Background Long-term exposure to ultraviolet irradiation fromsunlight causes premature skin aging (photoaging), characterizedin part by wrinkles, altered pigmentation, and loss of skintone. Photoaged skin displays prominent alterations in the collagenousextracellular matrix of connective tissue. We investigated therole of matrix-degrading metalloproteinases, a family of proteolyticenzymes, as mediators of collagen damage in photoaging.
Methods We studied 59 whites (33 men and 26 women, ranging inage from 21 to 58 years) with light-to-moderate skin pigmentation,none of whom had current or prior skin disease. Only some ofthe participants were included in each of the studies. We irradiatedtheir buttock skin with fluorescent ultraviolet lights understandard conditions and obtained skin samples from irradiatedand nonirradiated areas by keratome or punch biopsy. In somestudies, tretinoin and its vehicle were applied to skin underocclusion 48 hours before ultraviolet irradiation. The expressionof matrix metalloproteinases was determined by in situ hybridization,immunohistology, and in situ zymography. Irradiation-induceddegradation of skin collagen was measured by radioimmunoassayof soluble cross-linked telopeptides. The protein level of tissueinhibitor of matrix metalloproteinases type 1 was determinedby Western blot analysis.
Results A single exposure to ultraviolet irradiation increasedthe expression of three matrix metalloproteinases — collagenase,a 92-kd gelatinase, and stromelysin — in skin connectivetissue and outer skin layers, as compared with nonirradiatedskin. The degradation of endogenous type I collagen fibrilswas increased by 58 percent in irradiated skin, as comparedwith nonirradiated skin. Collagenase and gelatinase activityremained maximally elevated (4.4 and 2.3 times, respectively)for seven days with four exposures to ultraviolet irradiation,delivered at two-day intervals, as compared with base-line levels.Pretreatment of skin with tretinoin (all-trans-retinoic acid)inhibited the induction of matrix metalloproteinase proteinsand activity (by 70 to 80 percent) in both connective tissueand outer layers of irradiated skin. Ultraviolet irradiationalso induced tissue inhibitor of matrix metalloproteinases-1,which regulates the enzyme. Induction of the inhibitor was notaffected by tretinoin.
Conclusions Multiple exposures to ultraviolet irradiation leadto sustained elevations of matrix metalloproteinases that degradeskin collagen and may contribute to photoaging. Treatment withtopical tretinoin inhibits irradiation-induced matrix metalloproteinasesbut not their endogenous inhibitor.
Ultraviolet irradiation from the sun has deleterious effectsin human skin, including sunburn, immune suppression,1 cancer,and premature aging (photoaging). Sunburn and immune suppressionoccur acutely in response to excessive exposure to the sun,whereas skin cancer and photoaging result from accumulated damagecaused by repeated exposures. Skin cancer, the most prevalentform of cancer in humans,2 typically occurs in skin that isphotoaged.3 Photoaged skin is characterized by wrinkles, laxity,uneven pigmentation, brown spots, and a leathery appearance.4In contrast, chronologically aged skin that has been protectedfrom the sun is thin and has reduced elasticity but is otherwisesmooth and unblemished.5
Histologic and ultrastructural studies have shown that alterationsin photoaged skin are found in dermal connective tissue.6,7The dermis lies below and provides mechanical support for theouter, protective layer of skin, the epidermis. The extracellularmatrix in the dermis is composed primarily of type I collagen,with lesser amounts of type III collagen, elastin, proteoglycans,and fibronectin. Collagen fibrils are responsible for the strengthand resiliency of skin.8
Dermal damage induced by ultraviolet irradiation is principallymanifested histologically as the disorganization of collagenfibrils9 and the accumulation of abnormal elastin-containingmaterial.5 Biochemical evidence of connective-tissue alterationsin photoaged skin includes reduced levels of types I and IIIcollagen precursors10 and cross-links,11 an increased ratioof type III to type I collagen,12 and an increased level ofelastin.13 Improvement in the appearance of photoaged skin aftertreatment with tretinoin (all-trans-retinoic acid) is associatedwith the restoration of type I procollagen to levels approachingthose in skin that has not been exposed to sun.14 These observationshave led to the belief that the aged appearance of sun-exposedskin results from alterations in the structure and compositionof collagen and elastin in the dermal extracellular matrix.Mechanisms responsible for skin connective-tissue damage causedby ultraviolet irradiation are not known.
Matrix-degrading metalloproteinases are a family of proteolyticenzymes that specifically degrade collagens, elastin, and otherproteins in connective tissue and bone.15,16 Matrix metalloproteinasesare critical for matrix remodeling during development and woundhealing.17 Their activities are regulated by tissue inhibitorsof matrix metalloproteinases.18 The expression of tissue inhibitorof matrix metalloproteinases type 1 often parallels that ofmatrix metalloproteinases, to prevent excessive matrix degradation.19,20
In a previous study, we demonstrated by biochemical methodsthat a single exposure to ultraviolet irradiation induces threematrix metalloproteinases in human skin in vivo — collagenase,92-kd gelatinase, and stromelysin-1 — and that pretreatmentof skin with tretinoin inhibits the induction of all three.21The combined actions of collagenase, 92-kd gelatinase, and stromelysincan fully degrade skin collagen.15,16,17 This raises the possibilitythat matrix metalloproteinases induced by ultraviolet irradiationare largely responsible for dermal damage in photoaging andthat tretinoin, by inhibiting the induction of matrix metalloproteinases,may prevent this damage. We performed the current study to demonstratethat matrix metalloproteinases induced by ultraviolet irradiationare expressed in the dermis (the site of damage in photoaging),where they degrade skin collagen; that the expression of matrixmetalloproteinases remains maximally elevated after multipleexposures to ultraviolet irradiation; and that pretreatmentof skin with tretinoin inhibits the expression of matrix metalloproteinasesdue to ultraviolet irradiation.
Methods
Ultraviolet Irradiation and Tissue Samples
We studied 59 white adults (33 men and 26 women; mean age, 35.3years; range, 21 to 58), without current or prior skin diseaseand with light-to-moderate pigmentation. We irradiated buttockskin with four fluorescent ultraviolet lights (F36T12 ERE-VHO).A Kodacel filter (TA401/407; Kodak, Rochester, N.Y.) was mounted4 cm in front of the tubes to remove wavelengths of less than290 nm (ultraviolet C). The intensity of irradiation was monitoredwith the use of a phototherapy radiometer (model 443, InternationalLight, Newburyport, Mass.) and a photodetector (SED240/UVB/W,International Light). Spectroradiometry was performed with theOL 754 system (Optronic Laboratories, Orlando, Fla.). The totalirradiation (290 to 800 nm) 17 inches from the light sourcewas 1.49x10-3 W per square centimeter. The distribution of poweroutput was 47 percent ultraviolet B (290 to 320 nm), 18 percentultraviolet A2 (320 to 340 nm), 9 percent ultraviolet A1 (340to 400 nm), and 26 percent visible and near-infrared (400 to800 nm). The dose causing minimal erythema was determined 24hours after irradiation. Irradiated and nonirradiated skin sampleswere obtained from each subject by keratome or punch biopsy,as previously described.22 Tretinoin and its vehicle (70 percentethanol, 30 percent polyethylene glycol, and 0.05 percent butylatedhydroxytoluene) were applied to skin, to which a protectivecovering was then applied, 48 hours before exposure to ultravioletirradiation. For studies with multiple time points or treatments,tissue was obtained from each subject for each time point ortreatment. Twenty-five of the 105 subjects who donated skinspecimens for studies described in a prior report21 also donatedskin specimens for the studies reported here. The study wasapproved by the institutional review board at the Universityof Michigan, and all subjects provided written informed consent.
Measurements of Matrix Metalloproteinases
Skin specimens from irradiated and nonirradiated sites werehomogenized in 20 mM TRIS–hydrochloric acid (pH 7.6) and5 mM calcium chloride and centrifuged at 3000 x g for 10 minutes.Supernatants were used to measure collagenase and gelatinaseactivity by hydrolysis of tritium-labeled fibrillar collagen17(100 µg per assay) and gelatin zymography23 (20 µgper assay), respectively.
In Situ Hybridization
Digoxigenin-containing sense and antisense riboprobes to detecthuman collagenase, 92-kd gelatinase, and stromelysin-1 messengerRNA (mRNA) were synthesized with the use of T3 and T7 ribonucleicacid polymerases. Frozen skin sections (5 µm) were mounted,fixed, treated, and hybridized as described elsewhere.24 Hybridizationsignals were detected immunohistochemically with the use ofalkaline phosphatase–conjugated antidigoxigenin antibody.
Immunohistologic Analysis
Immunohistologic analysis of collagenase, gelatinase, and stromelysinwas performed as described elsewhere.14 Collagenase and gelatinasewere detected with affinity-purified polyclonal IgG antibody(Cambio, Cambridge, United Kingdom). Stromelysin was detectedwith mouse monoclonal antibody (a gift from Dr. Constance E.Brinckerhoff, Dartmouth Medical School, Hanover, N.H.). SheepIgG and mouse IgG (Sigma Chemical, St. Louis) were used as controls.
In Situ Zymography
In situ zymography with the use of fluorescein isothiocyanate–gelatinand resorufin–casein substrates has been described elsewhere.25The same procedures were followed with the use of fluorescein–collagen(Elastin Products, Owensville, Mo.) as substrate, except thatfluorescein–collagen (4 mg per millimeter) was coateddirectly onto slides, without the addition of agarose.
Radioimmunoassay for Type I Collagen Telopeptide
Punch-biopsy specimens were homogenized in 150 mM sodium chloride,50 mM TRIS (pH 7.5), 0.02 percent sodium azide, 2 mM phenylmethylsulfonylfluoride, and 10 µg of aprotinin per milliliter and centrifugedat 25,000 x g for 30 minutes. Supernatants were used to measuresoluble type I collagen cross-linked telopeptides with the useof a commercial radioimmunoassay kit26 (Incstar, Stillwater,Minn.).
Western Blot Analysis
Levels of tissue inhibitor of matrix metalloproteinases-1 andstromelysin were determined in supernatants from skin homogenates,as described elsewhere.21 Samples (100 µg) were incubatedfor 16 hours with antibodies (Cambio). Immunoreactive stromelysinand tissue inhibitor of matrix metalloproteinases-1 were visualizedwith enhanced chemiluminescence.
Statistical Analysis
Data were analyzed with paired t-tests (for the telopeptidedata and the comparison of tretinoin-treated skin and vehicle-treatedskin) or repeated-measures analysis of variance and with Tukey'sstudentized range test27 (for the data on multiple exposuresand different doses of radiation). All P values are two-tailed,and differences were considered significant when P values wereless than or equal to 0.05.
Results
Ultraviolet Irradiation and Induction of Matrix Metalloproteinases
Collagenase mRNA was minimally detectable in nonirradiated skin(Figure 1A). Twenty-four hours after exposure to ultravioletirradiation, collagenase mRNA was induced in all keratinocytesthroughout the epidermis (Figure 1B). Ultraviolet irradiationalso induced collagenase mRNA in cells in the connective tissue,although to a lesser extent than in keratinocytes (Figure 1B,inset). Hybridization of irradiation-exposed skin with a senseprobe, a control for nonspecific hybridization, yielded no detectablesignal (data not shown). Collagenase protein was minimally detectablein nonirradiated skin (Figure 1C). Exposure to ultraviolet irradiationinduced the expression of collagenase protein throughout theepidermis and in connective tissue (Figure 1D). Immunoreactivityin the epidermis and dermis was detected intracellularly inkeratinocytes and fibroblasts, respectively, and extracellularly.Extracellular staining was especially noticeable in the dermis,where immunoreactivity was seen throughout the collagenous matrix(reddish-brown staining in Figure 1D). Staining of irradiation-exposedskin specimens with control IgG revealed minimal backgroundstaining (data not shown). The level of in situ collagenaseactivity was very low in nonirradiated skin (Figure 1E). Inirradiated skin, collagenase activity was detectable throughoutthe epidermis and dermis (Figure 1F). The addition of tissueinhibitor of matrix metalloproteinases-1 or a cation chelator(EDTA) to irradiated skin sections reduced the level of collagenbreakdown to control values (Figure 1E), as expected for collagendegradation by collagenase (data not shown). In sections ofirradiated skin incubated at 4°C as a control, collagenaseactivity was not detectable (data not shown).
Figure 1. Induction of Collagenase Messenger RNA, Protein, and Activity in Skin Specimens from Three Subjects Exposed to Ultraviolet Irradiation.
Buttock skin was irradiated with twice the dose of ultraviolet irradiation that caused minimal erythema. Specimens of irradiated and nonirradiated buttock skin were obtained from each subject 24 hours after irradiation. Collagenase messenger RNA (mRNA) was detected with the use of digoxigenin-riboprobe in situ hybridization in nonirradiated skin (Panel A) and irradiated skin (Panel B). Collagenase mRNA is stained purple by this technique. The inset in Panel A shows the dermis at greater magnification. The inset in Panel B shows collagenase mRNA (arrows) in cells in the dermis. The specimens are from one subject and are representative of the findings in nonirradiated and irradiated tissue from six subjects.
Collagenase protein was detected by peroxidase immunohistologic techniques in nonirradiated skin (Panel C) and irradiated skin (Panel D). Collagenase protein is stained reddish brown by this technique. The specimens are from one subject and are representative of the findings in nonirradiated and irradiated tissue from six subjects.
Collagenase activity was detected by in situ zymography in nonirradiated skin (Panel E) and irradiated skin (Panel F). The green color is fluorescein-labeled collagen, which was coated onto a glass slide. The skin section was laid on top of the slide and incubated for 24 hours to allow collagenase in the tissue to degrade the fluorescein-labeled collagen on the slide. Darkened areas, especially noticeable in the specimen of irradiated skin (Panel F), are due to the degradation of fluorescein–collagen substrate. The white lines demarcate the boundary between the epidermis (top) and the dermis (bottom). The specimens are from one subject and are representative of the findings in nonirradiated and irradiated tissue from five subjects.
The findings for 92-kd gelatinase and stromelysin in nonirradiatedand irradiated skin were very similar to those for collagenase.Ultraviolet irradiation induced the expression of mRNA for 92-kdgelatinase and stromelysin predominantly in the epidermis, whereasgelatinase and stromelysin proteins were induced in the connectivetissue and the epidermis (data not shown). Gelatinase activitywas induced throughout the dermis and epidermis, whereas stromelysinactivity was predominantly located in the dermis (data not shown).
Ultraviolet Irradiation and Degradation of Endogenous Collagen
The induction of dermal matrix metalloproteinase activity inskin exposed to ultraviolet irradiation would be expected tocause increased degradation of endogenous collagen. To examinethis hypothesis, we quantified soluble type I collagen C-terminalcross-linked telopeptides, a reliable measure of endogenousdestruction of type I collagen,26,28 in nonirradiated and irradiatedskin. The level of soluble cross-linked telopeptides was elevatedby 58 percent 24 hours after ultraviolet irradiation (mean [±SE]level, 30.2 ± 4.6 ng per 100 µg of protein in fivespecimens, vs. 19.0 ± 2.2 ng per 100 µg of proteinin five specimens of nonirradiated skin; P = 0.02) and remainedelevated for at least 72 hours (mean level, 27.0 ± 3.4ng per 100 µg of protein; P = 0.02).
Multiple Exposures to Ultraviolet Irradiation
Since photoaging results from the accumulation of damage causedby repeated exposures to ultraviolet light, we investigatedthe effect of single and multiple exposures on collagenase and92-kd gelatinase activity in vivo. After a single exposure,collagenase and gelatinase activity increased within 8 hours,peaked within 24 hours, and returned to nearly basal (nonirradiated)levels within 72 hours (data not shown). For studies of multipleexposures, each subject was exposed to ultraviolet irradiationat four separate sites, with each site exposed one, two, three,or four times to irradiation delivered at 48-hour intervals.Skin specimens were obtained from each irradiated site 24 hoursafter the last exposure at that site and were also obtainedfrom nonirradiated (control) sites for analyses. After a singleexposure to ultraviolet irradiation, collagenase and 92-kd gelatinaseactivity was elevated 4.4±0.2 times the values in nonirradiatedskin (in eight specimens) and 2.3±0.4 times (in fivespecimens), respectively (Figure 2). Collagenase and gelatinaseactivity remained maximally elevated after the second, third,and fourth exposures on days 3, 5, and 7, respectively (Figure 2).Thus, multiple exposures led to the sustained inductionof matrix metalloproteinases.
Figure 2. Sustained Elevations in Collagenase and 92-kd Gelatinase Activity after Multiple Exposures to Ultraviolet Irradiation.
Skin was obtained from five separate sites on the buttock. The first site was not irradiated, the second site was irradiated one time (on day 0), the third site was irradiated a second time (on day 2), the fourth site was irradiated a third time (on day 4), and the fifth site was irradiated a fourth time (on day 6). Skin was irradiated with half the dose that caused minimal erythema; doses were given at 48-hour intervals. Tissue specimens were obtained 24 hours after the last exposure and assayed for collagenase activity (in eight specimens) and 92-kd gelatinase activity (in five specimens). The values shown are means, with standard errors indicated by the bars. For each set of exposures, metalloproteinase activity was significantly higher in irradiated tissue than in nonirradiated tissue (P<0.05).
Pretreatment with Tretinoin
Topical pretreatment with tretinoin inhibits the induction ofmatrix metalloproteinases, detected biochemically, in skin exposedto ultraviolet irradiation in vivo.21 This finding suggeststhat tretinoin may prevent dermal damage. To do so, tretinoinmust block the induction of matrix metalloproteinases in thedermis. We used immunohistologic methods and in situ zymographyto localize the effects of tretinoin on the induced expressionof collagenase, 92-kd gelatinase, and stromelysin in skin exposedto ultraviolet irradiation.
The treatment of skin with tretinoin, or its vehicle alone,did not alter the low basal levels of collagenase, 92-kd gelatinase,or stromelysin proteins or activity (data not shown). However,treatment with tretinoin before exposure to ultraviolet irradiationsubstantially reduced the subsequent induction of all threematrix metalloproteinases. Tretinoin blocked the epidermal anddermal expression of collagenase, 92-kd gelatinase, and stromelysinproteins (data not shown) and activity (Figure 3A, Figure 3B,Figure 3C, Figure 3D, Figure 3E, and Figure 3F).
Figure 3. Induction of Collagenase, 92-kd Gelatinase, and Stromelysin Activity in Skin Specimens from Three Subjects Pretreated with Tretinoin and Exposed to Ultraviolet Irradiation.
In each subject, one site was treated with the vehicle and one site with 0.1 percent tretinoin 48 hours before ultraviolet irradiation with two times the dose that caused minimal erythema. Specimens of treated skin were obtained 24 hours after irradiation, and collagenase, 92-kd gelatinase, and stromelysin activity was assessed by in situ zymography.
Collagenase activity in irradiated skin pretreated with the vehicle (Panel A) and in irradiated skin pretreated with tretinoin (Panel B) was measured with the use of fluorescein-labeled collagen as substrate. The specimens are from one subject and are representative of the findings in tissue from five subjects. Gelatinase activity in irradiated skin pretreated with the vehicle (Panel C) and in irradiated skin pretreated with tretinoin (Panel D) was measured with the use of fluorescein-labeled gelatin as substrate. The specimens are from one subject and are representative of the findings in tissue from five subjects. Stromelysin activity in irradiated skin pretreated with the vehicle (Panel E) and in irradiated skin pretreated with tretinoin (Panel F) was measured with the use of resorufin-labeled casein as substrate. The specimens are from one subject and are representative of the findings in tissue from five subjects. Darkened areas, especially noticeable in Panels A, C, and E, are due to the degradation of colored substrates. The white lines demarcate the boundary between the epidermis (top) and the dermis (bottom).
Induction of Tissue Inhibitor of Matrix Metalloproteinases-1
Since the regulation of matrix metalloproteinases and the regulationof their tissue inhibitors is coordinated, we investigated whethertissue inhibitors of matrix metalloproteinases are induced inskin exposed to ultraviolet irradiation. Nonirradiated skinexpressed mRNA for tissue inhibitor of matrix metalloproteinasestypes 1 and 2. In irradiated skin, mRNA for tissue inhibitorof matrix metalloproteinases-1 was induced in a time- and dose-dependentmanner. Induction occurred within 8 hours after exposure, peakedwithin 16 to 24 hours, and remained elevated for at least 72hours (data not shown). The time course for induction of tissueinhibitor of matrix metalloproteinases-1 protein mirrored thatof its mRNA (data not shown). The expression of mRNA for tissueinhibitor of matrix metalloproteinases-2 was not induced byultraviolet irradiation (data not shown).
Tissue inhibitor of matrix metalloproteinases-1 was significantlyinduced by 1/10 the amount of ultraviolet irradiation thatcaused minimal erythema (0.1 minimal-erythema dose) and maximallyinduced by one minimal-erythema dose (Figure 4A). The inductionof stromelysin protein showed a similar dose dependence (Figure 4A).Pretreatment of skin for 48 hours with tretinoin did notalter either basal or irradiation-induced values for tissueinhibitor of matrix metalloproteinases-1 mRNA (data not shown)or protein levels (Figure 4B). In contrast, tretinoin significantlyinhibited the induction of stromelysin mRNA (data not shown)and protein in irradiated skin (Figure 4B).
Figure 4. Effect of Ultraviolet Irradiation (UV) and Treatment with Tretinoin on the Induction of Tissue Inhibitor of Matrix Metalloproteinases-1 (TIMP-1) and Stromelysin-1.
Panel A shows the relation between the dose of ultraviolet irradiation and induction of TIMP-1 and stromelysin-1. The level of protein in untreated skin was assigned a value of 1. Levels measured after treatment are expressed in relation to that level. Each subject was irradiated on seven separate sites of the buttock with one of the indicated dose levels. One dose was defined as the amount of irradiation causing minimal erythema. Specimens of treated skin were obtained 24 hours after irradiation, and the levels of TIMP-1 (in specimens from three subjects) and stromelysin-1 (in specimens from nine subjects) were determined by Western blot analyses. Asterisks denote values that differ significantly from those in nonirradiated specimens (P<0.05). Three of the subjects were the same for the two analyses.
Panel B shows the effect of tretinoin on the induction of TIMP-1 and stromelysin-1 in irradiated skin. In each subject, separate sites were treated with the vehicle or 0.1 percent tretinoin 48 hours before irradiation at two times the dose causing minimal erythema. Specimens of treated skin were obtained 24 hours after irradiation and analyzed for TIMP-1 (in specimens from seven subjects) and stromelysin-1 (in specimens from nine subjects) by Western blot analyses. Three of the subjects were the same for the two analyses. The induction of stromelysin-1 was significantly decreased in specimens treated with tretinoin, as compared with those treated with the vehicle alone (P<0.001).
Bands from representative Western blot analyses are shown at the top of each panel. The values shown are means, with standard errors indicated by the bars.
Discussion
We found that ultraviolet irradiation induced matrix metalloproteinasesin the epidermis and dermis (these substances degrade collagenin the dermis) and that their induction was sustained with multipleexposures. These results support the concept that matrix metalloproteinasesare primary mediators of connective-tissue damage in skin exposedto ultraviolet irradiation and of the premature aging of skinthat results.
Figure 5 shows a model of the pathophysiology of dermal damagecaused by ultraviolet irradiation and leading to skin wrinklingin photoaging. The model does not address aspects of photoagingthat occur in the epidermis and lead to alterations in skinpigmentation and surface texture. We offer the hypothesis thatagents that block the induction of matrix metalloproteinasesby ultraviolet irradiation, such as tretinoin and its metabolicprecursor, retinol,29,30 or direct inhibitors of matrix metalloproteinasesmay prevent the dermal damage that leads to photoaging. An evaluationof this hypothesis will require long-term clinical trials.
Figure 5. Hypothetical Model of the Pathophysiology of Dermal Damage and Photoaging Induced by Ultraviolet Irradiation.
Exposure to levels of ultraviolet (UV) light that cause no detectable sunburn induces the expression of matrix metalloproteinases (MMPs) in keratinocytes (KC) in the outer layers of skin, as well as fibroblasts (FB) in connective tissue; these metalloproteinases degrade collagen in the extracellular matrix of the dermis. The extent of matrix destruction is limited by the simultaneous induction of tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), which partially inhibits the activity of matrix metalloproteinases. The breakdown of collagen is followed by synthesis and repair, which, as with all types of wound healing, is imperfect and leaves subtle, clinically undetectable deficits in the organization or composition of the extracellular matrix, or both. Matrix damage, followed by imperfect repair, occurs with each ensuing exposure to the sun, leading to the accumulation of altered matrix (solar scar) and, eventually, observable photoaging (wrinkles). The upper panels with the blue background depict the processes examined in our study, and the upper and lower panels with the yellow background depict hypothetical processes that are consistent with our results.
Although irradiation-induced expression of the genes for collagenase,92-kd gelatinase, and stromelysin-1 occurred predominantly inthe epidermis, matrix metalloproteinase proteins and their enzymaticactivity were abundant in both the dermis and the epidermis.Therefore, a substantial fraction of the matrix metalloproteinasessynthesized in the epidermis was transported to the dermis.This was not an unexpected finding, since collagenase, 92-kdgelatinase, and stromelysin-1 are secreted proteins, and thebasement-membrane zone, which separates the epidermis from thedermis, readily allows the passage of proteins between the twocompartments.31,32
Collagenases are the only mammalian proteases capable of hydrolyzingfibrillar collagen, within its triple helical domain, and arerequired for normal collagen turnover in adult animals.33 Oncecleaved, collagen is further broken down by gelatinases andstromelysins. In situ hydrolysis of collagen, which we observedin skin exposed to ultraviolet irradiation, therefore presumablyreflects the combined activities of collagenase and other proteinases,including 92-kd gelatinase and stromelysin-1, which were alsoelevated in the dermis in skin exposed to ultraviolet irradiation.
Type I collagen fibrils are stabilized by intermolecular covalentcross-links,34 which connect N- or C-terminal telopeptide domainsto central triple helical domains on adjacent type I collagenmolecules. Cross-linked telopeptides are released in solubleform after collagen has been broken down.26,28 Levels of typeI collagen C-terminal telopeptide cross-links were significantlyhigher in skin exposed to ultraviolet irradiation than in nonirradiatedskin. Taken together, the results of in situ zymography andcross-linked telopeptide measurements demonstrated that metalloproteinasesinduced by ultraviolet irradiation degrade dermal collagen invivo.
Collagenase activity and 92-kd gelatinase activity, measuredafter four consecutive exposures to ultraviolet irradiationat 48-hour intervals, were induced to the same maximal levelas that observed after a single exposure. Thus, there was noevidence of a lessened response over time. These studies, involvingwhites with light-to-moderate skin color, used a dose of ultravioletirradiation that does not cause reddening of the skin. The doseused is equivalent to 5 to 15 minutes of exposure to noondaysun. In practical terms, this means that brief exposure to sunevery other day should maintain elevated levels of matrix metalloproteinases,in the absence of skin reddening, in comparable persons. Althoughdirect evidence is lacking, this mechanism may lead to persistentbreakdown of skin connective tissue and accelerated prematureskin aging.
Pretreatment of skin with tretinoin substantially inhibitedthe induction of collagenase, 92-kd gelatinase, and stromelysin-1,in both the epidermis and the dermis, in skin exposed to ultravioletirradiation. The transcription of the genes for collagenase,92-kd gelatinase, and stromelysin-1 is dependent on the transcriptionfactor activating protein 1 (AP-1),35,36,37,38 which is activatedby ultraviolet irradiation and antagonized by tretinoin in humanskin.21 Pretreatment of skin with tretinoin did not inhibitinduction of tissue inhibitor of matrix metalloproteinases-1.By inhibiting the induction of matrix metalloproteinases butnot the induction of tissue inhibitor of matrix metalloproteinases-1,tretinoin alters their ratio in favor of the inhibition of matrixmetalloproteinase. This may account for our observation thattretinoin substantially inhibited the induction of matrix metalloproteinaseactivities. The transcription of tissue inhibitor of matrixmetalloproteinases-1, like that of the matrix metalloproteinases,37,38,39is regulated by AP-1.19 The fact that tretinoin does not inhibitthe induction of tissue inhibitor of matrix metalloproteinases-1suggests that other transcription factors activated by ultravioletirradiation, besides AP-1, stimulate the expression of tissueinhibitor of matrix metalloproteinases-1 in human skin. It isalso possible that residual AP-1 activity in tretinoin-treated,ultraviolet-irradiated skin (approximately 30 percent of thatin untreated skin21) may be sufficient for the full inductionof tissue inhibitor of metalloproteinases-1 by ultraviolet irradiation.
Topical tretinoin improves the appearance of photoaged skinby reducing fine lines and wrinkles40 and lightening brown spots.41Topical tretinoin preparations are available by prescriptionin the United States and several other countries for the treatmentof visible photoaging. Our data suggest that tretinoin may alsohave a role in preventing the aspect of photoaging attributableto matrix metalloproteinase-mediated dermal damage (i.e., wrinkling).We used alcoholic solutions of tretinoin, not prescription products,in our study, and it was designed to test the concept that tretinoinmay prevent photoaging rather than to assess its effectivenessin practice.
Whether topical tretinoin can in fact prevent photoaging mustbe determined by carefully controlled clinical trials. Suchtrials should also address the merits of topical tretinoin inrelation to sunscreens, which are generally believed to provideprotection against photoaging, and the widely held belief, basedon anecdotal observations, that tretinoin increases the sensitivityof the skin to sun.
Supported in part by grants from the Babcock Endowment for DermatologicalResearch, the Dermatology Foundation, and Johnson & Johnson,which was not involved in the design or conduct of the studyor in the analysis, interpretation, or reporting of the results.The retinoic acid preparation used in this study was preparedin our laboratory and is not sold.
Drs. Fisher and Voorhees are named inventors on an approvedpatent application as well as a pending patent application (withDr. Kang) concerning methods of preventing sun-induced skinaging. They will receive royalties under the University of Michigan'sIntellectual Property Policy, in the event that a commerciallicense is signed and a product is sold. This article describesresearch that was part of the basis of the approved and pendingpatent applications.
We are indebted to Carolyn Petersen and Suzan Rehbine for theprocurement of tissue specimens, Nandini Duraiswamy for technicalassistance, Laura VanGoor for the preparation of graphic material,Ted Hamilton for statistical analyses, and Anne Chapple foreditorial assistance.
Source Information
From the Departments of Dermatology (G.J.F., Z.Q.W., S.C.D., S.K., J.J.V.) and Pathology (J.V.), University of Michigan Medical School, Ann Arbor. Presented in part at the annual meeting of the Society for Investigative Dermatology, Washington, D.C., May 1–4, 1996.
Address reprint requests to Dr. Fisher at Medical Science I, Rm. 6447, 1150 W. Medical Center Dr., Department of Dermatology, University of Michigan, Ann Arbor, MI 48109-0609.
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