Sialylation regulates myofibroblast differentiation of human skin fibroblasts
© The Author(s). 2017
Received: 11 January 2017
Accepted: 11 March 2017
Published: 18 April 2017
Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts.
The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-β1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment.
A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast differentiation in LP fibroblasts was restored by a sialidase inhibitor.
Desialylation of CD44 with increased sialidase during the process to senescence reduced the localization of CD44 in lipid rafts after TGF-β1 stimulation, leading to the inhibition of myofibroblast differentiation. Thus, regulation of sialylation may be an attractive strategy for the prevention and regenerative therapy of age-related skin diseases, cosmetic skin alterations, and chronic wounds caused by delayed healing in elderly people.
KeywordsMyofibroblast differentiation Sialylation Sialidase CD44 Lipid rafts Aging
With the dramatic expansion of the size of the elderly population (those over 65 years old), the risk of age-related pathologies, such as chronic wounds, and the associated clinical and socioeconomic burdens are increased. Therefore, research on the effects of aging on wound healing is a relevant topic. In response to injury, myofibroblasts, which are characterized by increased contractile ability and expression of α-smooth muscle actin (α-SMA) , differentiate from resident fibroblasts after proliferation and migration to the site of injury [1–3]. Myofibroblasts play important roles in the successful formation of granulation tissue and matrix remodeling in wound healing . Therefore, myofibroblasts are considered key players in the delayed healing seen in elderly people. So far, it has been demonstrated that skin fibroblasts isolated from elderly people have impaired migration, premature senescence, impaired proliferative response, and defects in matrix generation as compared with skin fibroblasts from young people [5, 6]. Furthermore, it has been shown that aged fibroblasts resist myofibroblast differentiation . However, the molecular mechanisms underlying these dysfunctions in fibroblasts with aging have not been clarified.
Carbohydrate antigens (also called glycans) expressed on the cell surface as components of glycoproteins change depending on the cellular state (developmental and pathological state), and contribute significantly to fundamental biological functions, such as cell differentiation, cell adhesion, cell-cell interaction, and regulation of signaling pathways . Recent reports have demonstrated that cell surface glycans on fibroblasts change during aging and the senescent process; for instance, reduced sialylation on glycans was observed during cellular senescence in human lung fibroblasts [9–12]. Recently, our previous glycome analysis using lectin microarrays demonstrated changes in the total membrane glycosylation of glycoproteins in human skin fibroblasts with aging and the senescent process . However, it is not known whether the changes in glycosylation have any effects on fibroblast function and senescence.
Here, we have investigated whether the changes in glycosylation during the process to senescence have functional effects on skin fibroblasts and have demonstrated novel mechanisms underlying age-related defects in myofibroblast differentiation.
TIG-3S cells (fetal-derived human skin fibroblasts) were purchased from Health Science Research Resources Bank (Osaka, Japan); the population doubling level (PDL) was 23. Cells were grown in Dulbecco’s modified Eagle medium (Wako Pure Chem. Ind. Co. Ltd., Osaka, Japan) containing 10% fetal bovine serum (Cell Culture Technologies, Gravesano, Switzerland), and penicillin-streptomycin (Gibco, Grand Island, NY, USA). Cells were passaged at a confluence of 80–90% and seeded at a density of 2500–5000 cells/cm2. All cells were studied at a confluence of 80–90%. PDLs were calculated at each passage using the following equation: n = (log2X – log2Y), where n = PDL, X = number of cells at the end of one passage, and Y = number of cells seeded at the beginning of one passage. In this study, fibroblasts with PDL 35–45 and PDL 73–78 were used as early-passage (EP) and late-passage (LP) fibroblasts, respectively. Cellular senescence occurred after PDL 80 as reported previously . An in vitro aging model based on cellular senescence has previously been described and validated as a model of age-related alterations in human aortic smooth muscle cells, human vascular endothelial cells, and human fibroblasts [5, 6, 14–16]. Therefore, here we defined the LP fibroblasts as in vitro aged cells at the stage of pre-senescence, just before the process to cellular senescence. Doubling time was calculated by the formula PDL/time for increasing PDL. For myofibroblast differentiation, cells were cultured in serum-free medium containing 10 ng/ml human transforming growth factor (TGF)-β1 (Peprotech, Rocky Hill, NJ, USA). For depletion of sialic acid, cells were incubated with 2 mM GalNAc-α-O-benzyl (BGN; Sigma-Aldrich, St. Louis, MO, USA) or 100 U/ml sialidase from C. perfringens Type V (New England Biolabs, Ipswich, MA, USA) for at least 24 h and throughout the experiments. For inhibition of sialidase, cells were incubated with 2 mM 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (zanamivir; TCI, Tokyo, Japan) throughout the experiments.
Senescence-associated β-galactosidase (SA-β-Gal) assay
SA-β-Gal activity was analyzed using a senescence detection kit (BioVision Inc., Milpitas, CA, USA) according to the manufacturer’s instructions. Briefly, cells were washed twice with phosphate-buffered saline (PBS), exposed to fixation solution for 10 min, and then incubated overnight in freshly prepared staining solution. After staining, cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI), and then microscopic examination of four fields was performed. By counting the number of SA-β-Gal-positive cells based on their blue color and the total number of cells stained with DAPI using ImageJ software (National Institutes of Health, Bethesda, MD, USA), the percentage of SA-β-Gal-positive cells was calculated to estimate the percentage of senescent cells.
The cells were cultured to confluence in 35-mm dishes. Next, a sterile 200-μl pipette tip was used to place a single wound across the diameter of each monolayer, and the medium was replaced to remove cell debris. The cells were then incubated for 8 h with medium containing serum. At 0 and 8 h, images of each monolayer were captured using a microscope coupled with a digital camera (Nikon, Tokyo, Japan). Cell migration into each wound after 8 h was compared with that observed in the same wounded monolayer at 0 h.
Fluorescence-activated cell sorting (FACS) analysis
In general, trypsinization reduces the abundance of some cell surface antigens. Therefore, to avoid this effect, cells were harvested with Accutase® cell detachment solution (Gibco), and dissociated single cells were incubated with primary antibodies diluted in FACS buffer (0.5% (w/v) bovine serum albumin (BSA) and 0.1% (w/v) sodium azide in PBS) for 30 min on ice. After washing, the cell suspension was incubated with Alexa Fluor® 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA) diluted (1:400) in FACS buffer for 30 min on ice. Cell sorting and analysis were performed using a FACSAria™ Cell Sorter (Becton Dickinson, Franklin Lakes, NJ, USA). We used the following primary antibodies: monoclonal mouse anti-CD44 (dilution 1:100; R&D Systems Inc., Minneapolis, MN, USA), polyclonal rabbit anti-epidermal growth factor receptor (EGFR; dilution 1:100; GeneTex Inc., Irvine, CA, USA), and polyclonal rabbit anti-NEU1 (dilution 1:100; Thermo Fisher Scientific, Waltham, MA, USA). For lectin staining, FITC-conjugated Wisteria floribunda agglutinin (WFA; dilution 1:100; Vector Laboratories, Peterborough, UK), FITC-conjugated peanut agglutinin (PNA; dilution 1:100; J-Oil Mills, Tokyo, Japan), biotin-conjugated Maackia amurensis lectin II (MAL-II; dilution 1:100; Vector Laboratories), biotin-conjugated Erythrina cristagalli agglutinin (ECA; dilution 1:100; J-Oil Mills), and FITC-streptavidin (dilution 1:100; BioLegend, San Diego, CA, USA) were used. Mean fluorescence intensities (MFIs) were calculated by subtracting the intensities of the controls.
For the analysis of TGF-β1 signaling, cell culture medium was replaced with serum-free medium for 24 h, and the cells were stimulated for 5 min with 10 ng/ml human TGF-β1. Cells were lysed with lysis buffer (50 mM Tris HCl pH 7.4, 150 mM NaCl, and 1% (v/v) Triton™ X-100) containing protease and phosphatase inhibitor cocktails (Roche, Indianapolis, IN, USA). For the ECA lectin pull-down assay, total cell lysates were incubated with biotin-conjugated ECA at 4 °C overnight. Then lysates were mixed with streptavidin-conjugated magnetic beads (Invitrogen, Carlsbad, CA, USA) and incubated for an additional 6 h. For immunoprecipitation (IP), cells were lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.1% (v/v) SDS, 1% (v/v) sodium deoxycholate, and 1% (v/v) NP-40) containing protease and phosphatase inhibitor cocktails, and IPs were performed with the appropriate antibody and protein G magnetic beads (Veritas, Tokyo, Japan). Total lysates and precipitated proteins were separated by SDS-PAGE using a gel of the appropriate percentage and then transferred onto PVDF membranes (Merck Millipore, Billerica, MA, USA). After blocking, the membranes were incubated with the following primary antibodies: polyclonal rabbit anti-extracellular signal-regulated kinase 1/2 (ERK1/2; dilution 1:1000; Cell Signaling Technology, Danvers, MA, USA), polyclonal rabbit anti-phosphorylated ERK1/2 (pERK1/2; Thr183/185; dilution 1:1000; Cell Signaling Technology), monoclonal mouse anti-CD44 (dilution 1:1000), polyclonal rabbit anti-EGFR (dilution 1:1000; Abcam, Cambridge, UK), monoclonal mouse anti-α-SMA (dilution 1:1000; Abcam), polyclonal rabbit anti-NEU1 (dilution 1:1000), and monoclonal mouse anti-β-actin (dilution 1:10,000; Sigma-Aldrich). The membranes were then incubated with the appropriate peroxidase-conjugated secondary antibodies (dilution 1:30,000; Cell Signaling Technology), washed, and developed with ECL™ Prime reagents (GE Healthcare, Piscataway, NJ, USA).
Cells were fixed with 4% (w/v) paraformaldehyde and washed. Next, cells were permeabilized and blocked with PBS containing 0.2% (v/v) Triton™ X-100, 1% (w/v) BSA, and 5% (v/v) normal goat serum. After washing, cells were incubated with an anti-α-SMA antibody (dilution 1:100) at 4 °C overnight. After washing, cells were stained with an Alexa Fluor® 488-conjugated secondary antibody (dilution 1:400; Molecular Probes) and then counterstained with DAPI. For the observation of CD44, monosialotetrahexosylganglioside (GM1), and NEU1 on the cell surface, immunostaining was performed under non-permeabilized conditions using an anti-CD44 (dilution 1:100) or an anti-NEU1 primary antibody (dilution 1:100), an Alexa Fluor® 488-conjugated secondary antibody (dilution 1:400), and Alexa Fluor® 594-conjugated cholera toxin B subunit (dilution 1:200; Molecular Probes), which is known to bind to the raft-enriched ganglioside GM1. Immunofluorescence images were taken with a fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Immunofluorescence images of colocalized CD44 with GM1 were taken with a confocal laser scanning microscope (Leica Microsystems). For the quantification of colocalized CD44 with GM1, the numbers of GM1-CD44 colocalized cells based on their yellow color and the total numbers of cells stained with DAPI at three fields (average total cell numbers at one field were 27 cells) were counted and then the percentage of GM1-CD44 colocalized cells was calculated.
Real-time polymerase chain reaction (PCR)
List of primer sets for real-time polymerase chain reaction
Western blot images were densitometrically analyzed using ImageJ software (National Institutes of Health). Values are expressed as means ± standard deviation (SD) from three or four independent experiments. The Student’s t test for independent samples was used for statistical analysis.
Sialylation of glycoproteins decreases in skin fibroblasts during the process to senescence
Myofibroblast differentiation is inhibited by reduced sialylation with BGN
It is known that skin fibroblasts exhibit defects in several processes with aging, such as migration and myofibroblast differentiation [5–7]. First, we examined the effect of decreased sialylation using BGN on migration ability. As shown in Figure S4d (Additional file 4), migration was not different between control and BGN-treated EP fibroblasts, indicating that the decreased sialylation has no effect on migration ability. Next, we examined the effect of decreased sialylation on myofibroblast differentiation induced by TGF-β1, which is a key mediator of wound healing and is known to regulate myofibroblast differentiation [1, 23]. Immunocytochemical staining showed that TGF-β1 stimulation induced α-SMA-positive myofibroblast differentiation in control EP fibroblasts, but not in BGN-treated EP fibroblasts (Fig. 2c). Furthermore, Western blot analysis of α-SMA confirmed that myofibroblast differentiation was inhibited in BGN-treated EP fibroblasts (Fig. 2d and e). Thus, these results indicated that the reduction of sialylation by BGN inhibited myofibroblast differentiation induced by TGF-β1 stimulation.
Raft localization of CD44 after TGF-β1 stimulation is inhibited by reduced sialylation with BGN
Myofibroblast differentiation is inhibited by sialidase
To clarify the mechanism underlying the reduced myofibroblast differentiation of sialidase-treated EP fibroblasts, first we examined the expression levels of EGFR and CD44. The expression levels (total and cell surface) of EGFR and CD44 did not significantly change after the sialylation was reduced by sialidase treatment (Additional file 7: Figure S7a and b). Next, we examined raft localization of CD44 in sialidase-treated EP fibroblasts. Immunocytochemical staining showed that the ratio of colocalized CD44 with the raft marker GM1 in sialidase-treated EP fibroblasts was lower than that in control EP fibroblasts, indicating that the localization of CD44 in lipid rafts was inhibited in sialidase-treated EP fibroblasts (Fig. 4e and f). Furthermore, biochemical analysis indicated that the interaction between EGFR and CD44 in lipid rafts after TGF-β1 stimulation was inhibited by sialidase treatment (Fig. 4g). IP of the CD44 assay using ECA and MAL-II demonstrated that sialylation of CD44 was reduced by sialidase treatment (Additional file 7: Figure S7c). Taken together, these results clearly indicate that sialic acid is required for the localization of CD44 in rafts after TGF-β1 stimulation followed by myofibroblast differentiation.
Age-dependent reduction of myofibroblast differentiation is restored by a sialidase inhibitor
Myofibroblasts, which differentiate from migrated fibroblasts, play pivotal roles in maintaining skin homeostasis and orchestrating physiological tissue repair and skin regeneration. Therefore, dysfunctions in fibroblasts such as decreased proliferation, migration, and myofibroblast differentiation lead to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The molecular mechanisms of fibroblast dysfunctions during aging have not yet been elucidated. In this study, we proposed a novel mechanism for myofibroblast differentiation (Fig. 6e). HA is synthesized via HAS2 after TGF-β1 stimulation contributing to CD44 relocalization to lipid rafts; the association between EGFR and CD44 then activates MAPK/ERK signaling required for myofibroblast differentiation in non-aged fibroblasts . In in vitro aged fibroblasts, increased NEU1 sialidase expression contributes to desialylation of CD44, which is unable to associate with raft-localized EGFR even after TGF-β1 stimulation, affecting MAPK/ERK signaling and inhibiting myofibroblast differentiation. Thus, we propose that the reduction of sialylation in fibroblasts with aging is a risk factor for age-related pathologies, including chronic wounds in elderly people.
Sialic acids, with relatively strong electronegative charges, on glycans contribute to many cellular functions including migration and proliferation; hence, changes in sialylation levels have been considered to be functionally involved in developmental and pathological states [8, 27]. Recent reports using lectin microarrays have demonstrated changes in sialylation of glycoproteins during senescence in human mesenchymal stem cells and human skin fibroblasts [13, 28]. Here, we show that sialylation of glycoproteins in human skin fibroblasts was decreased during the process to senescence via increased sialidase NEU1 expression. Therefore, changes in sialylation may contribute to dysfunctions in in vitro aged skin fibroblasts, such as premature senescence and decreased migration and proliferation [5, 6], although the molecular mechanisms underlying those dysfunctions with aging have not been clarified. Next, in our study, we examined how reduced sialylation might affect skin fibroblast function. Our results demonstrated that the reduction of sialylation with BGN or sialidase treatment has no effect on cellular senescence, proliferation, or migration (Additional file 4: Figure S4 and Additional file 6: Figure S6). It is well known that glycans other than sialic acid play functional roles in proliferation and migration in several cell types including fibroblasts [8, 29, 30], but their role in aging has not been elucidated. In our previous study, we demonstrated changes in glycosylation of glycoproteins, such as N- and O-glycans, in human skin fibroblasts during aging and the senescent process . Therefore, glycans other than sialic acid may have functional roles during those processes. Further studies are required to clarify whether changes in glycosylation with aging have an effect on skin fibroblasts.
In our study, myofibroblast differentiation was inhibited in LP fibroblasts, even though cell surface levels of EGFR and CD44 did not change in serum-free culture conditions or normal culture conditions in non-confluent cells (Additional file 8: Figure S8a). Furthermore, there were no differences in HAS2 expression between EP and LP fibroblasts after TGF-β1 stimulation (Additional file 8: Figure S8b). It has been demonstrated that reduction of EGFR and HA levels during senescence in confluent dermal fibroblasts inhibits myofibroblast differentiation in serum-free conditions . Here, we show that CD44 sialylation affects myofibroblast differentiation. It is known that the ability of myofibroblast to differentiate is cell context-dependent . Therefore, the differences between our results and past reports may be due to the type of fibroblasts and cell culture conditions used. We also show that reduction of CD44 sialylation during the process to senescence affected raft localization of CD44 after TGF-β1 stimulation. Because CD44 sialylation negatively regulates HA binding , reduced mobility of CD44 due to the binding between desialylated CD44 and HA may inhibit CD44 relocalization to lipid rafts. Furthermore, raft localization of CD44 may depend on the interactions between sialic acid and raft-resident proteins, and sialic acid-binding lectins may be good candidates. At the moment, it is unknown what kinds of sialylated structures on CD44 are critical for the function of CD44, but it is considered that sialyl T antigen (Siaα2-3Galβ1-3GalNAc-) is included on CD44 from the results using sialidase or sialidase inhibitor (Fig. 5b and Additional file 7: Figure S7c). It has been demonstrated that sialyl T antigen in the raft-localized protein, such as integrin β4, is important for integrin β4-mediated signaling . Thus, sialyl T antigen may be functional on raft-localized CD44 for the signal transduction in lipid rafts. Regardless, further studies are required to clarify the mechanisms underlying the inhibition of myofibroblast differentiation with aging due to the affected mobility of desialylated CD44 and to identify the candidate partners of CD44 in lipid rafts and functional sialylated structures on CD44.
Sialidase NEU1 has been shown to be involved in epithelial to mesenchymal transition in pancreatic cancer due to activation of matrix metalloproteinase 9-EGFR signaling after reduction of EGFR sialylation [34, 35]. NEU1 is involved in the activation of dendritic and macrophage cells by desialylation of Toll-like receptor 4, and in the recruitment of leukocytes by removal of sialyl residues from the activation epitopes on the adhesion molecules of endothelial cells . Hence, an increase in NEU1 may cause chronic inflammation, resulting in the initiation and progression of several diseases such as cancers, rheumatoid arthritis, and atherosclerosis. Therefore, an increase in NEU1 expression with aging can be a risk factor for such diseases in elderly people. In our study, we show an increase in NEU1 expression in skin fibroblasts during the process to senescence. NEU1 has substrate specificity for α2-3 sialyl linkage . Indeed, in our study (Fig. 1c and d and Fig. 5b) and our previous report , a decrease in α2-3 sialyl linkage with aging was indicated from the decrease in the binding of MAL-II. Therefore, NEU1 and also NEU1-recognizing glycans, such as α2-3 sialyl linkages, may be attractive targets for the prevention and therapy of age-related skin diseases, cosmetic skin alterations, and chronic wounds caused by delayed healing in elderly people.
In this study, we showed that raft localization of CD44 after TGF-β1 stimulation was reduced by desialylation with increased sialidase during the process to senescence, leading to the inhibition of myofibroblast differentiation in skin fibroblasts. Our results suggest that regulation of sialylation in fibroblasts may be an attractive strategy for the prevention and regenerative therapy of age-related skin diseases, cosmetic skin alterations, and chronic wounds caused by delayed healing in elderly people.
Bovine serum albumin
Erythrina cristagalli agglutinin
Epidermal growth factor receptor
Extracellular signal-regulated kinase
Fluorescence-activated cell sorting
Maackia amurensis lectin II
Mitogen-activated protein kinase
Mean fluorescence intensity
Polymerase chain reaction
Population doubling level
Transforming growth factor
Wisteria floribunda agglutinin
α-smooth muscle actin
This work was supported by JSPS KAKENHI Grant Numbers 25670182 and 26671017 (Grant-in-Aid for Challenging Exploratory Research) and 16 K08263 (Grant-in-Aid for Scientific Research (C)), and by grants from the Ministry of Health Labor and Welfare (H25-saisei-shitei-013).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
NS designed and performed the experiments, analyzed data, and wrote the paper. YI wrote the paper. MT designed the experiments and wrote the paper. All authors read and approved the final version of the manuscript.
The authors declare that they have no competing interests.
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