Abstract
Background/Aim: Skin regeneration is the intrinsic ability to repair damaged skin tissues to regaining skin well-being. Processes of wound healing, a major part of skin regeneration, involve various types of cells, including keratinocytes and dermal fibroblasts, through their autocrine/paracrine signals. The releasable factors from keratinocytes were reported to influence dermal fibroblasts behavior during wound-healing processes. Here, we developed a strategy to modulate cytokine components and improve the secretome quality of HaCaT cells, a nontumorigenic immortalized keratinocyte cell line, via the treatment of cordycepin, and designated as cordycepin-induced HaCaT secretome (CHS). Materials and Methods: The bioactivities of CHS were investigated in vitro on human dermal fibroblasts (HDF). The effects of CHS on HDF proliferation, reactive oxygen species-scavenging, cell migration, extracellular matrix production and autophagy activation were investigated by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide cell viability assay, dichloro-dihydro-fluorescein diacetate, the wound-healing assay, reverse transcription polymerase chain reaction and immunofluorescent microscopy. Finally, Proteome Profiler™ Array was used to determine the composition of the secretome. Results: CHS induced fibroblast proliferation/migration, reactive oxygen species-scavenging property, regulation of extracellular matrix synthesis, and autophagy activation. Such enhanced bioactivities of CHS were related to the increase of some key cytokines, including C-X-C motif chemokine ligand 1, interleukin 1 receptor A, interleukin 8, macrophage migration-inhibitory factor, and serpin family E member 1. Conclusion: These findings highlight the implications of cordycepin alteration of the cytokine profile of the HaCaT secretome, which represents a novel biosubstance for the development of wound healing and skin regeneration products.
Skin serves as the body’s first barrier against all harmful substances from the environment, protecting the body from chemicals, radiation, microbes, and mechanical forces. It also prevents internal fluid loss and importantly regulates body temperature (1, 2). Thus, the condition of the skin is critically important for human well-being. Histologically, the skin can be divided into three vertically aligned layers: the epidermis, the dermis, and the hypodermis. However, when skin is damaged and wounded, cutaneous wound healing plays a major part in skin regeneration, mainly processed through the dynamic sequences of synergistic interaction of various cell types localized within both the epidermis and dermis. Among them, the interaction between keratinocytes and fibroblasts plays the most crucial role in such healing processes. Despite being separated by basement membrane with minimal direct cell-to-cell contact, keratinocyte–fibroblast interaction is mostly carried on by releasable factors in an autocrine/paracrine manner to regulate cell proliferation and extracellular matrix (ECM) remodeling (3). Keratinocytes were also widely reported to influence and regulate the expression of dermal fibroblast genes via keratinocyte-derived soluble factors found in its set of secreted proteins referred to as the secretome (3-6). Therefore, the study of the effects of keratinocyte secretome and the factors it contains on dermal fibroblasts should be beneficial for the development of novel therapies to maintain skin integrity.
Autophagy, a lysosome-dependent recycling mechanism, plays an important role in wound healing, especially in dermal fibroblasts. In general, autophagy plays a role in the aging process and skin homeostasis under harmful conditions generated by external stimuli to repair cellular machineries (7). Autophagy is also activated during proliferative and remodeling phases of dermal fibroblasts, and helps prevent oxidative stress, promoting their survival and maintaining their normal functions (8). Autophagy has also been reported to regulate ECM and matrix metalloproteinase (MMP) mechanisms. Microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A), the key autophagic marker, was found to be up-regulated in vivo, and highly accumulated at the margin of the wound during the healing processes in rat models (9). Defective autophagic activity of fibroblasts was found to induce the degradation of ECM components via the expression of MMP1 and MMP3 (8, 10). Thus, autophagy might be a relevant cellular mechanism for skin regeneration, and it may be regulated by keratinocyte secretome factors.
However, the use of primary keratinocytes as a source of secretome may be problematic in both research and large-scale production due to their short lifespan and limited workable passages. HaCaT, spontaneously immortalized human keratinocytes, are a renowned substitute for primary keratinocytes in skin research fields (11). They are a nontumorigenic monoclonal cell line that is not only capable of continuing proliferation, but also exhibits almost identical genotype and phenotype to those of primary keratinocytes, including contact inhibition and anchorage-dependent growth in cell culture, the capability to form well-constructed epidermis in mouse model transplantation (12, 13), and the ability to produce keratinocyte-derived soluble factors/cytokines (14). Colombo et al. (15) found that HaCaT produced cytokines as expected in primary keratinocytes, with some slight differences in expression of the C-C motif chemokine ligand family. Despite such differences, the secretome of HaCaT is sufficient to be used in studies in place of primary keratinocyte secretome. An additional induction/stimulation protocol should be developed and applied to enhance the production of factors/cytokines and improve the quality of the HaCaT secretome.
Cordycepin (3′-deoxyadenosine), a unique nucleoside analog that is naturally found in Cordyceps militaris, has been reported to exhibit a wide-range of biological activities, in particular regulatory effects on human cytokines production and release, such as eotaxin, fibroblast growth factor 2 (FGF2), insulin like growth factor 2 (IGF2), interleukin (IL)-12, and interferon gamma (16). Cordycepin also increased the expression of type 2 helper (Th2) T-cell cytokines, IL4, and IL10 in mouse splenocytes (17). IL10, IL1 receptor antagonist (IL1RN) and transforming growth factor beta 1 (TGFB1) were up-regulated by cordycepin treatment in lipopolysaccharide-activated macrophages (18). Therefore, the cytokine profile of the HaCaT secretome might be altered and its quality improved by cordycepin treatment.
From the above, we hypothesized that HaCaT cells may be influenced by cordycepin treatment during culture and yield a secretome with different factor/cytokine compositions that consequently has a distinct biological influence regarding skin regeneration and wound healing of human dermal fibroblasts (HDFs). Therefore, in this study, we collected cordycepin-induced HaCaT secretome (CHS), and explored its biological effects on HDFs, including antioxidant activity, extracellular matrix mechanisms, autophagy activation, and wound-healing capacity. We also further investigated composition changes of CHS, which may be responsible for such different biological effects compared to the normal HaCaT secretome.
Materials and Methods
Cell line and cell culture. HaCaT cells and HDFs were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium, containing 10% (v/v) heat-inactivated fetal bovine serum, 1% (v/v) nonessential amino acids, 1% (v/v) L-glutamine and 1% (v/v) penicillin-streptomycin and were used for cell culture. Cells were incubated in a humidified incubator in atmosphere with 5% CO2 at 37°C for 2 days or until cells reached 80% confluence.
Collection of normal HaCaT secretome (NHS) and CHS. The toxicity of cordycepin to HaCaT cells was determined by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method. HaCaT cells were cultured in a 96-well plate and treated with different concentrations (1.25, 2.5, 5, 10, 20 and 50 μM) of cordycepin (Merck, Darmstadt, Germany) for 24 h. Then MTT solution was added to cell culture medium at 0.5 mg/ml final concentration and cells were incubated for another 3 h at 37°C in the dark. The medium was removed, and the formazan crystals were solubilized in dimethyl sulfoxide. The optical density (OD) was then measured at 570 nm, using a microplate reader (BMG Labtech, Ortenberg, Germany). The OD at 570 nm under control conditions (no cordycepin added) was taken as representing 100% viability. The concentration of cordycepin that did not reduce HaCaT cell viability was then used for the collection of CHS.
HaCaT cells were cultured in T-175 flasks until reaching 80% confluence. The cells were then treated with a sub-toxic dose of cordycepin (2.5 μM) for 24 h. The medium was discarded, and cells were washed twice with phosphate-buffered saline (PBS). They were then incubated overnight in Dulbecco’s modified Eagle’s medium without FBS and phenol red to allow the cells to secrete extracellular factors/cytokines to the medium, to produce the CHS. To collect NHS, the protocol described above was followed without the cordycepin treatment step. Both NHS and CHS were collected by centrifugation at 4,500×g for 10 min to remove residual HaCaT cells and was kept at −80°C.
Treatment of HDFs with secrotome. The effect of NHS and CHS on the viability of dermal fibroblast cells was determined by the MTT colorimetric method. HDFs were cultured in a 96-well plate and treated with different concentrations of NHS and CHS prepared by dilution in Dulbecco’s modified Eagle’s medium without FBS and phenol red (0, 6.25, 12.5, 25, 50 and 100%) for 24 h. Cell viability was then determined by MTT assay as described above. The OD at 570 nm under control conditions (0% of NHS or CHS) was taken as 100% viability.
Detection of reactive oxygen species (ROS) in HDFs by dichloro-dihydro-fluorescein diacetate (DCFH-DA) assays. HDFs were cultured in a black 96-well plate and treated with different concentrations of either NHS or CHS (6.25, 12.5 and 25%) and 5 mM N-acetyl cysteine (NAC) as positive control for 24 h. Then the cells were washed with PBS and incubated with 10 μM DCFH-DA at 37°C for 1 h in the dark and washed twice with PBS again. The cells were then challenged with 1 mM of hydrogen peroxide (H2O2) at 37°C for 10-30 min. The fluorescence intensity of DCF was measured using a Varioskan™ LUX multimode microplate reader (Thermo Scientific, Waltham MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Results were expressed as percentage of that of the control (0% CHS/NHS) (19).
Wound-healing assays using HDFs. HDFs were cultured in a 6-well plate until reaching 90% confluence of the cell monolayer. A wound gap was created by directly scratching the cell monolayer with a sterile 200 μl autopipette tip, washed with PBS twice to remove excess cells. Cells were then treated with 12.5% of either NHS or CHS to be compared with the control condition without secretome. Images of the cell monolayer were taken under a microscope at 0, 24 and 48 h. The wound-healing efficiency was quantitatively assessed by measuring the number of dermal fibroblasts migrating into the scratched area (20). The experiment was performed in triplicate and three images of each condition were analyzed.
Reverse transcription polymerase chain reaction (RT-PCR). HDFs were treated with different concentrations of CHS (0, 6.25, 12.5, 25, 50 and 100%) for 24 h, and collected. Total RNA was extracted using NucleoSpin RNA kit (Macherey-Nagel, Dueren, Germany), according to the manufacturer’s protocol. Then 1 μg of RNA was converted to complementary DNA using ReverTra Ace™ qPCR RT Kit (Toyobo, Osaka, Japan). RT-PCR was carried out in a C1000 Touch Thermocycle (BioRad, Hercules, CA, USA) for 35-40 cycles using 2x Taq Master Mix (Vivantis Technologies, Selangor, Malaysia) with specific primers as listed in Table I. PCR products were separated by agarose gel electrophoresis and visualized under UV after staining with RedSafe™ Nucleic Acid Staining Solution (iNtRON Biotechnology, Gyeonggi-do, Republic of Korea). The relative expression level of each target gene was quantified by normalization with the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using NIH Image J 2.0 software (National Institutes of Health, Bethesda, MA, USA).
Primer sets used for reverse transcription–polymerase chain reaction for gene-expression analysis.
Immunofluorescence microscopy. HDFs were cultured on coverslips and treated with 12.5% of NHS and CHS for 24 h. The cells were then washed twice in PBS, fixed with 4% paraformaldehyde in PBS at room temperature for 30 min, washed three times for 5 min each, and permeabilized with 0.2% Triton X-100 in PBS for 20 min. After blocking with 10% FBS in PBS for 1 h, the cells were incubated with antibody to marker of proliferation Ki-67, collagen type I alpha 1 chain (COL1A1) or MAP1LC3A (Merck) overnight at 4°C. They were then washed three times in PBS and incubated in fluorescein isothiocyanate-conjugated anti-rabbit IgG (Merck) for 1 h. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA), and the stained cells were observed under a ZOE Fluorescent Cell Imager (BioRad). Images were analyzed using NIH Image J 2.0 software (21).
NHS and CHS composition determined by cytokine array. The presence of cytokines within NHS and CHS was determined using Proteome Profiler™ Array (catalog no. ARY005B; R&D Systems, Minneapolis, MN, USA), according to the manufacturer’s protocol with adjusted 15-min exposure. The dot densities both between and among arrays were measured, with the background signals subtracted, and normalized with the referenced spots using NIH Image J 2.0 software.
Statistical analysis. Statistical analyses were performed using SPSS Statistics, version 16.0 (SPSS Inc., Chicago, IL, USA). All experiments were performed in triplicate and repeated three times. Data are expressed as the mean±standard deviation. Significant differences among treatments were determined by one-way analysis of variance, followed by Tukey’s test at p<0.05.
Results
The effects of NHS and CHS on HDF viability. HaCaT cells were cultured with different concentrations of cordycepin (from 1.25-50 μM), and the cytotoxicity of cordycepin was then assessed by MTT assay. Cordycepin at 2.5 μM was found to be to the optimal sub-toxic concentration to be used in CHS preparation since cell proliferation was increased and no significant change in cell morphology was observed (Figure 1A). After NHS and CHS preparation, both types secretome were applied to HDF cultures at different concentrations (0-100% by volume) to evaluate their effects on HDF cell viability. While HDFs cultured in different concentrations of NHS showed no difference in cell viability, the proliferation of HDFs cultured in different concentrations of CHS increased in a dose-dependent manner (Figure 1B), reflecting the beneficial effects of CHS on HDFs. The expression of MKI67, a marker of proliferation, was also assessed. HDFs cultured with CHS showed higher levels of MKI67 mRNA and protein expression than those cultured with NHS and without any secretome (Figure 1C and D), which was conformed with previously monitored cell viability. Hence, CHS was obviously more beneficial for HDFs and its skin cell rejuvenation effects were evaluated in further experiments.
The determination of optimal cordycepin concentration for use in cordycepin-induced HaCaT secretome (CHS) production and effect of CHS on human dermal fibroblast (HDF) proliferation. A: 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) cell viability assay and microscopic images of HaCaT cells in response to cordycepin. Cordycepin at 2.5 μM was the optimal concentration since it promoted HaCaT proliferation without any morphological alteration. Scale bar=200 μm. Significantly different from the control (0 μM) at: *p<0.05 and **p<0.01. B: MTT cell viability assay of HDFs in response to normal HaCaT secretome (NHS) and CHS. While NHS had no impact on HDF proliferation, CHS promoted HDF proliferation in a dose-dependent manner. Significantly different from the control (0%\) at: *p<0.05 and **p<0.01. C: Densitometric analysis for mRNA expression of MKI67. It was highly up-regulated in NHS- and CHS-treated HDFs compared to those without any secretome. Notably, CHS-treated HDFs showed the highest MKI67 expression. MKI67 expression in CHS-treated HDFs was also significantly higher than that of NHS-treated HDFs. D: Representative fluorescence-microscopy images showing the highest MKI67 fluorescence intensity was in CHS-treated HDFs. Scale bar=100 μm. Significantly different at: **p<0.01 from control, non-treated HDFs; #p<0.05 and ##p<0.01. The experiments were performed in triplicate.
CHS exhibited superior ROS-scavenging property. The overproduction and accumulation of ROS, referred to as free radicals or oxidants, can lead to oxidative stress that can cause skin damage and aging (22). To evaluate the effectiveness of CHS as an antioxidant, HDFs were cultured with different concentrations of NHS or CHS (6.25, 12.5 and 25%) and 5 mM of NAC, a conventional ROS scavenger, and were then challenged with 1 mM H2O2 to stimulate mitochondrial ROS generation. The antioxidant efficiency of CHS was determined by scavenging the hydroxyl radical (•OH) visualized by DCF relative to the fluorescent intensity with NAC. Prior to H2O2 challenge, CHS was found not to increase relative DCF fluorescence, indicating how it did not cause any oxidative stress to HDFs. After challenging with H2O2, CHS was also found to significantly reduce relative DCF fluorescence at a level comparable to that of NAC, while no statistical significance was found with NHS-treated HDFs (Figure 2A). The expression levels of a number of key genes involved in intracellular ROS-scavenging were also measured. The mRNA expression levels of superoxide dismutase 1 (SOD1), glutathione peroxidase (GPX1) and catalase (CAT) were found to be elevated in dose-dependent manner in HDFs cultured with CHS (Figure 2B).
Cordycepin-induced HaCaT secretome (CHS) led to up-regulation of intracellular reactive oxygen species (ROS)-scavenging pathways. A: Quantitative analysis of dichloro-dihydro-fluorescein diacetate assay of normal HaCaT secretome (NHS)- and CHS-treated human dermal fibroblasts (HDFs) showing ROS-scavenging capability comparable to that of the antioxidant N-acetyl cysteine (NAC). Significantly different at p<0.01 from: **control, non-treated HDFs; ##non-treated HDFs with H2O2 stimulation. B: Densitometric analysis for the expression of genes involved in intracellular ROS-scavenging pathways. CHS promoted the expression of superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1) and catalase (CAT). Significantly different from the control (0%) at: *p<0.05 and **p<0.01. The experiments were performed in triplicate.
CHS effectively promoted wound healing in HDFs. Skin regeneration is associated with the restoration of dermis caused by migration and proliferation of fibroblasts (23). In vitro wound-healing assays were performed by scratching the HDF monolayer with a sterile autopipette tip to mimic cutaneous skin damage, then treating with 12.5% by volume of NHS or CHS. Images of HDF migration into the scratch area were captured and the number of cells in the scratch area was counted and compared to the control condition without secretome treatment. Both NHS and CHS were found to elicit faster cell migration than the control condition. As expected, HDFs treated with CHS exhibited the fastest cell migration, implying its greater wound-healing potential (Figure 3).
Cordycepin-induced HaCaT secretome (CHS) promotes wound healing in human dermal fibroblasts (HDFs) compared to normal HaCaT secretome (NHS) and control without any secretome. A: Representative images of wound-healing assays. Scale bar=200 μm. B: Quantitative analysis of migrating HDF cells in the scratched area in response to NHS and CHS treatment at 0, 24 and 48 h post wounding. Significantly different at: **p<0.01 from control, non-treated HDFs; #p<0.05 and ##p<0.01. The experiments were performed in triplicate.
CHS regulated ECM components in HDFs. HDFs produce several ECM proteins to nourish and support the foundation of skin. Collagen and deposition of other ECM components are required to maintain skin condition and facilitate wound repair. To monitor whether CHS affected the production and degradation of ECM components, the mRNA expression of genes encoding ECM components collagen type I alpha 1 chain (COL1A1), COL1A2, COL3A1 and elastin, as well as those encoding two enzymes, MMP1 and MMP3, was investigated in HDFs cultured with CHS. As shown in Figure 4A, the expression of the genes for ECM components was up-regulated, while the expression of both MMPs, responsible for the degradation of collagen and elastin, respectively, were down-regulated, indicating that CHS has the capability to promote ECM production and reduce the tendency for ECM degradation. Additionally, most of these genes exhibited a dose-dependent response to the concentration of CHS.
Cordycepin-induced HaCaT secretome (CHS) regulated the expression of extracellular matrix-related elements at both the transcriptional and translational levels. A: Densitometric analysis for mRNA expression of extracellular matrix-related genes. The mRNA expression of collagen type I alpha 1 chain (COL1A1), collagen type I alpha 2 chain (COL1A2), collagen type III alpha 1 chain (COL3A1) and elastin (ELN) were up-regulated. In contrast, the mRNA expression of matrix metalloproteinases MMP1 and MMP3 was down-regulated. Expression of most genes was dose-dependent manner. Significantly different from control, non-treated HDFs at: *p<0.05 and **p<0.01. B. Representative fluorescence-microscopy images showing fluorescence of COL1A1 protein. Scale bar=100 μm. DAPI: 4′,6-Diamidino-2-phenylindole. The greatest fluorescence was present in CHS-treated HDFs. However, the difference between NHS- and CHS-treated HDFs was not statistically significant. **Significantly different from the control (0%) at p<0.01. The experiments were performed in triplicate.
To further confirm the effect on ECM regulation at the protein level, the expression of COL1A1 was compared in HDFs treated with 12.5% by volume of NHS or CHS or without any secretome via immunofluorescence microscopy. The highest production of COL1A1 was found in HDFs cultured with CHS, as expected (Figure 4B).
CHS activated autophagy in HDFs. Autophagy, an intrinsic process of cellular self-digestion, plays an important role in skin response under stress conditions to maintain cellular homeostasis (24). To demonstrate how CHS modulates autophagy in HDFs, the mRNA expression of genes encoding several proteins crucial in autophagy were investigated in CHS-treated HDFs. The expression of sequestosome 1 (SQSTM1), autophagy-related 5 (ATG5), beclin 1 (BECN1) and MAP1LC3A tended to increase with increasing CHS concentration. Among them, the mRNA expression of MAP1LC3A was drastically up-regulated in a dose-dependent manner (Figure 5A). Expression of MAP1LC3A protein was correspondingly significantly increased in CHS-treated HDFs compared to those treated with NHS and without any secretome using immunofluorescence microscopy. Both transcriptional and translational evidence emphasized the autophagy activation property of CHS in HDFs.
Cordycepin-induced HaCaT secretome (CHS) activated the expression of autophagy-related genes in human dermal fibroblasts (HDFs) at both the transcriptional and translational levels. A: Densitometric analysis for mRNA expression of autophagy-related genes indicated the up-regulation of sequestosome 1 (SQSTM1), autophagy related 5 (ATG5), beclin 1 (BECN1) and microtubule-associated protein 1 light chain 3 alpha (MAP1LC3A). The most obvious up-regulation was found in LC3 expression. Significantly different from the control (0%) at: *p<0.05 and **p<0.01. B: Representative fluorescence-microscopy images showing the highest fluorescence intensity of MAP1LC3A protein was in CHS-treated HDFs. Scale bar=100 μm. Significantly different at: **p<0.01 from control, non-treated HDFs; #p<0.05 and ##p<0.01. The experiments were performed in triplicate.
Cordycepin altered the composition of the HaCaT secretome, leading to superior biological effects of CHS on HDF. Based on the biological activity of HaCaT secretome in HDFs in this study, CHS exhibited predominantly beneficial effects on skin cells. The determination of the differences in composition between CHS and NHS helped shed light on the superior skin cell benefits. The levels of cytokines present in NHS and CHS were detected using Proteome Profiler™ Array. There were six types of cytokines found in HaCaT secretome, both NHS and CHS, including C-C motif chemokine ligand 2 (CCL2), chemokine C-X-C motif ligand 1 (CXCL1), IL1RN, IL6, CXCL8 (also known as IL8), macrophage migration-inhibitory factor (MIF) and serpin family E member 1 (SERPINE1). Among them, the levels of CXCL1 and IL1RN were significantly higher in CHS. CXCL8, MIF and SERPINE1 were also found to be slightly increased by cordycepin induction Additionally, CCL2 and IL6 were found at minimal levels compared to other cytokines in both NHS and CHS (Figure 6).
Densitometric analysis for the determination and comparison of cytokine components of normal HaCaT secretome (NHS) and cordycepin-induced HaCaT secretome (CHS). 1: C-C motif chemokine ligand 2 (CCL2); 2: chemokine C-X-C motif ligand 1 (CXCL1); 3: interleukin 1 receptor antagonist (IL1RN); 4: interleukin 6 (IL6); 5: CXCL8; 6: macrophage migration-inhibitory factor (MIF); 7: serpin family E member 1 (SERPINE1). All were found in both NHS and CHS but among these, CXCL1, IL1RN, ICXCL8, MIF and SERPINE1 were found to be at higher levels in CHS.
Discussion
Skin regeneration is the innate ability of the skin to restore damaged compartments of skin tissue to maintain skin function and integrity (23). Wound healing, a vital process of skin regeneration, requires the collaborative actions of many cell types in phases of hemostasis, inflammation, re-epithelialization, and contraction/tissue remodeling, which are governed by cytokines and matrix signals at the wound site (25). Among all cell lineages, the interaction between skin-resident cells during the inflammatory phase of the repair/regeneration process, primarily keratinocytes and dermal fibroblasts, is required in order for cell proliferation and maturation to assist wound healing (26). In addition, keratinocytes and fibroblasts coordinate their activity to restore normal tissue homeostasis after injury through double paracrine signaling loops known as crosstalk or dynamic reciprocity, mostly mediated by secretable substances. Several studies have revealed the regulatory involvement of keratinocyte-released factors in the expression of genes in dermal fibroblasts. The co-culture of fibroblasts and keratinocytes was reported to up-regulate 243 genes and down-regulate 100 genes in dermal fibroblasts compared to their independent culture (27), including genes responsible for ECM synthesis (4, 28). Thus, the investigation of the impact of the keratinocyte secretome and its constituent components on dermal fibroblasts may aid in the development of innovative therapies to maintain skin integrity. However, variable effects of keratinocyte-released factors were also reported in different studies, depending on the source and origin of keratinocytes. Sato et al. (3) found that the keratinocyte secretome greatly enhanced the production of lipid mediator prostaglandin E2, involved in the wound-healing response in fibroblasts due to the induction of cyclo-oxygenase 2 mRNA expression by pro IL1α released from keratinocytes (3). Ghaffari et al. (4) found that pro IL1 in keratinocyte secretome reduced pro-a1(I) collagen expression and increased MMP1 expression in dermal fibroblasts at mRNA and protein levels to prevent fibrotic conditions. Bukowska et al. (6) reported that the viability of dermal fibroblasts was stimulated by keratinocyte secretome collected from forkhead box N1 (FOXN1)-active keratinocytes. Therefore, the possibility to improve the quality of keratinocyte secretome via adding regulators is worth exploring and may prove to be beneficial for further applications.
In this study, we used HaCaT cells as the source of secretome to relieve the concerns associated with using primary keratinocytes, such as donor-to-donor variability in growth characteristics and in-vitro responses, different plating efficiencies, the short lifetime in culture, and changes in proliferation and differentiation characteristics with increasing number of passages, which may complicate experimental data interpretation (15). We applied cordycepin to HaCaT culture aiming to alter the composition of released cytokines contained in the secretome. The results suggest that cordycepin treatment might affect the biological properties of HaCaT secretome in many aspects. CHS significantly induced the proliferation and migration of dermal fibroblasts, indicating how CHS potentially facilitates dermal restoration through migration and proliferation of fibroblasts to close the wound site and re-establish the skin’s barrier function. Accordingly, the increase in proliferation and migration is in agreement with literature that the secretome influences the success of tissue healing during skin regeneration (23, 29, 30). Besides the increase of cell viability, CHS can potentially regulate ECM synthesis in HDFs. The up-regulation of collagen types 1 and 3, and elastin was reported to support the production of such ECM components for deposition in the wound in a precise reticular pattern, resulting in better ECM restoration (31, 32). The presence of CCL2, and the up-regulation of both chemokine CXCL1 and MIF in the CHS may be responsible for the increase of cell viability, migration and ECM production. CCL2, a powerful macrophage chemoattractant, improved wound healing in mice with diabetes caused by streptozotocin through generating growth factors that promoted cell proliferation and protein synthesis. CCL2 therapy boosted the generation of endothelial progenitor cells at wound sites and accelerated wound closure rates, neovascularization, and ECM synthesis, which ultimately aided diabetic cutaneous wound healing (33). CCL2 was also reported to promote pulmonary fibroblast proliferation and migration (34). CXC chemokines, such as CXCL1, are recognized for their ability to directly induce angiogenesis, allow cell migration into a wound, and provide the necessary metabolic support for the rapidly multiplying cells in a wound (35). Interestingly, cordycepin itself was also reported to bind and activate adenosine receptor, which subsequently induced dermal fibroblast migration and promoted wound healing in vitro via the stimulation of WNT/β-catenin signaling (36). Such ability of cordycepin to bind and activate adenosine receptor may possibly lead to the modulation of HaCaT secretome since the activation of adenosine A2 receptor was found to enhance the expression of CXCL1 in keratinocytes (37). MIF, a key pro-inflammatory cytokine that integrates the immune, neuronal and endocrine systems, is known to regulate the synthesis and release of other cytokines and interferons during wound-healing processes (38). Keratinocyte MIF production in vivo and in vitro was induced by certain uncommon environmental influences such as UV radiation. UVB exposure was demonstrated to increase MIF protein level in normal human keratinocytes (39) and up-regulate cutaneous MIF mRNA expression in a mouse model (40). The up-regulation of MIF facilitated the adaptation to a hostile environment by inhibiting p53-mediated gene activation and apoptosis (41, 42). MIF has also been proven to enhance the proliferation and migration of HDFs (43). In contrast, the expression of MMP1 and MMP3 was down-regulated by CHS, promoting the availability of ECM components during wound healing. Since MMP1 and MMP3 production was reported to be induced by IL1β (44, 45), the enhancement of IL1RN level in CHS might lead to the down-regulation of MMPs. IL1RN is a naturally occurring inhibitor of IL1β by competitively binding to IL1β receptor. As a result, IL1β bioactivities, such as the activation of MMP formation, are also reduced, but this does not trigger any intracellular agonist effects. (46, 47).
ROS also contribute to the wound-healing process. When ROS levels are low, they help to prevent infections and speed up wound healing by producing cell-survival signaling, but when their levels are high, they cause oxidative stress, which damages cells and promotes an inflammatory state (48). If redox imbalance occurs due to the levels of ROS exceeding the capacity of endogenous antioxidants to scavenge them, the healing processes are interrupted, therefore the focus on management of ROS and antioxidant levels to control redox equilibrium is essential (49). Here, we reported that the antioxidant potential of CHS was comparable to that of NAC and was regulated through the up-regulation of genes involving in intracellular ROS-scavenging mechanisms, including SOD1, GPX1 and CAT. From literature, IL1β has been shown to be responsible for •NO and O•2 radical production and disturbed the antioxidant enzyme system in bovine chondrocytes (50), leading to a delayed increased of GPX and a decrease of CAT activity, resulting in an accumulation of H2O2 in mitochondria. Therefore, the high level of IL1RN present in CHS might possibly inhibit ROS production and accumulation in HDFs caused by intracellular IL1β.
As one of the vital mechanisms in both fibroblast proliferation and ECM remodeling, autophagy also plays a significant role in wound healing. We found that CHS up-regulated genes involving in autophagy including SQSTM1, ATG5, BECN1, and MAP1LC3A, indicating that acceleration of wound healing partially involves with autophagy modulation, which might be mediated by CXCL1 and MIF. According to some reports, CXCL1 is crucial in controlling metastasis and autophagy. With CXCL1 overexpression, the expression levels of autophagy-related proteins were found to be noticeably increased in breast cancer cells, which subsequently promoted autophagy and cell migration (51, 52). Additionally, MIF was stated to activate autophagy via the AMP-activated protein kinase/mammalian target of rapamycin signaling pathway, consequently increasing the expression of proteins associated with autophagy (such as LC3BI/LC3BII, BECN1, and ATG5) to help protecting mesenchymal stem cells from apoptosis (53). The increase of autophagic flux also promotes ECM production (54, 55) and inhibits MMP expression (56). Besides the wound-healing perspectives, considering their lengthy lifespan, HDFs are vulnerable to both intrinsic and extrinsic harm. Skin damage and aging have been linked to changes in fibroblast autophagic flux (57). Additionally, the microenvironment may be impacted by autophagy and its age-related dysfunction in chronic, UV-induced, and premature aging phenotypes (58). Dysregulation of the proteasome and autophagy-induced aging of skin is thought to be responsible for the loss of proteostasis and to cause skin deterioration and aging. Tashiro et al. (59) reported that the fibroblast treatment with pepstatin A (leu/pep), a significant lysosomal protease inhibitor, caused autophagy disruption and resulted in decreased levels of type I procollagen, hyaluronan, and elastin, as well as elevated levels of MMP1 which could potentially lead to the loss of dermal integrity. From the genes involved in autophagy which were up-regulated by CHS treatment, CHS has potential anti-aging properties and may help resolve impaired autophagic flux, reduction of ECM components and collapse of dermal structure. Although autophagy is important in skin regeneration and wound healing, it is still debatable whether it is advantageous for the treatment of skin problems because autophagic imbalance can give rise to some undesirable skin concerns. Hypertrophic scar (HS) is a serious skin fibrotic disease characterized by hypercellularity and excessive ECM component deposition, resulting in irregularly shaped nodules, congestive appearance, and an uneven skin surface, accompanied by paresthesia such as itching and pain (60). It was reported to involve excessive autophagy activation. Shi et al. (61) found that autophagosome as well as LC3 protein were elevated in HS tissues, with the involvement of BCL2 apoptosis regulator-xL, and the knockdown of LC3 protein improved the appearance of fibrotic scar in rabbit. Recently, Liu et al. (62) investigated dynamic alterations of autophagy during HS development and reported that autophagic activity increased in the initial and stabilized stage of HS formation. In addition, treatment with autophagy inhibitors potentially prevented the development of HS in the rabbit model, whereas autophagy inducers caused the reverse effect. However, it should be noted that mechanisms of autophagy may work differently in HS and normal skin, and a number of studies have pointed out how essential autophagy is to skin wellbeing. Therefore, the development of a novel strategy to indicate and maintain well-balanced autophagic activity in skin is the key for autophagy-related therapeutic applications.
Considering other components enhanced in CHS, CXCL8, and SERPINE1 may contribute to the superior effects of CHS in HDF regeneration. CXCL8 is a chemoattractant cytokine that was reported to influence dermal fibroblast phenotypic behavior. CXCL8 induced a migratory phenotype in HDFs with a lack of focal adhesion (63). Thus, CXCL8 mediates the migration of fibroblasts and facilitates wound healing (64) Lastly, SERPINE1 is an important regulator of the pericellular proteolytic and fibrinolytic cascades in wound-healing processes. Although, there was no obvious evidence how this cytokine assists fibroblasts in wound healing, SERPINE1 who shown to take part in keratinocyte adhesion and migration during wound healing (65).
Notably, the co-culture of human epidermal keratinocyte and dermal fibroblast was reported to induce prostaglandin E2 production and cyclo-oxygenase 2 activity due to the mediation of pro-IL-1α released from keratinocytes (3). Such cell–cell interaction was proposed to be involved in wound healing and ECM organization after injury. In contrast, pro-IL1α was not detected in CHS in this study, possibly due to the absence of dermal fibroblasts in the culture environment during CHS collection. Therefore, HaCaT might independently produce a secretome with a different cytokine profile from that in the co-culture scenario. The effect of keratinocyte secretome on collagen synthesis by dermal fibroblasts was found to be inconsistent. Granulocyte colony-stimulating factor inhibited collagen expression in dermal fibroblasts and was identified in a co-culture system (66). The absence of granulocyte colony-stimulating factor in CHS obtained from stand-alone HaCaT cultivation might be due to the lack of HDF-stimulation signals indicating excessive collagen production.
Although the findings of this study elucidated the possible biological influences of CHS on HDFs to assist skin regeneration and wound healing, some future perspective should be mentioned. To illustrate a clearer regulatory network of CHS, the effects of such secretome on other cell types involved in wound healing, such as immune cells and endothelial cells, still need to be evaluated. Specific study focusing on particular cytokines that were up-regulated by cordycepin such as CXCL1 and IL1RN would also be beneficial to fill the gaps in the big picture of the system. Ultimately, further in vivo study, is necessary to bridge the gap of the lack of correlation between in vitro and clinical situations and make the translation of this finding to therapeutic applications more practical.
Conclusion
CHS collected from HaCaT cultured in cordycepin possessed the capability to assist skin regeneration and wound healing through its bioactivities on dermal fibroblasts, including induction of cell proliferation and migration, ROS-scavenging, regulatory effects on ECM production, and autophagy modulation. The exceptional bioactivity of CHS may be related to the positive alteration of some secretome components, including CXCL1, IL1RN, CXCL8, MIF and SERPINE1. Therefore, the cordycepin treatment strategy was efficient at improving the quality of HaCaT secretome and has potential implications in the further improvement of skin regeneration in advanced novel therapies (Figure 7).
Schematic illustration hypothesizing the possible biological activity of cordycepin-induced HaCaT secretome (CHS) in promoting skin regeneration. ATG5: Autophagy-related 5; BECN1: beclin 1; CAT: catalase; CCL2: C-C motif chemokine ligand 2; COL1A1: collagen type I alpha 1 chain; COL1A2: collagen type I alpha 2 chain; COL3A1: collagen type III alpha 1 chain; CXCL: chemokine C-X-C motif ligand; ELN: elastin; GPX1: glutathione peroxidase 1; IL: interleukin; IL1RN: interleukin 1 receptor antagonist; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MIF: macrophage migration-inhibitory factor; MMP: matrix metalloproteinase; SERPINE1: serpin family E member 1; SOD1: superoxide dismutase 1; SQSTM1: sequestosome 1. ↑ Up-regulation; ↓ down-regulation.
Acknowledgements
This work was supported by Agricultural Research Development Agency (ARDA), Suranaree University of Technology (SUT), Thailand Science Research and Innovation (TSRI), and National Science, Research, and Innovation Fund (NSRF) (project code 90464).
Footnotes
Authors’ Contributions
Phongsakorn Kunhorm provided the concept and design of the study, performed experiments, analyzed and interpreted data, and prepared the article for publication. Nipha Chaicharoenaudomrung analyzed and interpreted the data and prepared the article for publication. Parinya Noisa provided the concept and design of the study, analyzed and interpreted data, wrote the article, prepared the article for publication and gave the final publication approval of the article.
Conflicts of Interest
The Authors declare that there are no conflicts of interests.
- Received January 6, 2023.
- Revision received January 27, 2023.
- Accepted February 6, 2023.
- Copyright © 2023 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
