Activation of mTORC1 in fibroblasts accelerates wound healing and induces fibrosis in mice

Xiao Hu1,*, Hanbin Zhang2, Xiaojian Li1, Yeyang Li1, Zhenguo Chen2,*

Abstract

Wound healing is a multicellular process that involves the coordinated efforts of several cell types, including keratinocytes, fibroblasts, and endothelial cells. This process is also regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines. The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth, proliferation, and differentiation. A recent study showed that mTORC1 activation in epithelial cells dramatically enhanced epithelial cell proliferation, migration, and cutaneous wound healing; however, the roles of mTORC1 in fibroblasts during wound healing remain unknown. Here, we generated genetically mutated mice with activated mTORC1 in fibroblasts by conditionally deleting the mTORC1 inhibitor, TSC1. Activation of mTORC1 in fibroblasts significantly increased fibroblastic cell proliferation and contractile α-SMA expression; thus, promoting wound closure. Elevated mTORC1 activity also adversely induced excessive collagen production, leading to excessive scaring and fibrosis. Importantly, both accelerated wound healing and fibrotic phenotypes were largely reversed by the mTORC1 inhibitor, rapamycin. These observations were also replicated in primary human dermal fibroblasts. These results collectively demonstrated that mTORC1 activity in skin fibroblasts was a critical orchestrator in cutaneous wound healing and scarring.

Keywords: Wound healing; mTORC1; Myofibroblast; Fibrosis; Rapamycin
 
Introduction

The ability of skin to act as a barrier is primarily determined by cells that maintain the continuity and integrity of skin, and restore it following full-thickness wounding. One of the most important architects of such processes is the fibroblast. Upon wounding, fibroblasts are activated and become myofibroblasts that express the highly contractile protein, α-smooth muscle actin (SMA).1, 2 On one hand, myofibroblastic cells exhibit contractile properties due to the expression of α-SMA in microfilament bundles or stress fibers. Such properties play a major role in the contraction and maturation of granulation tissue, which are an efficient and important part of wound closure.3, 4 On the other hand, those cells synthesize and deposit extracellular matrix (ECM) components, including collagen and fibronectin, which eventually replace the provisional matrix and provide structural integrity to the wound.5-7 In the final maturation phase, the majority of fibroblastic cells are removed by programmed cell death.4 Thus, insufficient myofibroblast activation and activity prevents normal wound healing, while too many myofibroblasts working for too long commonly leads to excessive scarring or fibrosis.8-10
The mechanistic target of rapamycin (mTOR) is a highly conserved Ser/Thr protein kinase that forms two distinct functional complexes, including mTOR complex 1 (mTORC1) and mTORC2.11, 12 mTORC1 is the sensitive target of rapamycin while mTORC2 is insensitive. mTORC1 integrates diverse signals, including those from nutrients, growth factors, energy, and stresses to regulate cell growth, proliferation, and metabolism. The tuberous sclerosis complex 1/2 (TSC1/2) suppresses mTORC1 by inhibiting Rheb, a RAS-related small GTPase.11, 12 Therefore, the loss of TSC1/2 commonly causes cells and tissues to constitutively activate mTORC1, contributing to the tumor phenotype.
Although mTOR inhibitors have been frequently used for the prevention of allograft rejection in clinical solid organ transplantations, a number of adverse effects have been reported for the mTOR inhibitor, sirolimus (rapamycin), including wound healing complications.13-16 However, a recent study involving patients with moderate wound sizes (3-mm skin biopsy punch wounds) showed that the derivative of sirolimus, everolimus, did not impair wound closure in renal transplant patients.17 Such discrepancies indicated that the roles of mTOR signaling in skin wound healing requires further validation.
Downregulation of the miR-99 family members caused upregulation/activation of the AKT/mTOR signaling pathway, which in turn activated cell proliferation and migration to facilitate wound closure.18 Importantly, epithelial-specific activation of mTORC1 by conditional deletion of the Tsc1 gene in epithelia accelerated wound healing.19 However, the roles of mTORC1 in fibroblasts in wound healing remains unknown. Here, we show that mTORC1 activation in fibroblasts also promoted cutaneous wound healing, while unexpectedly, it induced excessive scarring and fibrosis. Additionally, we show that both of those properties can be largely reversed by rapamycin.

Materials and methods

Mice, husbandry, and genotyping

Tsc1-LoxP mice with the Tsc1 exons 17 and 18 flanked by two loxP sites had been applied in our previous studies.20, 21 Mice carrying a tamoxifen-inducible Cre-recombinase (Cre/ERT) under the control of a fibroblast-specific regulatory sequence from the proα2(I) collagen gene [B6.Cg-Tg(Col1a2-Cre/ERT)] were described previously,22 and were obtained from The Jackson Laboratory (stock no.016237, now 029235). Homozygous Tsc1-LoxP (Tsc1LoxP/LoxP) mice were mated with Col1a2-Cre/ERT mice to yield mice heterozygous for Tsc1-LoxP and heterozygous for Col1a2-Cre/ERT. Such heterozygotes were then bred with mice homozygous for Tsc1-LoxP to obtain mice homozygous for Tsc1-LoxP and heterozygous for Col1a2-Cre/ERT. Those mice carried a fibroblast-specific deletion of Tsc1 (Col1a2-Cre/ERT+, Tsc1LoxP/LoxP), and were used for further experiments. DNA isolated from tail biopsies was used for genotyping, which was performed using PCR as described previously, or following The Jackson Laboratory’s instructions. All animal experiments were approved by the Guangzhou Red Cross Hospital Committee on the Use and Care of Animals, and were performed in accordance with the Committee’s guidelines and regulations. To delete Tsc1, a stock solution of tamoxifen (4-hydroxitamoxifen, Cat.H7904, Sigma-Aldrich) in DMSO (100 mg/ml) was diluted in corn oil to 10 mg/mL. Six-week-old littermate mice with the above-described double mutations were given intraperitoneal injections of the tamoxifen suspension (0.1 mL of 10 mg/mL), or corn oil as control (Tsc1-Ctrl) for 10 days to conditionally delete the Tsc1 gene (termed Tsc1-cKO in this study). Mice homozygous for Tsc1-LoxP without Col1a2-Cre/ERT were also treated with tamoxifen as negative Ctrl mice, and there was no any significant difference in physical activity or wound healing dynamics between Tsc1-Ctrl and negative Ctrl mice, which can exclude the effects of tamoxifen on wound healing. Tsc1 deletion was further confirmed by immunofluorescence and western blotting to detect the protein level of TSC1 and phospho-S6 (S235/S236).

Wound surgery

Wounding experiments were carried out 7 days after the last injection of tamoxifen or corn oil on Tsc1-cKO or Tsc1-Ctrl mice, respectively. Mice were anesthetized by intraperitoneal injection of 10 μL/g chloral hydrate (5%), and the back skin was shaved, depilated with a hair removal spray, and cleaned with alcohol. Using a sterile 4-mm biopsy punch, four bilateral full-thickness skin wounds were created on the back skin with two wounds on each dorsorostral side. The underlying muscle was uninjured, and wounds were made with a minimum of 6 mm of uninjured skin between adjacent wounds. Wounds were photographed at 0, 3, 7, and 10 days after wounding using a Panasonic DMC-FX33 digital camera. Wound diameter was measured using Photoshop CS6 software to calculate the wound area, and the percentage of wound closure at each time point was calculated according to the following formula: [1 – (current wound area/initial wound area)] × 100. Mice were euthanized 7, 14, and 21 days after wounding and wound tissue specimens were collected for further analysis.

Hematoxylin and eosin (H&E) staining and immunofluorescence (IF)

Wound tissue specimens were fixed in 4% paraformaldehyde and processed using paraffin wax and standard methods. Five wound tissue cross-sections (3 μm, taken 100 μm apart) were stained with H&E (Cat.HHS16, HT110116, Sigma-Aldrich) for histomorphometric analysis, including measurement of wound diameter, epithelial thickness, epithelial area and granular area. For immunofluorescence, cross sections were dewaxed in xylene and rehydrated by successive immersion in descending concentrations of alcohol. After blocking in 5% normal goat serum/PBS for 20 min, sections were incubated with primary antibodies overnight at 4 °C, followed by Alexa Fluor 488- or 594-labeled secondary antibodies (Cat.111-545-003, 111-585-003, Jackson Immunoresearch) for secondary incubations. 4, 6-diamidino-2-phenylindole (DAPI, Cat.P36935, Invitrogen) was used to visualize the nuclei. Immunofluorescent images were obtained using a FluoView FV1000 confocal microscope (Olympus). The primary antibodies used for immunofluorescence are summarized in Table 1.

Western blotting (WB)

Wound tissue biopsies were triturated and lysed on ice, and boiled in sodium dodecyl sulfate (SDS) loading buffer. The protein extracts were then subjected to 6%-12% SDS-PAGE and electrotransferred to nitrocellulose membranes (Cat.10600001, GE Healthcare Life Sciences). The membranes were blocked in 5% nonfat milk for 1 h at room temperature, followed by incubation with the indicated primary antibodies overnight at 4°C, and secondary incubation with horseradish peroxidase (HRP)-conjugated IgG (H+L) (Cat.111-035-003, 115-035-003, Jackson Immunoresearch) for 1 h at room temperature. The protein signals were visualized using an enhanced chemiluminescence kit (Cat.NEL105001EA, PerkinElmer). The primary antibodies used for WB are summarized in Table 1.

Cell apoptosis assay

Cell apoptosis was evaluated in wound cross sections with a TUNEL assay using the DeadEnd Fluorometric TUNEL System (Cat.G3250, Promega). Positive labeling in the fibroblast zone was recorded by a confocal microscope. Van Gieson collagen staining and collagen maturity assessment Wound collagen synthesis was assessed using van Gieson staining (Cat. DC0046, Nanjing Jiancheng Bioengineering), in which mature collagen fibers exhibited a deep red color and immature collagen fibers a pink color. The maturity of collagen fibers was scored according to the following criteria: 0, pale pink color; 1, pink color; 2, red color; 3, deep red color.

Hydroxyproline assay

The hydroxyproline assay was performed to quantify collagen synthesis in wound tissues, as described previously.23 Wound tissues were homogenized in saline and hydrolyzed with 2 M NaOH, followed by the determination of hydroxyproline using chloramine T and Ehrlich’s reagent, following the conditions established previously.24 Values were calculated according to a hydroxyproline standard curve, and were expressed as micrograms of hydroxyproline per milligram of protein.

Rapamycin treatment.

Tsc1-cKO and Tsc1-Ctrl mice were intragastrically administered rapamycin (1 mg/kg/d, Cat.2A-13346-10, Cayman), a dose that had been demonstrated effectively inhibiting the mTORC1 activity in vivo in our previous study, 20 or vehicle (corn oil with DMSO) for 21 days after wounding. As described above, wounds were photographed at 0, 7 and 10 days after wounding to determine the effect of rapamycin treatment on wound closure. Mice were killed and wound tissue biopsies were collected on day 7 after wounding for immunofluorescent staining, or on day 21 for van Gieson staining and hydroxyproline assays.

Primary human dermal fibroblast culture and related assays

To culture primary human dermal fibroblasts, skin samples donated by the patients undergoing circumcision were harvested, minced into small pieces, and incubated in PBS containing Dispase (Cat.17105041, ThermoFisher Scientific) for 1 h. After removing the epidermis, the dermis underwent further incubation in 1× PBS containing collagenase (Cat. C0130, Sigma-Aldrich) for another hour. Cells were pelleted by centrifugation, washed twice and grown in DMEM containing 10% FBS. Fibroblasts at passages 2-5 were transfected with siRNA targeting Tsc1 or negative control (NC) siRNA using Lipofectamine 3000 (Cat. L3000001, invitrogen). 12 h later, transfection compounds were replaced with fresh complete medium with/without 100 nM rapamycin, and cultured for another 48 h. Cells were finally subjected to WB, IF, and cell proliferation by Cell Counting Kit (CCK-8, Cat. CK04, Dojindo).

Statistics

All experiments were performed in triplicate. Data are expressed as the mean ± SEM. Differences in the percentage data were analyzed by the chi square test. Between-group differences in measurable data were analyzed using the Student’s t-test, and the ANOVA test for more than two groups (SPSS 13.0). A P-value < 0.05 was considered statistically significant.

Results

Generation of Tsc1-conditional KO mice

To generate mice with fibroblast-specific deletion of Tsc1, we crossed Tsc1-LoxP mice with mice containing the Col1α2-Cre/ERT recombinase gene under the control of a proα2(I) collagen promoter. The activation of Cre/ERT relies on the presence of tamoxifen. Mice homozygous for the Tsc1-LoxP and hemizygous for the Col1α2-cre/ERT (Tsc1LoxP/LoxP, Col1α2-cre/ERT) were treated with (to delete Tsc1, Tsc1-conditional KO [Tsc1-cKO] mice) or without tamoxifen (for controls, Tsc1-Ctrl mice), as described in the Materials and Methods. Deletion of the Tsc1 gene was verified by PCR of tail DNA, which showed a 360bp Tsc1 deletion band (Figure 1A). Because TSC1 is an upstream inhibitor of mTORC1, Tsc1 deletion should result in the activation of mTORC1. Indeed, co-immunofluorescence of the phosphorylated level of S6 (p-S6, S235/S236), a downstream target of mTORC1, and vimentin, a marker for fibroblasts (which also stained smooth muscle cells and endothelium), in unwounded skins revealed the enhancement of p-S6 expression in the fibroblasts in Tsc1-cKO mice compared with those in Tsc1-Ctrl mice (Figure 1, B and C). No distinguishable difference in the expression pattern was observed in the epidermis or follicles of Tsc1-cKO mice versus -Ctrl mice (Figure 1B). Furthermore, western blotting confirmed that TSC1 protein levels almost vanished, while the p-S6 (S235/S236) level increased in skin tissues of Tsc1-cKO mice, compared to Tsc1-Ctrl mice (Figure 1, D and E). The level of p-Akt (S473), a downstream marker for mTORC2, remained stable (Figure 1, D and E). Those results suggested that the Cre-LoxP strategy deleted the Tsc1 gene and caused mTORC1 activation in the fibroblasts of Tsc1-cKO mice.

mTORC1 activation in fibroblasts promotes wound repair.

To investigate the effects of mTORC1 activation in fibroblasts during cutaneous wound closure, 8-week-old Tsc1-Ctrl and Tsc1-cKO mice were subjected to a dermal punch wound model for wound healing, after which a 10-day period of wound closure was monitored. As shown in Figure 2A and B, Tsc1-cKO mice had a marked acceleration in wound closure compared to Tsc1-Ctrl mice. On day 3 following wounding, Tsc1-cKO mice had displayed significantly accelerated wound healing (P < 0.05). On day 10 following wounding, Tsc1-cKO mice exhibited almost 100% wound closure, compared to 70% wound closure in Tsc1-Ctrl mice (P < 0.01). In addition, histomorphometric examinations of cross-sections of day 7 wounds further revealed the enhanced wound closure in Tsc1-cKO mice. The wound diameter, epithelial thickness, epithelial area and granular area in Tsc1-cKO mice were significantly decreased compared to controls (P < 0.05; Figure 2, C-G). Collectively, those results suggested that mTORC1 activation in fibroblasts induced by Tsc1 deletion accelerated wound healing.

mTORC1 activation enhances fibroblast proliferation and α-SMA expression.

Three wounds from two Tsc1-cKO mice displayed “volcano-like” healing on day 7 after wounding, with a higher wound closure than the adjacent intact tissues. However, such findings were not observed in Tsc1-Ctrl mice (Figure 3, A and B). This phenomenon led to the hypothesis that the accelerated wound closure in Tsc1-cKO mice maybe due to increased fibroblast proliferation or decreased fibroblast apoptosis. As expected, seven days after wounding, Tsc1-cKO mice exhibited increased fibroblast cell proliferation (P < 0.01), as revealed by Ki-67 immunostaining (Figure 3, C and D). However, a comparable level of fibroblast cell apoptosis was observed between Tsc1-cKO and Tsc1-Ctrl mice, as revealed by DeadEnd Fluorometric TUNEL assay (Figure 3, E and F). Such findings were consistent with the results presented in the Figure 1C that Tsc1-cKO and -Ctrl mice had comparable levels of S473-phosphorylated Akt, whose signaling controls cell survival. Those results also suggested that it was not decreased fibroblast apoptosis, but increased fibroblast proliferation that accelerated wound healing in Tsc1-cKO mice. We then examined the effects of activation of mTORC1 on fibroblastic α-SMA protein expression. Immunofluorescent staining revealed dramatically elevated α-SMA protein levels in day 7 wounds of Tsc1-cKO, compared with Tsc1-Ctrl mice (P < 0.05; Figure 3, G and H). Those results suggested that mTORC1 activation enhanced fibroblast proliferation and α-SMA synthesis.

mTORC1 activation in fibroblasts increases collagen synthesis, resulting in excessive scarring and fibrosis.

We next investigated the effects of mTORC1 activation on wounds after healing. We assessed scarring status in the wounds of Tsc1-cKO and -Ctrl mice. Examination of day 21 wounds revealed increased collagen deposition and excessive scarring in Tsc1-cKO mice, as detected by van Gieson collagen staining (Figure 4A). Staining also indicated a higher maturity of collagen fibers in Tsc1-cKO mice (P < 0.05; Figure 4B). Consistently, Tsc1-cKO mice had significantly elevated hydroxyproline levels at day 21 after wounding (Figure 4C). In addition, western blot analyses also showed that the protein level of Col1 was largely increased in Tsc1-cKO mice versus Tsc1-Ctrl mice (Figure 4D). Collectively, those results suggested that the effects of mTORC1 activation could persist following wound closure, and led to excessive scarring and fibrosis.

mTORC1 antagonist rapamycin reverses the wound-healing and fibrotic phenotypes in Tsc1-cKO mice.

We sought to assess whether the mTORC1 inhibitor, rapamycin, could restore the observed phenotypes in Tsc1-cKO mice. For this purpose, Tsc1-cKO and litter-mated control mice were treated with rapamycin for 21 days, and the parameters for wound healing and fibrogenesis were monitored. Rapamycin treatment effectively recovered the level of phosphorylated S6, but did not alter the wound closure kinetics of control mice; however, it reversed the accelerated wound healing process of Tsc1-cKO mice (Figure 5, A and B). Moreover, rapamycin also reduced increased fibroblast cell proliferation (Figure 5, C and D), and decreased the number of α-SMA-expressing myofibroblasts (Figure 5, E and F) in Tsc1-cKO mice. In addition, enhanced collagen deposition and excessive scarring and fibrogenesis in Tsc1-cKO mice were also largely ameliorated by rapamycin (Figure 5, G and H). Collectively, those results demonstrated that inhibition of mTORC1 by rapamycin restored the wound-healing histomorphology, and attenuated the scarring and fibrotic phenotypes in Tsc1-cKO mice.

Rapamycin restores the phenotypes of mTORC1 activation in primary human dermal fibroblasts.

We finally sought to explore whether the above observations could be potentially translatable to human wound healing. Primary human dermal fibroblasts were transfected with negative control (NC)- or Tsc1-siRNA, followed by treatment with rapamycin or vehicle for 48 h. Immunoblotting showed that rapamycin treatment significantly attenuated the mTORC1 activation by Tsc1 knockdown, as well as the elevated protein levels of Col1 isoforms and α-SMA (Figure 6, A and B). Rapamycin treatment also markedly rescued the enhanced cell proliferation by Tsc1 knockdown, as revealed by CCK-8 assay (Figure 6C). Furthermore, Tsc1 knockdown promoted myofibroblastic transition in primary human dermal fibroblasts, manifested by the higher expression of α-SMA and greater stress fiber formation (Figure 6D), indicative of a higher cell migration activity following mTORC1 activation. Rapamycin treatment recovered this phenotype (Figure 6D). These in vitro data on primary human dermal fibroblasts suggest translation significance of our observations to human wound healing.

Discussion

In the present study, we investigated the role of mTORC1 signaling in fibroblasts during the wound healing process and scarring. By generating mutant mice with fibroblast-specific deletions of the Tsc1 gene, and using those mice in a full-thickness skin wound model, we showed that mTORC1 activation in fibroblasts dramatically accelerated the wound healing process by enhancing fibroblastic cell proliferation and contractile α-SMA expression. However, elevating the mTORC1 activity induced excessive collagen production, leading to excessive scaring and fibrosis. Importantly, both in vivo and in vitro data suggested that accelerated wound healing and the fibrotic phenotypes were largely restored after decreasing mTORC1 activity with rapamycin. Those observations demonstrated that mTORC1 activity was an important orchestrator in cutaneous wound healing and fibrogenesis.
Upon injury to the skin, the provisional fibrin clot ECM is formed to induce hemostasis, which then must be replaced by a stronger collagen ECM to maintain skin integrity.25 The ECM change requires proliferation and in-migration of activated fibroblastic cells (myofibroblasts) from adjacent tissues. The presence of α-SMA represents the most reliable marker for the myofibroblastic phenotype.3, 4
We found that mTORC1 activation markedly increased the number of Ki-67- and α-SMA-positive fibroblastic cells, indicating that the proliferative activity and migratory capacity of those cells may be controlled by mTOR. Re-epithelialization is an essential component of wound healing and is used as a defining parameter for successful wound closure.1 Re-epithelialization is mediated by keratinocytes, a major cellular component of the epidermis and impaired epithelialization leads to chronic wounds.26, 27 Fibroblasts are the primary cells expressing cytokines, including TGF-β, FGF-2, EGF, and PDGF,28 which are critical regulators of epithelialization during wound healing. In this context, it is possible that mTOR activity in the fibroblastic cells may facilitate wound closure by regulating fibroblastic and epithelial cell proliferation and migration; combined with its ability to regulate paracrine signals involved in cellular communication processes that contribute to the wound microenvironment.
Although it is exciting to observe that elevated mTORC1 activity in fibroblasts accelerated wound closure, Tsc1-cKO mice exhibited excessive scarring and fibrotic phenotypes following wound closure, indicating that mTORC1 activation may induce undesirable effects. Although the myofibroblasts are essential for the recovery of skin integrity, they dramatically disappear in a prominent wave of apoptosis during the resolution phase of healing to reduce the cellular scar.4 In this regard, mTORC1 activation may hinder the clearance of myofibroblasts, leading to excessive collagen production and scarring. However, the activity of Akt (p-Akt, S473), which is a downstream target of mTORC2 that regulates cell survival,29 remained stable in Tsc1-cKO mice. Those findings indicated that cell apoptosis may not be the factor.
Rather, cell autophagy is likely to be involved, as evidence has emerged in recent years showing that such processes are highly involved in wound healing.30-32 mTOR is a well-established critical regulator of autophagy.33, 34 It is also possible that mTORC1 activation prevented active myofibroblasts from de-differentiating to be quiescent fibroblasts.35 Thus, mTORC1 activity should be differentially controlled during different phases of healing to reduce side-effects. In the early phase of healing (~7-10 days after wounding), elevated mTORC1 activity (by potential molecular inhibitors of TSC1/2) may accelerate the wound healing process, while in the later phases of healing, mTORC1 activity should be downregulated to avoid excessive collagen production and scarring. For the latter, mTORC1 inhibitors such as rapamycin or everolimus should be considered as our data showed that the fibrotic phenotypes in the Tsc1-cKO mice can be effectively alleviated by such compounds.
In summary, by using a genetically defined mouse model, we demonstrated that elevated mTORC1 activity in fibroblasts significantly accelerated the wound healing process, which also induced excessive scaring and fibrosis in the later phases of healing. Importantly, both the in vivo and in vitro fibrotic phenotypes were effectively attenuated via inhibition of mTORC1 activity by rapamycin. Those results may provide a rationale for future prevention or treatment of chronic or fibrosis-prone wounds.
 
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