UVB-induced cutaneous photodamage/photoaging is characterized by qualitative and quantitative deterioration in dermal extracellular matrix (ECM) components such as collagen and elastic fibers. Disappearance of microfibrillar-associated protein 4 (MFAP-4), a possible limiting factor for cutaneous elasticity, was documented in photoaged dermis, but its function is poorly understood. To characterize its possible contribution to photoprotection, MFAP-4 expression was either augmented or inhibited in a human skin xenograft photodamage murine model and human fibroblasts. Xenografted skin with enhanced MFAP-4 expression was protected from UVB-induced photodamage/photoaging accompanied by the prevention of ECM degradation and aggravated elasticity. Additionally, remarkably increased or decreased fibrillin-1-based microfibril development was observed when fibroblasts were treated with recombinant MFAP-4 or with MFAP-4-specific siRNA, respectively. Immunoprecipitation analysis confirmed direct interaction between MFAP-4 and fibrillin-1. Taken together, our findings reveal the essential role of MFAP-4 in photoprotection and offer new therapeutic opportunities to prevent skin-associated pathologies.
Skin, the outermost barrier of the body, plays an important role in protection against environmental assaults including UV radiation which has been documented to be associated with the increased incidence of photoaging and photocarcinogenesis, in part due to the marked destruction of the stratospheric ozone layer over the past decades1,2,3. Photodamage/photoaging is a term describing the time-dependent changes that occur in chronically sun-exposed skin which appears to be an acceleration of the intrinsic aging process that occurs even in sun-protected skin4. Skin photodamage/photoaging has been reported to be physiologically correlated with several alterations including the increased disorganization of elastic fibers and the reduction of collagens in the dermal ECM5,6,7,8,9,10,11,12 as well as the increased levels of keratins 6 and 16 and the deterioration of keratin intermediate filaments in the epidermis13,14,15,16. Elastic fibers, as well as collagen fibers, are components of the dermal ECM that primarily account for the fibrous mechanism(s) controlling cutaneous elasticity5,6,17,18. In addition to the degeneration of elastic fibers in chronologically and/or photoaged skins that have been reported to stem from increased activities of matrix metalloproteinase (MMP)-12 and/or elastase19,20,21,22, the accumulation of dystrophic elastotic material in the reticular dermis, referred to as solar elastosis, is also commonly observed in photoaged skin23,24,25. With regard to the incidence of solar elastosis, UVB radiation has been demonstrated both in vivo and in vitro to up-regulate tropoelastin gene expression and protein abundance in fibroblasts and in keratinocytes, which results in an aberrant accumulation of dermal elastic fibers and elastin content6,9,11,12,26. However, the mechanisms underlying the alteration of elastic fibers in photoaged skin, including their production, accumulation and degradation, have not been fully characterized to date.
Elastic fibers, in spite of their lower abundance compared to collagen fibers, are larger structures of the ECM that control the elastic properties of connective tissues which consist of two major components, microfibrils and tropoelastin. One of the major structural constituents of microfibrils is fibrillin-1, a large (∼350 kDa) cysteine-rich glycoprotein, whose amount has been reported to be significantly decreased in tissues and in cells from patients with Marfan syndrome who demonstrate ocular, cardiovascular, and skeletal abnormalities27,28. In addition, the fibrillin-1 monomer has been documented to be assembled both linearly and laterally to form the frame of microfibrils29, followed by its association with various other proteins, including latent TGF-β-binding proteins (LTBPs), fibulins, microfibril associated glycoproteins and elastin microfibril interface located protein-1, to produce mature microfibrils30. On the other hand, tropoelastin, a 60–70 kDa protein that has lysine-containing cross-linking and hydrophobic domains, is subjected to a process of well-regulated self-aggregation called coacervation that is induced by specific interactions of each hydrophobic domain under optimized conditions31. Coacervation can be stimulated by an increase in temperature and is thought to be an important prerequisite process for cross-linking32,33,34. It was proposed that tropoelastin binds microfibrils followed by coacervation to be cross-linked by lysyl oxidase (LOX)29.
Apart from microfibrils and tropoelastin, MFAP-4 has been considered as a human homologue of 36 kDa microfibril-associated glycoprotein (MAGP-36) due to its high level of an Arg-Gly-Asp (RGD) sequence homology, its fibrinogen-like domain and its similar molecular weight, which was initially discovered in the porcine aorta and has been detected in the elastic tissue of various animals35,36,37,38,39. An immunohistochemical study demonstrated that MAGP-36, which is localized around elastic fibers in the rat aorta and is rich in elastin-associated microfibrils, had disappeared in photoaged dermis and could be found in the accumulation of disintegrated elastic fibers in the lesional skin of pseudoxanthoma elasticum, an elastin-related disorder40. That report strongly suggested that MAGP-36 is a microfibrilar-associated protein, although little is known about its role(s) in human elastic tissues.
In this study, a human skin xenograft model in combination with a lentiviral vector was used to assess the role of MFAP-4 in human skin. Despite that a lot of studies on skin photoaging have been conducted using animal models and human skin substitutes, it has been suggested that these results may be misleading because of the differences in inferior architectures, such as the relative thin epidermal layer and compromised barrier function between genuine human skin and the models and that the use of actual human skins or human skin xenografts is more appropriate for the study on skin photoaging. Therefore human xenografted photodamage/photoaging model which had been previously established was introduced in this study16. Our results demonstrate for the first time that MFAP-4 plays a critical role in fibrillin-1-based microfibril assembly and is associated with elastic fiber formation, resulting in the protection of the skin photodamage/photoaging.
Abundance of MFAP-4 is significantly decreased both in extrinsically photoaged skin and in intrinsically aged skin
In order to characterize the possible function of MFAP-4 to protect skin from photodamage and/or aging, expression of MFAP-4 was evaluated in a photoaging skin model induced by repeated UVB exposure and in intrinsically aged skin. Human skin xenografted onto severe combined immunodeficiency (SCID) mice was exposed to 1 to 2 minimal erythema dose (MED) UVB radiation for 6 weeks (wks) and total RNAs were extracted for transcript expression analysis. Skin punch biopsies from the ventral upper arms of healthy Caucasian females in their twenties and fifties were also used for total RNA extraction. Quantitative RT-PCR analysis showed that expression of MFAP-4 was significantly lower in the photoaging skin model, which is consistent with a previous report40 (Fig. 1a). Similarly, expression of MFAP-4 was also significantly impaired in intrinsically aged skin (Fig. 1b), suggesting a potential role for MFAP-4 in protection against cutaneous aging. In consistent with mRNA transcript evaluation, immunohistochemical comparison using the dermis at the ventral upper arms (sun-protected areas) of Caucasian skin specimens clearly demonstrated that MFAP-4 protein localisation detected over the dermis in their thirties was remarkably diminished in their sixties. Interestingly its protein levels were found to be almost abolished in the dermis at the dorsal lower arms (sun-exposed areas) of the donors in their sixties (Fig. 2).
Over-expression of MFAP-4 protects the skin from chronic UVB-induced photodamage
Following the observation of the down-regulation of MFAP-4 expression both in photodamaged/photoaged skin and in intrinsically aged skin, a lentiviral vector over-expressing MFAP-4 was subepidermally injected into the xenografted skin to investigate whether cutaneous over-expression of MFAP-4 prevents photodamage/photoaging. MFAP-4 expression in the skin treated with a lentiviral vector encoding MFAP-4 was confirmed to remain higher than in skin over-expressing a control reporter gene by Western blotting analysis before and after UVB irradiation for 4 wks (data not shown). In addition, immunohistochemical analysis using xenografted skin just after UVB irradiation for 8 wks demonstrated the even higher protein contents of MFAP-4 in the skin treated with a lentiviral vector encoding MFAP-4 than in the skin over-expressing a control reporter gene (Fig. 3b), suggesting that higher expression of MFAP-4 was maintained during the experimental period.
During the course of chronic UVB irradiation, en face representative images of xenografted skin demonstrated that furrow formation was induced gradually in the control reporter gene-transduced skin (Fig. 3c). This was in agreement with the quantitative analyses of skin surface roughness and skin elasticity which demonstrates significant increases in both parameters (Sa and Sz) at 10 and 14 wks (after UVB exposure for 4 and 8 wks) in the repeated UVB-exposed skin compared to the non-UVB irradiated control transfected with the control reporter gene (Fig. 3d). Additionally, the impaired value of pure elasticity (Ur/Ue) was also recorded at 10 wks (after UVB exposure for 4 wks) (Fig. 3e). The increase in skin surface roughness was kept for at least 6 wks after the final UVB irradiation. In contrast, the UVB-induced surface roughness was found to be remarkably diminished in skin over-expressing MFAP-4 according to en face images and skin surface roughness parameters (Sa and Sz) demonstrating significantly lower values compared to those in UVB-exposed skin over-expressing the control reporter gene (Fig. 3c, d). Consistently, the value of Ur/Ue was significantly higher in skin over-expressing MFAP-4 than in UVB-exposed skin over-expressing the control reporter gene at 10 wks (after UVB exposure-driven for 4 wks) (Fig. 3e), adding more evidences to the protective role of MFAP-4 against UVB-induced photodamage/photoaging.
MFAP-4 is essential to elastic fiber assembly
The finding of the protective role of MFAP-4 led us to examine the impact of MFAP-4 on elastic fiber formation. Fig. 4a shows Luna staining of human skin xenografted on SCID mice. Elastic fibers in the control reporter gene-transfected skin exposed to UVB for 8 wks were remarkably diminished compared to those in non-UVB exposed skin over-expressing the control reporter gene. On the other hand, skin treated with the MFAP-4-over-expressing vector was protected from the repetitive UVB exposure-driven degradation of elastic fibers. Interestingly, the UVB induced up-regulation of MMP-12 activity, which has been reported to be secreted from dermal fibroblasts to deteriorate elastic fibers19,20,21,22, was significantly suppressed in the skin by over-expression of MFAP-4 (Fig. 4b).
Following that in vivo analysis, further detailed analysis on the mechanisms underlying the elastic fiber assembly promoted by MFAP-4 was performed using normal human dermal fibroblasts (NHDFs). NHDFs treated with a non-specific siRNA in the presence or absence of human recombinant MFAP-4 or NHDFs treated with a siRNA specific for MFAP-4 were cultured for 8 days to allow NHDFs to assemble elastic fibers. After confirmation of the marked reduction of MFAP-4 transcript and protein levels by treatment with the MFAP-4-specific siRNA (Fig. 5a, b), immunohistochemical analysis with anti-human tropoelastin and anti-human fibrillin-1 antibodies was performed. The association of tropoelastin and fibrillin-1, which are both essential to generate elastic fibers, was found to be reduced on the cells with suppressed MFAP-4 protein contents compared to cells treated with a non-specific siRNA (Fig. 5c), whereas total protein levels of fibrillin-1 in the supernatant of cultured NHDFs was not changed by the administration with the MFAP-4-specific siRNA (Fig. 5b). On the other hand, increases in their assemblies were observed following treatment with human recombinant MFAP-4, which resulted in the enhancement of elastic fiber assembly. Consequent to the diminished organization of elastic fibers by the MFAP-4-specific siRNA, the levels of the MMP-12 transcript expression and protein was stimulated by the repression of MFAP-4 expression (Fig. 5d, e). To confirm the role of MFAP-4 in the acceleration of elastic fiber formation, NHDFs were cultured for 8 days in non-fetal bovine serum (FBS) starved conditions with or without human recombinant MFAP-4. Immunohistochemical staining of elastic fiber components demonstrated that the colocalisation of tropoelastin and fibrillin-1 in NHDFs was close to the limit of detection in control cells not treated with human recombinant MFAP-4, whereas their protein abundance following treatment with human recombinant MFAP-4 was enhanced (Fig. 5f).
Additionally, to elucidate which cellular components interact directly or indirectly with MFAP-4, concentrated supernatants from NHDFs were immunoprecipitated with an anti-MFAP-4 antibody, followed by Western blotting analysis with anti-fibrillin-1 or anti-tropoelastin antibodies. Corresponding signals were detected using an antibody specific for fibrillin-1 (Fig. 6a), whereas no signal was observed when an anti-tropoelastin antibody was used (data not shown). Furthermore, the colocalisation of MFAP-4 with fibrillin-1 was confirmed in NHDFs transfected with a non-specific siRNA by immunocytochemical analysis (Fig. 6b). Intriguingly, decreased signal of fibrillin-1 was observed in NHDFs transfected with an MFAP-4-specific siRNA synchronized with the depletion of MFAP-4, suggesting that MFAP-4 is in charge of the direct interaction with fibrillin-1 for the promotion of microfibril assembly.
MFAP-4 over-expression prevents UVB-induced collagen degradation as well as elastin deterioration
Given that the expression of pro-MMP-1 is induced by elastin peptides in skin fibroblasts41,42, the finding of the role of MFAP-4 in the promotion of elastic fiber assembly prompted us to also investigate its impact on the metabolism of collagen I. Immunohistochemical analysis with an anti-human collagen I antibody demonstrated that collagen I localisation was observed throughout the dermis in the control reporter gene-transfected skin without UVB exposure but was markedly degraded after UVB exposure for 8 wks (Fig. 7a). In contrast, the deposited collagen I in skin treated with a lentiviral vector encoding MFAP-4 was protected from UVB-induced degradation. In agreement with those immunohistochemical analyses, Western blotting analysis also showed that collagen I abundance in skin over-expressing MFAP-4 was higher than in skin over-expressing a control reporter gene before and after repeated UVB irradiation for 4 wks (Fig. 7b). Subsequently, further confirmation of the impact of MFAP-4 deficiency on MMP-1 expression was examined using NHDFs. NHDFs treated with a non-specific siRNA in the presence or absence of human recombinant MFAP-4 or NHDFs treated with an siRNA specific for MFAP-4 were cultured for 8 days. The expression of MMP-1 transcripts was significantly increased in NHDFs with impaired expression of MFAP-4 (Fig. 7c). Consistently, the secretion of active MMP-1 was also stimulated when MFAP-4 expression was knocked down (Fig. 7d).
Elasticity provided by elastic fiber formation is essential to maintain tissue flexibility and extensibility for many organs such as the large arteries, lungs, and skin30. Although fibrillin-1 microfibrils and tropoelastin protein represent the majority of elastic fiber components, it has been poorly understood how microfibrils are built following fibrillin-1 assembly and how microfibrils and tropoelastin interact during elastic fiber organization. Recently, it was shown that a disintegrin and metalloproteinase with thrombospondin motifs-like-6 protein directly binds to fibrillin-1 to promote the formation of fibrillin-1 microfibrils43. Another recent study revealed that fibulin-4 protein is indispensable for tethering LOX to tropoelastin to facilitate cross-linking44. Those reports are of great importance to comprehend the mechanisms underlying the construction of elastic fibers, whereas it remains to be determined how many other molecules are involved in elastogenesis to completely understand the process of elastic fiber formation. For example, MAGP-36, a homologue of MFAP-4 detected around elastic fibers in various animals, was reported to have disappeared in photoaged dermis but accumulated in disintegrated elastic fibers in the lesional skin of pseudoxanthoma elasticum, an elastin-related disorder40. These findings encouraged us to explore the function of MFAP-4 in human skin using an in vivo photodamaged/photoaged skin model with human skin xenografted on SCID mice in concert with a lentiviral vector to over-express MFAP-4 as well as in vitro NHDFs combined with siRNA technology.
One of the most significant problems that needed to be addressed in this study was the consideration of how MFAP-4 is involved in elastic fiber formation. It has been previously demonstrated that fibulin-4 and -5 are in charge of recruiting tropoelastins and its cross-linking enzymes onto microfibrils in order to accelerate elastic fiber assembly in collaboration with LTBP-2, which binds to heparin and heparin sulfate proteoglycans45,46,47,48,49. In addition, the abundance of fibulin-5 has also been documented to be decreased both in photoaged and in intrinsically aged dermis, suggesting that it plays a role in maintaining cutaneous elasticity50. Given that fibulins play roles in the assembly and cross-linking of tropoelastin monomers, it is reasonable to hypothesize that MFAP-4 contributes to the development of microfibrils rather than the tethering of LOX to tropoelastin which is mainly regulated by fibulins. We have clearly demonstrated that MFAP-4 interacts with fibrillin-1, whose production is also reported to be decreased in photoaged skin51, and not with tropoelastin for the organization of microfibrils which allows the participation of tropoelastin cross-linked with LOX for the formation of functional mature elastic fibers substantially required for skin homeostasis. Based on our findings, we propose a model for the contribution of MFAP-4 to the development of elastic fibers (Fig. 8). By virtue of the presence of MFAP-4, fibrillin-1 can be assembled to form microfibrils followed by the cross-linking of tropoelastins tethered with LOX to microfibrils, as supported by a previous study44. In addition to its role in the development of microfibrils, another important role of MFAP-4 in the suppression of MMP-12 activity is illustrated both in our in vivo and in vitro analyses. In parallel with previous studies indicating that elastin-derived peptides induce the expression of various kinds of MMPs (including MMP-12) in several types of cells52,53 and that UV radiation alone is sufficient to selectively degrade many elastic fiber associated proteins54, the enhancement of MFAP-4 is thought to be in charge of the suppression of MMP-12 activity as well as the promotion of microfibril formation which is essential for the organization of functional elastic fibers, resulting in the observed photoprotection.
It is also of interest to investigate the impact of the elastic fiber formation mediated by MFAP-4 on another major type of age-dependent-decreasing components, collagens, because it has been reported that MAGP-36 also binds to collagen I39. Our data indicate that over-expression of MFAP-4 protects against UVB-induced degradation of collagen I which is associated with the inhibition of photodamage/photoaging deterioration in xenografted human skin in vivo. Further, the amount of MMP-1, which is one of the collagenases expressed by dermal fibroblasts55, was significantly up-regulated in MFAP-4 knockdown NHDFs both at the transcript and the protein levels. Consistently, elevation of MMP-1 in dermal fibroblasts is also observed when the cells are exposed to broken collagens in organ cultures of human skin56. These findings led us to hypothesize that MFAP-4 has another role in the stabilization of collagen I. Given that the expression of pro-MMP-1 is stimulated by elastin peptides in skin fibroblasts41,42, it is reasonable to propose another role of MFAP-4 in the maintenance of collagens which is also important for skin homeostasis. In addition to the protection of collagen I by increased MFAP-4 expression, UVB-induced deterioration of collagen IV was also found to be hindered in our photodamage model of xenografted human skin (data not shown).
In conclusion, our data reveal for the first time that the protein levels of MFAP-4 are decreased both in extrinsic photoaged skin and in intrinsic aged skins, and that the enhancement of MFAP-4 expression protects the skin from UVB-induced photodamage/photoaging characterized by the degradation of fibers of elastin and collagens resulting in the aggravation of skin elasticity. More importantly, MFAP-4 plays essential roles not only in microfibril development by direct interaction with fibrillin-1 but also in the maintenance of ECM proteins, including collagen I, by an as yet undisclosed mechanism(s). These findings provide new insights into a fundamental comprehension of the mechanisms underlying microfibril formation resulting in skin homeostasis and also achieve a basis to develop an efficient strategy for treating UV-induced skin disorders.
Full-thickness abdominal skin from a healthy 41-year-old Caucasian female with type II skin undergoing abdominoplasty and breast skin from a healthy 46-year-old Caucasian female with type II skin undergoing breast reduction (University Pointe, Cincinnati, OH, USA), were used for skin xenografts. Three to four punch biopsies of skin at the ventral upper arm or the dorsal lower arm from healthy Caucasian females with type II skin in their twenties, thirties, fifties and sixties (Stephens and Associates, Carrollton, TX, USA) were also used for the investigation of mRNA transcript expression or protein localisation. Collection of the specimens was approved by the Institutional Review Board of the Cincinnati Children's Hospital Medical Center or the IntegReview Ethical Review Board (Austin, TX) and informed consent was obtained from the volunteers prior to the procedure.
Sixty female ICR-SCID mice, 4 to 6 wks old (Taconic Farms, Germantown, NY) were handled according to the guidelines of the Institutional Animal Care and Use Committee at Cincinnati Children's Hospital that approved the experiments and were kept under pathogen-free conditions throughout the experiments. Full-thickness abdominal and breast skins were xenografted onto SCID mice anesthetized and maintained using isofluorane/oxygen (2%/0.7L) throughout the surgery as described previously16. Briefly, after the dorsal skin of each mouse was cut to produce a wound bed approximately 2.0 to 3.0 cm in diameter, the full-thickness human skin was sutured in place with a reverse cutting precision monofilament PS-3, 6-0. Sensorcaine was applied to the graft bed edges to provide analgesia.
Chronic UVB exposure of the human skin xenografted onto SCID mice was started approximately 10 wks after the grafting when healing was complete, as previously described1. Briefly, a bank of two UVB lamps (340044-1 lamps; UVB, Upland, CA) with a filter yielding an emission peak near 302 nm was used, and the energy output was measured using a UV light meter, UV-340 MSR7000 (Lutron Electronic Enterprise Co., Ltd., Taipei, Taiwan). A progressive UVB exposure regimen was used starting at 40 mJ/cm2 (1 minimal erythema dose; MED) and was increased by 10 mJ/cm2 per week until week 3. The irradiation dose of 60 mJ/cm2 was then kept constant for the remaining period of exposure. The grafted human skin was irradiated 5 times weekly for 6 or 8 wks yielding total UVB doses of approximately 1.65 or 2.2 J/cm2, respectively. Before or after each UVB exposure, a 30 sec mechanical stretching was applied to stimulate the physiological movement of human skin.
Lentiviral vector design
Full length MFAP-4 was prepared from a skin cDNA library (Invitrogen, Carlsbad, CA) using the following primer sets.
The amplified PCR product was treated with Klenow enzyme before being cut with XbaI, and was then cloned as a half-blunted fragment into pBlueScript at the XbaI/SmaI sites. After the isolation of plasmid DNA, MFAP-4 in pBlueScript was released using StuI and XbaI and was then cloned into DeltaUX3M-9. Finally, the helper packaging construct pCMVR8.2, the transfer vector DeltaUX3M-9-MFAP-4, and a plasmid encoding vesicular stomatitis virus glycoprotein (VSV-G) were used for triple transfection as described elsewhere57. The transfer vectors DeltaUX3M-9-eGFP and DeltaUX3M-9-β-galactosidase were also prepared for reporter gene-expressing vectors and their titers were examined as described previously58.
Over-expression of MFAP-4 in xenografted human skin
After the certification of viral stocks to be RCL-free, approximately 25 μl VSV-G pseudotyped lentiviral vector encoding MFAP-4 or reporter genes (1.5×109 TU/ml) was intradermally injected into the completely healed human skin xenografted onto SCID mice twice with a week interval as described elsewhere59.
Skin profilometric measurement
Replicas of skin xenografts were prepared using the replica agent SILFLO (Flexico Developments, Potters Bar, UK) at the indicated points as described elsewhere16. In brief, skin replicas compressed using GC exafine (GC Corporation, Tokyo, Japan) were analyzed by the use of 3-dimensional optical measurement of each replica using PRIMOS Compact (GF Messtechnik GmbH, Berlin, Germany) as reported previously16. After the measurement, moiré fringes were eliminated and a rectangular area (4 to 5 mm×7 to 9 mm) was chosen in the central part of each replica in a plane analysis. Two 2-dimensional parameters, Sa and Sz, were used to assess the degree of photodamage. Sa is the average of absolute values of the area of mountains and of valleys reflecting wrinkles per certain area and Sz represents the arithmetical average of maximum peak to valley height of the roughness values of 5 consecutive sampling sections over the filtered profile.
Skin elasticity was analyzed using the Cutometer SEM575 (Courage & Khazaka Electronic, Koln, Germany) as previously documented60. Briefly, the center of each skin xenograft was used for 5 seconds application loads of 200 hPa, followed by a 2 seconds relaxation period. Two parameters, Ue (the immediate deformation) and Ur (the immediate refraction), were used as indicators of elasticity degree. Ur/Ue = pure elasticity ignoring creep.
MMP-12 activity assay
Whole skin was solubilized for MMP-12 activity measurement using a SensoLyteTM 520 MMP-12 Fluorimetric Assay Kit (AnaSpec Corporate, San Jose, CA).
NHDFs (Kurabo Corporation, Osaka, Japan) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) containing 5% (v/v) fetal bovine serum (FBS) at 37˚C in a humidified atmosphere containing 5% CO2.
NHDFs seeded on a cover glass in a cell culture plate were transfected using siRNA (a non-specific sequence for a control or a specific sequence for MFAP-4) and Lipofectamine 2000 reagent (Invitrogen) twice with 96 hours interval according to the manufacturer's instructions. Supernatants and cells were collected for Western blotting analysis and immunocytochemical and real-time quantitative RT-PCR analyses, respectively, 72 hours after the second transfection.
NHDFs, fixed in ice-cold methanol and subsequently treated for non-specific immunoreactivity blocking, were incubated with diluted rabbit anti-human tropoelastin antibody (Elastin Products Company, Owensville, MO), rabbit anti-human MFAP-4 antibody or mouse anti-human fibrillin-1 antibody (Millipore, Billerica, MA), and subsequently with diluted Alexa Fluo 488-labeled anti rabbit IgG antibody or Alexa Fluo 546-labeled anti mouse IgG antibody. The cells attached to the cover glass were mounted on a slide glass using ProLong Gold antifade reagent with DAPI (Invitrogen), and immunoreactivity was observed using a Leica DMR fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany).
Real-time RT-PCR analysis
Using real-time quantitative RT-PCR normalized against the expression of ribosomal protein large P0 (RPLP0) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the transcript expression of MFAP-4, MMP-12 or MMP-1 was determined in xenografted human skin 24 hours after the repetitive UVB exposure for 6 wks and in biopsied skin specimens from the ventral upper arms of young (20′s) and old (50′s) donors collected in RNAlater (Qiage, Valencia, CA). Total RNAs from each skin sample and from siRNA-transfected NHDFs were prepared using a RNeasy micro kit (Qiagen). cDNAs were synthesized by reverse transcription of total RNA using oligo dT and Moloney murine leukemia virus reverse transcriptase. Real-time quantitative RT-PCRs with TaqMan gene expression assays (Applied Biosystems, Foster City, CA, USA) were performed using an ABI Prism 7300 or 7500 sequence detection system (Applied Biosystems).
Supernatants from cultured NHDFs were concentrated using a 3 kDa-cut off membrane (Millipore), followed by the incubation with anti-human MFAP-4 antibody (AdipoGen, Incheon, Korea) or normal rabbit IgG. Protein G Sepharose beads (GE Healthcare, Waukesha, WI) were then added to the mixture and incubated before the collection of the beads by centrifugation. Immunoprecipitated proteins were suspended in 1X SDS sample buffer (Thermo Fisher scientific, Waltham, MA) for Western blotting analysis.
Western blotting analysis
Xenografted skin samples were lysed in cell lysis buffer (Cell Signaling Technology, Danvers, MA) containing 1 mM phenymethylsulfonyl fluoride (Sigma, St. Louis, MO). Twenty μg of each tissue extract or 12 μl of each culture medium from NHDFs were separated on 7.5% or 12% SDS gels (Bio-Rad Laboratories, Hercules, CA). Samples were transferred to PVDF membranes (Bio-Rad Laboratories) and were incubated with antibodies specific for human MFAP-4 (AdipoGen, Incheon, Korea), human collagen I (United States Biological, Swampscott, MA), human MMP-1 (AbD Serotec, Oxford, UK), human MMP-12 (Millipore), human fibrillin-1 (Elastin Products Company) or human β-actin (Sigma). Subsequent visualization of antibody recognition was performed using Enhanced Chemiluminescence Plus (GE Healthcare) according to the manufacturer's instructions.
Xenografted human skins and punch biopsy-derived human skins from the subjects in their thirties and sixties were fixed in 10% buffered formalin and embedded in paraffin. The immunoreactivities of MFAP-4 and collagen I were assessed using anti-human MFAP-4 (Abcam, Cambridge, UK) and anti-human collagen I (United States Biological), respectively, as described elsewhere16. Normal rabbit IgG was used as a negative control. The immunoreactivity of MFAP-4 and collagen I were visualized using a CSAII biotin-free catalyzed signal amplification system (DAKO) with aminoethylcarbazole.
Five μm thick sections of xenografted human skin were stained using conventional Luna staining for the visualization of fine elastic fibers.
The level of significance of differences among the groups was analyzed using Student t-test. Differences in the mean or raw values among the groups were considered significant when p < 0.05.
We thank Dr. Jun Yang (Key Clone Technologies) for technical expertise in plasmid modification and for critical discussions of vector design and Ms. Megan Kemski for her assistance with the maintenance of human skins xenografted onto SCID mice.
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/