Microfibril-Associated Protein 5 Contributes to the Elastic Fiber Abnormalities in Aged Skin

Abstract

Elastic fibers, which contribute to the flexibility of tissues such as the skin, alveoli, and arteries, have a long half-life and are not regenerated once formed during the fetal stage. Consequently, the degradation of elastic fibers due to aging or inflammation can significantly impact tissue function. In the dermis, degeneration of elastic fibers is characterized by degradation in photoaging, driven by UV radiation, and structural abnormalities of elastic fibers in intrinsic aging. However, the mechanisms driving the abnormalities associated with intrinsic aging remain incomplete. This study aimed to identify the factors involved in the elastic fiber abnormalities associated with intrinsic aging of the dermis. Through a comprehensive analysis of gene expression, this study focused on microfibril-associated protein 5 (MFAP5) as a candidate gene responsible for the elastic fiber abnormalities associated with intrinsic aging. Immunofluorescence staining revealed that aged fibroblasts highly expressed MFAP5 and strongly localized it to aggregated elastic fibers. Furthermore, the elimination of MFAP5 expression suppressed elastic fiber aggregation. The exogenous addition of MFAP5 induced thickening and disorganization of elastic fibers, effects that were not observed with the overexpression of MFAP5 in young fibroblasts, which merely express MFAP5. Moreover, MFAP5 inhibited the interaction between latent transforming growth factor β binding protein 4 and fibulin-5, which are crucial for elastic fiber formation. These results suggest that excess MFAP5 expression associated with aging causes abnormalities in elastic fibers. Understanding the role of MFAP5 in elastic fiber abnormalities highlights its potential as a therapeutic target for mitigating intrinsic dermal aging and improving skin elasticity.

INTRODUCTION

Elastic fibers, a type of extracellular matrix, are essential for the flexibility of tissues such as the skin, alveoli, and arteries. Elastic fibers are composite structures composed primarily of elastin and several associated proteins, which are synthesized during fetal development and do not regenerate thereafter.¹⁾ Consequently, alterations in elastic fibers due to aging or inflammation, such as degradation, fragmentation, or compositional changes, are associated with phenotypes characteristic of human aging, including wrinkles, reduced pulmonary function, and increased blood pressure.¹⁾ Severe alterations can lead to pathological conditions such as emphysema and atherosclerosis.²⁾

Aging-related changes in the skin are aggravated by various environmental factors, such as exposure to UV radiation and microbial interactions. Cutaneous aging is typically classified into intrinsic aging, also referred to as natural aging, and extrinsic aging, commonly termed photoaging in the dermis. Histological and ultrastructural studies have shown that changes in elastic fibers differ significantly between intrinsically aged and photoaged skins.^3,4) In extrinsic aging, primarily caused by UV radiation, elastic fibers undergo degradation and reduction through matrix metalloproteinases mediated by inflammatory responses, resulting in a reduction that ultimately contributes to the formation of deep wrinkles.⁵⁾ In contrast, in sun-protected areas, such as the dermis of the buttocks, elastic fibers do not decrease; instead, thickened fibers with a disrupted alignment are observed.^3,4) These elastic fiber abnormalities are thought to contribute to the formation of fine wrinkles; however, the underlying mechanisms remain unclear.

Elastic fibers are formed through the following steps: (i) self-assembly of elastin secreted into the extracellular space, (ii) deposition of the self-assembled elastin onto microfibrils, and (iii) cross-linking of elastin molecules intra- and intermolecularly by lysyl oxidase (LOX).⁶⁾ Previous studies using genetically modified mice have identified essential molecules required for elastogenesis.^1,6) However, the molecules involved in the elastic fiber abnormalities associated with intrinsic aging remain unidentified. Here, we found that the expression of microfibril-associated protein 5 (MFAP5), which encodes microfibril-associated protein-2 (MAGP-2), a reported component of elastic fibers,⁷⁾ increased with aging. Therefore, we investigated the involvement of the upregulation of MFAP5 in the elastic fiber abnormalities with aging. To avoid confusion between gene and protein names, this study will uniformly refer to both the gene and protein as MFAP5.

MATERIALS AND METHODS

Cell Culture

Neonatal or adult normal human dermal fibroblasts (NHDF-Neo and NHDF-Ad) were obtained from Lonza K.K. (Tokyo, Japan). A series of TIGs, derived from human skin fibroblasts, were obtained from JCRB Cell Bank (Osaka, Japan). All cells were maintained in minimum essential medium-α (FUJIFILM Wako Pure Chemical Corporation (Wako), Osaka, Japan) supplemented with 10% fetal bovine serum (Biosera, Boussens, France) and 1 mM l-glutamine (Wako) at 37°C and 5% CO₂. The donor ages of the cells used are as follows: NHDF-Neo, neonatal; NHDF-Ad, 88 years; TIG103, 69 years; TIG107, 81 years; TIG120, 6 years; and TIG121, 8 months. NHDF-Neo stably expressing MFAP5-HA was generated via lentiviral transduction, as described previously.⁸⁾

Immunoblotting

Cells were seeded in 35 mm dishes and allowed to grow until they reached confluence. Following washes with phosphate-buffered saline (PBS), the cells were incubated in a serum-free medium. After 24 h, the conditioned medium and cell lysates were collected (see Supplementary Materials for further details) and subjected to immunoblotting according to previously described methods.⁹⁾ The antibodies used in this study are as follows: anti-tropoelastin antibody (Supplementary Materials); anti-MFAP5 antibody (Proteintech Japan, Tokyo, Japan); horseradish peroxidase-conjugated anti-GAPDH antibody; anti-DYKDDDK (FLAG) tag antibody; anti-HA tag antibody; anti-V5 tag antibody (all from Wako); and anti-His-tag mAb (Clone: OGHis) (MBL, Tokyo, Japan). Ponceau S solution was obtained from Beacle, Inc. (Kyoto, Japan).

Immunofluorescence

After the cells reached confluence, they were cultured for an additional 9 d. Cells were immunostained with an anti-tropoelastin antibody (diluted 1 : 1000), anti-MFAP5 antibody (diluted 1 : 500), and anti-fibrillin-1 antibody (diluted 1 : 200, Merck Millipore, MA, U.S.A.), as described previously.¹⁰⁾ After incubation with a secondary antibody, the cells were stained with Hoechst 33258 (Dojindo Laboratories, Kumamoto, Japan). The cells were mounted using Fluoromount/Plus (Diagnostic Biosystems, CA, U.S.A.), followed by observation with a confocal laser scanning microscope (FV3000, Olympus), and images were captured.

RT-Quantitative PCR (qPCR) and Gene Microarray

cDNA was synthesized from total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan) according to the manufacturer's recommendations. qPCR assays were performed using KAPA SYBR FAST qPCR Kit (Nippon Genetics, Tokyo, Japan) and primers (listed in Supplementary Table 1) and were normalized to the GAPDH gene. Microarray analysis on a 3-dimensional gene DNA chip was outsourced to Toray Industries Inc. (Tokyo, Japan).

Gene Expression in the Human Dermis across Different Age Groups

Gene expression data from dermal samples (GSE1081022) were obtained directly from the GEO database (NCBI, Bethesda, MD, U.S.A.). Expression levels of each gene in the dermis were converted to z-scores, and differential expression was assessed using Dunnett’s multiple comparison test. The analysis was performed by dividing the dataset into 3 groups: newborns (<89 weeks after birth) (n = 27), individuals aged 20 (n = 25), and individuals older than 60 (n = 18).

RNA Interference

Cells were seeded on a 35-mm dish with a 13-mm coverslip. After 24 h, the cells were transfected with negative control small interfering RNA (siRNA) or siRNAs targeting MFAP5 or latent TGF-β-binding protein 4 (LTBP4) (all from Integrated DNA Technologies, IA, U.S.A.) using Lipofectamine RNAiMAX (Thermo Fisher Scientific, MA, U.S.A.) according to the manufacturer’s instructions. The sequences of the siRNAs are listed in Supplementary Table 2. After the cells reached confluence, they were cultured for an additional 9 d. Subsequently, the coverslips were removed for immunostaining, and total RNA was extracted from the remaining cells.

Immunoprecipitation

The 293FT cells (Thermo Fisher Scientific) were transfected with LTBP4-3xFLAG/His, fibulin-5 (FBLN5)-V5/His, or MFAP5-HA/His expression vectors (Supplementary Materials) using PEI MAX (Polysciences, PA, U.S.A.). After transfection, the cells were cultured in serum-free Dulbecco’s modified Eagle’s medium (Wako) for 48 h. The conditioned media were subjected to immunoprecipitation with anti-DYKDDDK tag or anti-HA tag antibody beads (Wako), followed by immunoblotting according to the manufacturer’s recommendations.

For the competitive inhibition experiment, DYKDDDK beads conjugated with LTBP4S-3xFLAG/His were incubated at 4°C for 16 h with culture supernatants containing a fixed amount of FBLN5-V5/His, either with or without the addition of MFAP5-HA/His culture supernatant. After extensive washing with PBS, proteins bound to the beads were eluted using 4% sodium dodecyl sulfate and collected as samples.

Statistical Analysis

The significance of differences was determined using a 2-tailed Student’s t-test and GraphPad Prism 10 (GraphPad Software, San Diego, CA, U.S.A.), unless otherwise described. Differences between the groups were considered significant at p < 0.05.

RESULTS

MFAP5 Expression Increases with Aging

First, we examined whether elastic fibers exhibit abnormalities with aging. Immunofluorescence staining showed web-like fibers in NHDF-Neo and NHDF-Ad. In NHDF-Neo, thin and regularly aligned fibers were observed, while in NHDF-Ad, thicker and disordered fibers were noted, with some of these thicker fibers appearing as aggregated structures (Fig. 1A). Note that both fibroblasts were derived from sun-protected areas of the skin.

Fig. 1. MFAP5 Expression Is Upregulated in the Dermis with Aging

(A) Immunofluorescence staining of tropoelastin in NHDF-Neo and NHDF-Ad. The nuclei were counterstained with Hoechst dyes. Scale bar: 50 μm. (B) Results of DNA microarray analysis. The table lists the top 10 genes upregulated in NHDF-Ad compared to NHDF-Neo. (C) RT-qPCR analysis of indicated mRNA expressions in NHDF-Neo (Neo) and NHDF-Ad (Ad). Data represent the mean and S.E.M. of 3 independent samples. Data represent the relative expression of GAPDH as internal control. p-Values are indicated in the graph and were calculated using Student’s t-test. (D) Gene expression analysis in human dermis with aging (GSE1081022). Data represent the z-score of the average expression for each gene scaled across each column. p-Values are indicated in the graph and were calculated using one-way ANOVA followed by Dunnett's post hoc test. Newborn (n = 27), Age 20 (n = 25), and Age <60 (n = 18).

Next, we analyzed gene expression between these cells using a DNA array. Figure 1B shows the top 10 genes highly expressed in NHDF-Ad. Among these genes, we focused on MFAP5, which has been reportedly associated with elastic fibers.¹¹⁾ RT-qPCR demonstrated that MFAP5 exhibited a particularly pronounced increase, with expression levels approximately 14-fold higher in NHDF-Ad compared to NHDF-Neo (Fig. 1C). The expression of microfibril-associated protein 2 (MFAP2), a family gene of MFAP5, and fibrillin-1 (FBN1) was significantly increased, but not ELN and LOX.

Moreover, an analysis using a dataset of gene expression changes in the human dermis revealed that MFAP5 expression increased with aging, while MFAP2, ELN, FBN1, and LOX expressions decreased (Fig. 1D).

MFAP5 Accumulates in Thick Elastic Fibers Formed by Aged Fibroblasts

To validate MFAP5 protein expression in these cells, immunoblotting of the conditioned medium and cell lysate of each cell was performed (Fig. 2A). Immunoblotting showed strong MFAP5 expression in the conditioned medium of NHDF-Ad, whereas MFAP5 was below the detection limit in both the cell lysate and the conditioned medium in NHDF-Neo. In contrast, tropoelastin, the core component of elastic fibers, was detected at significantly higher levels in both the cell lysate and the conditioned medium of NHDF-Neo compared to those of NHDF-Ad. Immunofluorescence staining demonstrated that MFAP5 strongly localized to tropoelastin-positive fibers, particularly the thick fibers in NHDF-Ad (arrow in Fig. 2B), but was barely detectable in NHDF-Neo (Fig. 2B). Further, immunofluorescence staining using fibroblasts from donors of different ages revealed that MFAP5 expressed and colocalized with web-like tropoelastin-positive fibers in the fibroblasts from younger donors, while fibroblasts from aged donor exhibited thick, aggregated tropoelastin-positive fibers with robust MFAP5 localization (Fig. 2C).

Fig. 2. MFAP5 Strongly Co-localized with Aggregated Tropoelastin-Positive Fibers

(A) Immunoblotting with the antibody against MFAP5 and tropoelastin in NHDF-Neo and NHDF-Ad. GAPDH was detected as a loading control. The transferred proteins were visualized by staining with Ponceau S. (B, C) Immunofluorescence staining with MFAP5 and tropoelastin antibodies in the indicated cells derived from young or aged fibroblasts. The nuclei were counterstained with Hoechst dyes. Scale bar: 50 μm. (D) NHDF-Ad was transfected with control siRNA or MFAP5 siRNA. RT-qPCR analysis for MFAP5 mRNA expression. Data represent the fold expression of MFAP5 relative to the non-silencing siRNA (siCont). (E) Immunofluorescence staining of tropoelastin and MFAP5 in NHDF-Ad transfected with the indicated siRNA. The nuclei were counterstained with Hoechst dyes. Scale bar: 50 μm.

MFAP5 Facilitates the Aggregation of Elastic Fibers

To investigate the role of MFAP5 in the elastic fiber abnormalities with intrinsic aging, we suppressed MFAP5 gene expression in NHDF-Ad and confirmed the efficacy of MFAP5 knockdown using RT-qPCR (Fig. 2D). Immunofluorescence staining showed a reduction or disappearance of MFAP5 in the cells transfected with MFAP5 siRNA. Furthermore, the suppression of MFAP5 expression reduced aggregated elastic fibers (Fig. 2E). This inhibition of aggregation was restored by the addition of the conditioned medium from parental NHDF-Ad (Supplementary Fig. 2).

To examine the effect of MFAP5 on elastic fiber thickening and aggregation, we generated NHDF-Neo stably expressing MFAP5 (MFAP5-HA). Immunoblotting confirmed MFAP5 expression in both cell lysates and conditioned medium, with no changes in tropoelastin expression compared to empty vector controls (pLVSIN-HA) (Fig. 3A). Immunofluorescence revealed that tropoelastin-positive fibers strengthened and elongated alongside MFAP5-positive fibers (Fig. 3B). When conditioned medium from MFAP5-expressing cells was added to NHDF-Neo cells, MFAP5 accumulated locally on the cell surface, and tropoelastin-positive fibers were observed as locally aggregated and co-localized with MFAP5 compared to controls (Fig. 3C).

Fig. 3. Exogenous Addition of MFAP5 Induces Aggregation of Elastic Fibers

(A) Immunoblotting with the antibody against MFAP5 and tropoelastin in NHDF-Neo stably expressing MFAP5-HA and pLVSIN-HA (empty vector). GAPDH was detected as a loading control. The transferred proteins were visualized by staining with Ponceau S. (B) Immunofluorescence staining of tropoelastin and MFAP5 in NHDF-Neo stably expressing MFAP5-HA and pLVSIN-HA on Days 3, 5, and 9 after becoming confluent. The nuclei were counterstained with Hoechst dyes. Scale bar: 50 μm. (C) Immunofluorescence staining of tropoelastin and MFAP5 in NHDF-Neo supplemented with culture supernatant from NHDF-Neo stably expressing MFAP5 for 9 d after becoming confluent. The nuclei were counterstained with Hoechst dyes. Scale bar: 50 μm.

MFAP5 Inhibits the Interaction between LTBP4 and FBLN5

The interactome analysis has indicated that MFAP5 binds to LTBP4, which conducts the orderly deposition of tropoelastin via interaction with FBLN5,¹²⁾ predicting that MFAP5 could affect tropoelastin deposition. Therefore, we confirmed the interaction between MFAP5 and LTBP4. Immunoprecipitation using an anti-FLAG antibody revealed that MFAP5 co-precipitated with LTBP4S, an isoform of LTBP4 that binds to FBLN5¹³⁾ (Figs. 4A, 4B). However, FBLN5 did not co-precipitate with MFAP5 (Fig. 4B). Furthermore, the interaction between LTPB4S and FBLN5 decreased in the presence of MFAP5 (Fig. 4C). Finally, we performed immunostaining of MFAP5 and tropoelastin in TIG121 (Fig. 4D) and NHDF-Ad (Fig. 4E) under LTBP4 knockdown (Supplementary Fig. 3). As previously reported,¹³⁾ tropoelastin deposition was inhibited by LTBP4 knockdown in TIG121 cells, while MFAP5 was observed on fibers. In contrast, in NHDF-Ad cells, tropoelastin deposition was observed even under LTBP4 knockdown, with co-localization of aggregated MFAP5. Furthermore, in both cell types, MFAP5 co-localized with fibrillin-1 fibers regardless of LTBP4 expression.

Fig. 4. MFAP5 Disrupts the Interaction between Fibrillin-5 and LTBP4S

The expression vectors were independently transfected into 293FT cells. The transfectants were incubated with serum-free media for 48–72 h, and the conditioned media were collected. The media were incubated with each other, and the reactants were subsequently subjected to immunoprecipitation using anti-FLAG antibody (A) or anti-HA antibody (B). (C) The media from LTBP4S transfectants were incubated with media from fibulin-5 transfectants in the presence of media from MFAP5 transfectants, and the reactants were subsequently subjected to immunoprecipitation using an anti-FLAG antibody. The immunoprecipitants were separated by SDS-PAGE and subjected to immunoblotting with indicated antibodies. * and # indicate nonspecific bands and IgG light chains, respectively. (D, E) Immunostaining of MFAP5, tropoelastin, and fibrillin-1 in TIG121 and NHDF-Ad cells under LTBP4 knockdown. Scale bar: 50 μm.

DISCUSSION

This study aims to define factors involved in the elastic fiber abnormalities associated with intrinsic aging in the human dermis. We found that MFAP5, a component of elastic fibers, shows increased expression with aging, localizes within aggregated elastic fibers, and that suppression of MFAP5 expression reduces this aggregation. Additionally, our findings suggest a potential mechanism in which MFAP5 binds to LTBP4, thereby inhibiting the proper deposition of tropoelastin on microfibrils.

MFAP5 is a microfibrillar component that serves as a scaffold for elastic fibers.¹⁴⁾ While MFAP5 expression peaks during tissue elastogenesis,¹⁵⁾ it is not essential for elastogenesis or microfibril formation.¹⁶⁾ Previous research has suggested that microfibril-associated MFAP5, but not its free form, promotes the macroassembly of elastic fibers.¹⁷⁾ Consistent with these findings, we observed that MFAP5 increasingly localized to microfibrils in a time-dependent manner, which corresponded with the strengthening of elastic fibers in NHDF-Neo stably expressing MFAP5. In the NHDF-Neo-supplemented culture supernatant containing MFAP5, a localized accumulation of MFAP5 was observed. Additionally, MFAP5 is co-localized with aggregated and disorganized elastic fibers. These findings suggest that the association of MFAP5 with microfibrils is involved in the orderly deposition of elastic fibers. The association of MFAP5 with microfibrils appears to be independent of LTBP4 and likely occurs through binding with fibrillin-1, a core component of microfibrils.⁷⁾ To elucidate the mechanism of MFAP5-mediated macroassembly of elastic fibers, further studies are needed to investigate the mechanisms by which MFAP5 associates with microfibrils in detail.

Tropoelastin binds to FBLN5, but not MFAP5,⁷⁾ in the extracellular space, promoting self-assembly,^18,19) following which it is deposited onto microfibrils through interactions with LTBP4 and FBLN5.¹³⁾ Our findings suggest that the imbalance among these 3 proteins caused by excessive “free form MFAP5” disrupts the orderly deposition of tropoelastin. Consequently, in regions with “free form MFAP5” accumulation, it is speculated that the complex of FBLN5 and tropoelastin, in the presence of excessive “free form MFAP5,” would not be regulated by LTBP4-mediated deposition, resulting in irregularities in both the size of the self-assembled aggregates and their deposition onto microfibrils.

In summary, our study highlights the involvement of MFAP5 upregulation in the age-related alteration of elastic fibers, a previously underexplored aspect of aging. Although further studies are needed to clarify the mechanism by which MFAP5 induces fiber abnormalities, our findings suggest that age-associated upregulation of MFAP5 may serve as a potential anti-aging target to address intrinsic dermal aging.

Acknowledgments

We are grateful to Izumi Ogura, Reina Fuji, Anna Yanagisawa, Sayako Takeuchi, and Honoka Okamoto for their technical assistance.

This work was partially supported by Lydia O’Leary Memorial Pias Dermatological Foundation (to F.S.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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