 2 h. Epidermal sheets were separated from thedermis by incubation in 0.1 M phosphate bu¡er (PB; pH 7.4) for 2 h at 371C and then rinsed in 0.2 M sodium cacodylate in PB (pH 7.4). Theepidermal sheets were incubated in a freshly prepared solution of 4 mML-DOPA (Wako, Osaka, Japan) in 0.1 M PB for 3 h, and ¢xed inKarnovsky’s solution. DOPA-positive melanocytes were counted in 30randomly selected microscope ¢elds, using a magni¢cation of 200� .

Extraction of peptides and ACTH assay The blood samples werecollected in ethylenediamine tetraacetic acid coated tubes with proteaseinhibitors, 1 mg per ml of leupeptin (Sigma), 10 mg per ml of soybeantrypsin inhibitor (Sigma), and 1 mg per ml of aprotinin, at the end ofexperiments. The samples were centrifuged at 10,000g for 20 min (41C)and immediately stored at ^801C. The plasma samples were extractedaccording to the manufacturer’s protocol (Peninsula, San Carlos, CA,USA). One milliliter of Extraction Bu¡er A (provided with the kit,Peninsula Cat. No. BUF-A1), containing 0.1% tri£uoroacetic acid, wasadded to 1 ml of plasma sample and centrifuged at 15,000g for 20 min at41C. The supernatants were then passed through a pre-equilibrated C18Sep column (Cat. No. RIK-SEPCOL, Peninsula) by washing with 100%acetonitrile (1 ml, once) followed by Bu¡er A (3 ml, three times). Threemilliliters of Elution Bu¡er B (Cat. No. BUF-B1), containing 60%acetonitrile in 1% tri£uoroacetic acid, was used to elute the peptide. Thecentral dorsal skin samples were placed in 0.5 M acetic acid with proteaseinhibitors and then heated (951C, 10 min). Furkert et al (1997) reported thathot acidic conditions lead to gel formation from skin homogenates. Weheated (951C, 10 min) the extract of skin in 0.5 M acetic acid as soon aspossible, and after the tissues were taken out, but not stored at ^801C.Using the Polytron tissue homogenizer, the heated skin broke very easily.The homogenates were centrifuged at 10,000g for 20 min at 41C. Thesupernatants were washed with petroleum ether (3 ml, ¢ve times).Whenthe homogenates were washed with petroleum ether, excluding lipids, thewater phase gradually became clear. Both plasma and skin eluates wereevaporated to dryness in a centrifugal concentrator and stored at ^801Cuntil assayed. Both plasma and skin samples were assayed for ACTH byenzyme immunoassay (Peninsula). The content of protein in the skin wasdetermined using a protein assay reagent (Bio-Rad).

In vitro experiments

Cell culture Normal human melanocytes and medium 154S were obtainedfrom Sankoh Pure Chemistry (Tokyo, Japan) and Kurabo Industry (Osaka,Japan), respectively. Human melanocytes were maintained in medium 154Ssupplemented with 0.5% fetal bovine serum, 0.2% bovine pituitary ex-tract, 3 ng per ml of recombinant human ¢broblast growth factor, 0.18 mgper ml of hydrocortisone, 5 mg per ml of bovine insulin, 5 mg per ml ofhuman transferrin, 10 ng per ml of phorbol-12-myristate-13-acetate, and 3mg per ml of heparin at 37 1C in a humidi¢ed incubator with 5% CO2 inair. Cells were plated at a density of 8000 per well in 96-well plates. Theywere incubated with ACTH or 10% serum obtained from stressed animals(obtained from experiment 1) and 10 nM corticostatin for 72 h in medium154S without supplements. On day 3 after plating, melanocytes were as-sayed for tyrosinase activity.

Tyrosinase activity Tyrosinase activity was determined using L-DOPA aftertreatment with ACTH or 10% of the serum obtained in experiment 2.Tyr-osinase activity was assayed according to the method described byTomitaet al (1992). Normal human melanocytes were plated at a density of 8000per well in 100 ml of medium in 96-well plates and cultured for 3 d. Thecells were washed with phosphate-bu¡ered saline and lysated with 45 ml of1% Triton X-100, and then incubated with 1mM L-DOPA for 1 h at 371C.The absorbance was measured at 475 nm with a spectrophotometer.

Statistics Data represent the mean7SD. Statistical di¡erences betweentwo groups were determined by a two-tailed Student’s test. In the case ofthree groups, di¡erences were analyzed by analysis of variance (ANOVAtest) and Sche¡e’s test. po0.05 was considered to be statistically signi¢cant.

166 INOUE ETAL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

RESULTS

Changes of plasma and cutaneous ACTH in stress Thechanges in the levels of plasma and cutaneous ACTH causedby stress were examined in experiment 1. We con¢rmed theproduction of ACTH in stressed mice as reported by Hashimotoet al (1988) in rats. As shown in Fig 1(A), both a high populationdensity and restraint stress for 3 d resulted in 2^3-fold (po0.05,po0.001) increases in plasma ACTH levels compared to thecontrol. Interestingly, we found that restraint stress alonesigni¢cantly (po0.05) increased skin ACTH levels (Fig 1B).

In vitro e¡ects of serum obtained from stressed animalsand ACTH on tyrosinase activity in cultured humanmelanocytes To examine the direct e¡ects of the serumfactors on the tyrosinase activity of melanocytes, we obtainedserum from animals undergoing high population density andrestraint stress (experiment 1). The serum obtained from stressedanimals signi¢cantly increased tyrosinase activity in humanmelanocytes compared to the serum from nonstressed animals(po0.01, Fig 2A). Increased tyrosinase activity by the serumobtained from stressed animals was inhibited in the presence ofcorticostatin in cultured human melanocytes (Fig 2A). ACTHsigni¢cantly increased tyrosinase activity at physiologic plasmaconcentrations (0.01^0.1 nM) under stressful conditions (Fig 2B).

E¡ects of stress on UVB-induced hyperpigmentation Thee¡ects of stress on UVB-induced hyperpigmentation wereexamined in experiment 2. The pigmentary response started onday 8 of repeated exposure to suberythemal UVB irradiation.The visible pigmentary response to UVB in dorsal skin wasenhanced in the stressed mice (Fig 3). The melanin indexgradually increased in proportion to the pigmentary response torepeated exposure (Fig 4). Delayed tanning was marked for 10mice per cage and after restraint stress at days 19 and 22 (Figs 3, 4).The induction of pigmentation by UVB was greater in stressedmice (Fig 4). The pigmentation persisted during repeated UVBexposure and then disappeared completely within 5 wk after thelast exposure. Exposure to repeated UVB irradiation caused anincrease in the number of DOPA-positive melanocytes. Thenumber of DOPA-positive melanocytes in irradiated dorsal skinalso signi¢cantly increased in stressed animals. Numerous DOPA-positive melanocytes with enlarged dendrites appeared in dorsalskin, which had a pigmentary response under stressful conditions(Fig 5A). Quantitative measurements of these melanocytes areshown in Fig 5(B). The number of DOPA-positive melanocytesper area of 10,000 mm2 signi¢cantly (po0.001) increased in thegroups of 10 mice per cage (14.075.4) and restraint stress(12.275.6) compared with the control (5.373.8). The cutaneousACTH level also tended to increase in stressed mice afterrepeated UV irradiation (10 mice per cage (0.75670.275) andrestraint stress (1.13470.691) versus the control (0.58870.169 ngper mg protein)).

Figure1. E¡ects of stress on ACTH levels. Plasma (A) and cutaneous(B) ACTH levels under control, high population density stress (10 percage), and restraint stress (restraint) conditions. Values represent the meanof 10 animals (7 SD). npo0.001.

Figure 2. E¡ects of serum obtained from stressed animals andACTH on tyrosinase activity in cultured human melanocytes. (A)E¡ects of serum obtained from stressed animals in the presence or absenceof corticostatin on tyrosinase activity. (B) Dose-dependent activation oftyrosinase byACTH.Values represent the mean7SD (n¼ 8). npo0.001.

STRESS AND PIGMENTATION 167VOL. 121, NO. 1 JULY 2003

In vivo e¡ects of corticostatin on the augmentation ofpigmentary response under stressful conditions afterrepeated UVB irradiation E¡ects of the inhibition of ACTHafter pretreatment with corticostatin on pigmentary responsewere examined in experiment 3. Pretreatment with corticostatinwas e¡ective in inhibiting the augmentation of delayed tanningunder stress (Fig 6). DOPA-positive melanocytes with highdendricity and a large cell body were observed under theseconditions. The dendricity and number of melanocytes werereduced in mice pretreated with corticostatin (Fig 7A).Quantitative measurements of DOPA-positive melanocytes areshown in Fig 7(B). The number of positive melanocytessigni¢cantly decreased on pretreatment with corticostatin(8.273.9 per 10,000 mm2) compared with restraint stress(19.976.0 per 10,000 mm2).

E¡ects of stress on skin color The e¡ects of stress on skincolor were examined without UV irradiation in experiment 4.In contrast to the UV-induced pigmentation, the melanin indexdecreased in stressed mice (Fig 8).The number of DOPA-positivemelanocytes was little a¡ected by stress alone (data not shown).

DISCUSSION

This study demonstrated that two types of stress augmented theUV-induced pigmentary response. There is a close relationshipbetween hormone levels and pigmentation in endocrine disorders(Espinoza et al, 1975; Strakosch and Gordon, 1978; Pears et al, 1992;

Ketterer and Frenk, 1995; Pereira et al, 1998). There has been noreport, however, on the relation of circulating ACTH to pigmen-tation under stressful conditions. ACTH is one of the factors thatare easily changed by stress (Hashimoto et al, 1988) and yet itfurther stimulates melanogenesis and tyrosinase activity in cul-tured human melanocytes (Hunt et al, 1994a; 1994b; Abdel-Maleket al, 1995;Wakamatsu et al, 1997). The possibility that one of thefactors that activated the function of melanocytes might be circu-lating with ACTH was investigated.Corticostatins are a family of arginine-rich, cysteine-rich pep-

tides that inhibit ACTH-stimulated corticosterone production inrat adrenal cell suspensions (Zhu and Solomon, 1992). Corticosta-tin had no e¡ect on the production of corticosterone stimulatedbyACTH (1^13) acetyl amide (a-MSH), but it did lower the pro-duction of corticosterone stimulated byACTH (1^18) amide (Zhuand Solomon, 1992).Therefore, corticostatin speci¢cally competeswith the ACTH (14^18) sequence to bind to the melanocortin re-ceptor. Pretreatment of mice with corticostatin was e¡ective atinhibiting the augmentation of the pigmentary response and thenumber of DOPA-positive melanocytes under stressful condi-tions (Figs 6, 7). These results may indicate that corticostatinblocks ACTH-stimulated corticosterone production by inhibit-ing the melanocortin-2 receptor in the adrenal gland. Further-more, in vitro, the serum obtained from stressed animalssigni¢cantly increased tyrosinase activity in human melanocytescompared to that of nonstressed animals. This increase was signif-icantly inhibited in the presence of corticostatin. Our datashowed that corticostatin was also e¡ective against melanocytesin culture as well as the adrenal gland and therefore the serumobtained from stressed animals was rich in ACTH as a factor in-creasing tyrosinase activity. This action of corticostatin in themelanocytes con¢rmed the expression of melanocortin 2 receptorexpressed in mouse skin (Ermak and Slominski, 1997) and sug-gested by Slominski et al, 1996b. These results suggest thatACTHas well as a-MSH released from the pituitary gland into circula-tion could provide important control mechanisms in the regula-tion of melanocyte function.The parameter of skin color changed and the number of

DOPA-positive melanocytes increased remarkably under stressfulconditions in guinea pigs (Kaminaga and Shinomiya, 1997). Incontrast to former studies, however, stress did not a¡ect the de-velopment of pigmentation (Fig 8) nor change the number ofDOPA-positive melanocytes in the absence of UV irradiation(data not shown). The discrepancy may be due to the conditionsof stress or the species of animal. Repeated stress alone increasednot only the plasma ACTH level but also the skin ACTH levelcompared to the unstressed control (Fig 1), but did not stimulatelocal pigmentation (Fig 8).In rodent skin, UVB and the topical application of b-MSH act

synergistically to promote melanogenesis (Bolognia et al, 1989;Slominski and Pawelek, 1998; Chakraborty et al, 1999). UV irra-diation directly activates the cutaneous synthesis of CRH, andPOMC-derived peptides, ACTH and a-MSH, are synthesized inthe skin (Slominski et al, 1993; 1996a; 2000; Schauer et al, 1994;Chakraborty et al, 1996; Wintzen and Gilchrest, 1996; SlominskiandWortsman, 2000). In accordance with the results of these re-ports, the activation of a localized HPA axis by UV irradiation

Figure 3. Photographs of mouse dorsal skin showing e¡ects of stress on pigmentary response after UV irradiation.

Figure 4. E¡ects of stress on pigmentary response after repeatedUVB irradiation.Values represent the mean of 10 animals (7SD).


also seems to result in melanogenesis and to increase the numberof DOPA-positive melanocytes in each group in this report. Thedi¡erence in cutaneous ACTH concentration between the pre-sence and absence of irradiation, under nonstressful conditions,probably results from the involvement of the HPA axis, which isonly derived from the skin. There is no report that shows that theACTH level is increased by psychologic stress in nonpituitary tis-sues such as liver, kidney, and stomach. However, Slominski et al(1998) demonstrated that £uctuations of skin and serum concen-trations of ACTH and b-endorphin could be regulated by thephase of the hair cycle under physiological condition. The peakvalue of cutaneous ACTH was 107 pg per mg protein at anagenVI (Slominski et al, 1998). Although we modi¢ed the extractionmethod, the ACTH level in the skin may possibly be higher yet,as Furkert et al (1997) reported that the skin is a tough tissue forextraction. Furkert et al (1997) described how the hot acidic con-ditions lead to gel formation from skin homogenates. Gel forma-tion was not observed under our modi¢ed method. Our dataindicated 305 pg per mg protein in normal conditions, 750 pgper mg protein in stressful conditions. The £uctuations of the cu-taneous ACTH level during the hair cycle in the hairy skin ofC57BL6 mice was lower than the cutaneous ACTH level by stressin the pigmented F1 hairless mice, and hence the e¡ect of the haircycle had a negligible e¡ect in this report. The increase in cuta-neous ACTH level not only due to environmental stimuli butalso physiologic function is very interesting. The concentrationof cutaneous a-MSH is una¡ected by hypophysectomy and bydaily injection of a-MSH in rat (Thody et al, 1983).Therefore, thisincrease of cutaneous a-MSH level by stress may not be due toaugmentation of the tanning response by stress. At this point,however, we cannot explain the origin of the increasing ACTHin skin under stressful conditions. Gilchrest et al (1996) speculatedthat MSH and ACTH peptides produced in the epidermis contri-

bute to pigmentation these peptides more than released from thepituitary gland.We cannot explain how the pigmentary responseis augmented under conditions of stress, however. Why wouldUV irradiation appear to augment the pigmentary response un-

Figure 5. E¡ect of stress on DOPA-stain-positive melanocytes after repeatedUV irradiation. (A) Microscopic magni¢-cation of DOPA-stain-positive melanocytesin mouse dorsal epidermal sheets showingtanning response after repeated UVB irra-diation for 22 d. Scale bars: 100 mm. (B) Thenumber of DOPA-positive melanocytesafter repeated UV irradiation. The columnsrepresent numbers of DOPA-positive mela-nocytes. The average cell number in 30 areasled to the number per 10,000 mm2.

Figure 6. E¡ects of corticostatin on the augmentation of pigmen-tary response under stressful conditions after repeated UVB irradia-tion.Values represent the mean of ¢ve animals (7SD).


der stressful conditions? In this study, even though all the mice ineach group were repeatedly irradiated equally, the level of tan-ning response di¡ered.

There are a number of reports concerning the participation ofcirculatory POMC-derived peptides in the pigmentary response(Holtzmann et al, 1982; 1983; Altmeyer et al, 1986; Ghanem et al,1989; Pears et al, 1992). In clinical conditions, hyperpigmentationhas been related to endocrine dysfunction (Ketterer and Frenk,1995). Patients with Addison’s disease or Nelson’s syndrome devel-oped hyperpigmentation and elevated plasma ACTH levels (Espi-noza et al, 1975; Strakosch and Gordon, 1978; Pereira et al, 1998).Elevations of circulating a-MSH and ACTH levels are assumed tobe responsible for the hyperpigmentation. Though an increase inplasma and cutaneous ACTH levels under stressful conditions didnot lead directly to pigmentation except in the presence of theseendocrine disorders, it may be one of the factors in the augmenta-tion of pigmentary response. UV irradiation acts to increase mela-nogenesis in melanocytes and causes skin pigmentation. It increasesthe melanocortin-1 receptor mRNA levels in Cloudman melano-ma cells (Bolognia et al, 1989; Chakraborty et al, 1991) and normalhuman melanocytes (Funasaka et al, 1998). ACTH as well as a-MSHhas an important role in the regulation of melanocyte function, ac-tivating the melanocortin-1 receptor (Wakamatsu et al, 1997; Thodyand Graham, 1998). The excess circulating ACTH due to stress mayserve as natural ligands at the melanocortin-1 receptor upregulatedby UV irradiation. The increase in plasma and cutaneous ACTHlevels may activate tyrosinase during UV irradiation and hencemay cause augmentation of the tanning response under stressfulconditions. Stress could have an e¡ect on the pigmentary responseby changing a number of physiologic and hormonal parameters.

Figure 7. E¡ect of corticostatin onDOPA-stain-positive melanocytes afterrepeated UV irradiation. (A) Microscopemagni¢cation of DOPA-stain-positive mela-nocytes in mouse dorsal epidermal sheetsshowing the inhibition by corticostatin ofpigmentary response after UV irradiation.Scale bars: 100 mm. (B) E¡ect of corticostatinon the number of DOPA-positive melano-cytes after repeated UVB irradiation. Thecolumns represent numbers of DOPA-posi-tive melanocytes.The average cell number in30 areas led to the number per 10,000 mm2.

Figure 8. E¡ects of stress on skin color. The data are representive ofthree experiments.Values represent the mean of 10 animals (7SD).


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