Akt/mTOR integrate energy metabolism with Wnt signal to influence wound epithelium growth in Gekko Japonicus

Wang, Qinghua; Mao, Zuming; Liu, Zhuang; Xu, Man; Huang, Shuai; Wang, Yin; Xu, Yanran; Qi, Longju; Liu, Mei; Liu, Yan

doi:10.1038/s42003-022-04004-5

Download PDF

Article
Open access
Published: 27 September 2022

Akt/mTOR integrate energy metabolism with Wnt signal to influence wound epithelium growth in Gekko Japonicus

Qinghua Wang^1,2^na1,
Zuming Mao¹^na1,
Zhuang Liu¹,
Man Xu¹,
Shuai Huang¹,
Yin Wang¹,
Yanran Xu¹,
Longju Qi³,
Mei Liu¹ &
…
Yan Liu ORCID: orcid.org/0000-0002-1378-1825¹

Communications Biology volume 5, Article number: 1018 (2022) Cite this article

1372 Accesses
2 Citations
2 Altmetric
Metrics details

Subjects

Abstract

The formation of wound epithelium initiates regeneration of amputated tail in Gekko japonicus. Energy metabolism is indispensable for the growth of living creatures and typically influenced by temperature. In this study, we reveal that low temperature lowers energy metabolism level and inhibits the regeneration of amputated tails of Gekko japonicus. We further find that low temperature attenuates the activation of protein kinase B (Akt) and mammalian target of rapamycin (mTOR) in regenerated tissues upon injury signals, and the inhibition of Akt hinders proliferation of the wound epithelium. Additionally, wingless/integrated (Wnt) inhibition suppresses epithelium proliferation and formation by inhibiting Akt activation. Finally, low temperature elevates the activity of adenylate-activated kinase (AMPK) pathway and in turn attenuates wound epithelium formation. Meanwhile, either mTOR downregulation or AMPK upregulation is associated with worse wound epithelium formation. Summarily, low temperature restricts wound epithelium formation by influencing energy sensory pathways including Akt/mTOR and AMPK signaling, which is also modulated by injury induced Wnt signal. Our results provide a mechanism that incorporates the injury signals with metabolic pathway to facilitate regeneration.

JNK-mediated Slit-Robo signaling facilitates epithelial wound repair by extruding dying cells

Article Open access 20 December 2019

Reactive oxygen species rescue regeneration after silencing the MAPK–ERK signaling pathway in Schmidtea mediterranea

Article Open access 13 January 2021

Identification of rare, transient post-mitotic cell states that are induced by injury and required for whole-body regeneration in Schmidtea mediterranea

Article 02 September 2021

Introduction

Regenerative capacity varies among different species. In mammals, limited regeneration capacity has been observed in skin, epithelial mucosa of digestive system, and liver. However, lower vertebrates show higher innate regenerative abilities, where complete regeneration of tissues and organs can be achieved and preserved throughout life. For example, salamanders regenerate limbs¹, zebrafishes regenerate their hearts², and lizards regenerate their tails³. Similarly, Gekko japonicus has a strong regeneration ability to regenerate its severed tail⁴. The injured site forms wound epithelium, and then develops a bud that grows and differentiates into skin, muscle, cartilage and neural tissues^4,5. Finally, the newborn tail shows morphological regeneration and functional recovery. Therefore, Gekko japonicus is a feasible animal model for regeneration research.

Tissue regeneration is an energy-intensive process. However, the role and mechanism of the metabolic pathways in regulating appendage regeneration remain elusive. We found that the regeneration ability of Gekko japonicus was severely inhibited when the surrounding temperature was below 10 °C. This effect was consistent with that reported by Milsom et. al, who proposed the metabolic level was an essential criterion for successful regeneration⁶. Then, we proposed that the low temperature treatment could lead to a state of low metabolic rate and failure of tail regeneration. This model could provide an alternative opportunity to investigate how the metabolic pathway to regulate regeneration process.

Several studies have shown that the protein kinase B/mammalian target of rapamycin (Akt/mTOR) and adenylate-activated kinase (AMPK) pathways are important metabolic regulatory pathways for energy sensing^7,8,9. In addition, phosphoinositide 3 kinase (PI3K) and phosphatase and tensin homologous protein (PTEN) are important modulator of Akt/mTOR pathway^10,11. mTOR, as a serine/threonine protein kinase, plays an important role in sensing nutritional level of cells and regulating metabolism and cell behavior including protein synthesis, cell growth, movement and proliferation^12,13,14. AMPK is a kind of conserved serine/threonine protein kinase that is very sensitive to cell energy state, as indicated by the ratio of AMP and ATP^10,15. However, the regulatory mechanism of the energy sensing pathways in lizard tail regeneration remains unclear.

The initial stage of gecko’s tail regeneration was mainly composed of two courses, one was wound epithelium covering and the other was blastema formation^4,5,16. The wound epithelium gradually became thicker and formed apical epithelia (AEC), which was similar in structure and function to the apical ectodermal ridge (AER) during limb bud development^17,18. The injury-induced signals triggered formation of AEC, meanwhile the AEC could secrete many growth factors to facilitate the proliferation and migration of other cells and orchestrate the regeneration process. The Wnt pathway is a well-recognized signal to regulate the initiation of regeneration in many tissues^19,20,21,22.

However, whether and how the Wnt signal modulates the metabolic events during regeneration of tail regeneration of lizard is unclear yet. Here, low temperature was found to hinder the formation of wound epithelium, which might due to activation of AMPK pathway. Then, AMPK induced inhibition of Akt/mTOR decreased the proliferation of wound epithelium. Furthermore, we revealed that Wnt pathway activation promoted Akt/mTOR activity and thus facilitated wound epithelium formation. Results above shed light on the interaction between the injury signals and metabolic pathway during regeneration.

Results

Metabolic impedance caused by low temperature constrains blastema growth

The innate ability in regeneration of amputated tail was based on the formation of wound epithelium and blastema in geckos⁵. Wild geckos experience temperature and metabolic variance during different seasons^6,23. Then, we asked whether low temperature affect regeneration process. We tested the tail regeneration at 28 °C, 10 °C and 17 °C respectively to investigate the effect. In 28 °C group, 80% of the geckos successfully initiated regeneration, while none and 3% geckos could initiate regeneration in 10 °C group and 17 °C group respectively (Fig. 1a, b). Then, we termed 28 °C group as normal group, 10 °C group and 17 °C group as low temperature groups. To testify the metabolic variances in groups of different temperature, the tissues of the geckos were collected and examined. ATP levels were significantly higher in the tail tissues from the 28 °C group compared to that from the 10 °C group and 17 °C group (Fig. 1c). However, no significant difference was observed in the livers of normal and low temperature groups (Fig. 1c). To understand this difference, metabolic characteristics, such as cholesterol (CHOL, Fig. 1d), triglycerol (TG, Fig. 1e), serum glucose (GLU, Fig. 1f), low-density lipoprotein cholesterol (LDLC, Fig. 1g), high-density lipoprotein cholesterol (HDLC, Fig. 1h), uric acid (UA, Fig. 1i), and Creatinine (CR, Fig. 1j), was analyzed to determine the levels of glucose, lipid and protein metabolism. These results revealed that low temperature decreased the systematic metabolism. In addition, the formation of wound epithelium that positively stained with WE6 antibody was observed in low temperature and control group. We found that the wound epithelium formation was hindered in the 10 °C group and 17 °C group in a temperature dependent manner (Fig. 1k).

**Fig. 1: Energy homeostasis was altered in the non-regeneration geckos.**

Akt/mTOR pathway activation is inhibited during tail regeneration under low temperature

A thin layer of wound epithelium appeared on the 7th day after tail amputation, which induced the blastama formation thereafter²⁴. Akt and mTOR are known to be involved in the regulation of growth, development and energy utilization^25,26. Therefore, the activity of Akt and mTOR were analyzed in the tail tissues at day 0, 3, 7 and 14 after injury in the low and normal temperature groups. The Akt activity is indicated by phosphorylation of Thr308 and Ser473²⁷. Our results revealed that Akt activation level in the normal temperature (28 °C) group was significantly augmented at the beginning stage of regeneration. The phosphorylation of Thr308 was enhanced on day 3 (Fig. 2a, b), day 7 (Fig. 2a, b) and day 14 (Fig. 2a, b), and a significant increase of Ser473 phosphorylation was also observed on day 3 (Fig. 2a, c). Meanwhile, we evaluated the mTOR activity by phosphorylation on Ser2448 that usually infers activation of mTORC1²⁸. The results proposed that mTOR activity was higher in tail tissues from 28 °C group than 10 °C group on day 0 (Fig. 2a, d), day 3 (Fig. 2a, d), day 7 (Fig. 2a, d). Furthermore, we detected the activity of P70 S6K, a downstream target of mTOR that is required for cell growth and cell cycle progression^29,30. The level of p-P70 S6K (Thr389) were significantly enhanced in geckos from normal temperature group on day 3 (Fig. 2a, e). In conclusion, the Akt, mTOR and P70 S6K activities were boosted in normal group during the early stage of blastama formation.

**Fig. 2: Akt/mTOR pathway was involved in the regeneration process.**

Inhibition of Akt lowers the proliferation of wound epithelium

To investigate the effect of Akt activity on tail regeneration, MK-2206, a Akt inhibitor³¹ was injected intraperitoneally (i.p.) in tail-amputated geckos followed timeline listed Fig. 3a. As an important indicator of regeneration initiation, the formation of wound epithelium was checked at day 7 after the surgery. As illustrated in Fig. 3b, c, regeneration was clearly suppressed in the Akt inhibition group. Geckos in the control group shed the scab and covered the injured site with smooth wound epithelium, while those in inhibition group were still wrapped with scab. Similar results were also observed in the morphological examination that revealed only scab and no wound epithelium in the inhibition group (Fig. 3d). Following confirmation of the morphological changes, the newborn tissues were collected and analyzed by western blotting, which showed that phosphorylation level of Akt Ser473 (Fig. 3e, f) and Akt Thr308 (Fig. 3e, g) were significantly decreased. In addition, a significant difference was also observed in the phosphorylation of mTOR Ser2448 (Fig. 3e, h), whereas no significant difference was detected in total Akt and mTOR. WE6 is a common marker of wound epithelium and BrdU incorporation is an efficient marker of cell proliferation. The BrdU positive cells were significantly decreased in the Akt inhibition group compared to that in the control group (Fig. 3i, j). These data suggest that Akt activation is essential for proliferation of blastema cell and formation of wound epithelium, ultimately affecting the regeneration of the injured tails.

**Fig. 3: Akt blocking lowered the proliferation of wound epithelium.**

Wnt/β-catenin signal regulates wound epithelium formation and Akt activity

The Wnt/β-catenin axis is pivotal in appendage regeneration^19,24. We investigated whether the Wnt inhibitors, IWP-4 and XAV939, affect the formation of wound epithelium and tail regeneration. The formation of wound epithelium was examined 7 days after surgery. Both, the formation of wound epithelium (Fig. 4a) and rate of regeneration (Fig. 4b) were clearly affected by IWP-4 or XAV939 treatment. Morphological examination showed the presence of ample amount of scab tissue, instead of wound epithelium, bundled at the injury site (Fig. 4c, lower panel). Cell proliferation was analyzed using BrdU staining, and WE6 served as a marker to outline the location of the wound epithelium. The results revealed that Wnt inhibition suppressed cell proliferation (Fig. 4c, upper panel, and 4d).

**Fig. 4: Wnt inhibition decreased the proliferation of wound epithelium.**

We have revealed that both low temperature or inhibitor induced Akt inhibition impaired the epithelium formation, and the Wnt signals were also implicated in the proliferation of epithelium cells. Then, we question whether Wnt signal regulates Akt activity during tail regeneration. The Wnt inhibitor, IWP-4 and XAV939 were applied in the experiments. We detected the effect of Wnt inhibitor by evaluating the level in β-Catenin and lymphoid enhancer-binding factor 1 (LEF1) in samples. LEF1, an effector transcription factor of Wnt pathway activation, plays an important role in regulating cell proliferation, differentiation and apoptosis. IWP-4 and XAV939 efficiently decreased the level of LEF1 (Fig. 5a, b), β-catenin (Fig. 5a, c), which indicated that the two inhibitors might negatively regulate β-catenin dependent Wnt pathway. We further investigated whether Wnt blocking results in Akt inhibition. The results showed that the phosphorylation of Akt were both inhibited at the sites of Serine 473 (Fig. 5a, d) and Threonine 308 (Fig. 5a, e). These findings implied that Akt activity was regulated by Wnt/β-catenin during regeneration. Then, we asked whether Wnt signal directly influence Akt activity. Previous study on the effect of Wnt signal on appendage regeneration revealed that Wnt signal could induce the expression of fibroblast growth factors¹⁹, which facilitated the proliferation of blastema cells. We tested this effect of Wnt agonist (SKL2001 or CHIR) in cultured blastema cells, and found the Akt phosphorylation was augmented (Supplementary Fig. 1a–c). Meanwhile, we also observed that FGF8, FGF10 and FGF20 were up-regulated upon treatment of SKL2001, while inhibition of Akt activity attenuated the upregulation of FGFs (Supplementary Fig. 1d), which is consistent with the hypothesis as other reports. These factors might contribute to the Akt activity.

**Fig. 5: Akt phosphorylation was inhibited by Wnt antagonist during tail regeneration.**

We further investigated whether Wnt agonist could overcome the low temperature-induced deficiency in tail regeneration. We detected the effect of Wnt agonist on the formation of WE6 positive epithelium at low temperature. Compared with geckos under normal temperature (Supplementary Fig. 2a), Wnt agonist could stimulate epithelium formation in low temperature condition, which showed in Supplementary Fig. 2b, c. However, Wnt agonist was not able to proceed and complete the regeneration at low temperature (Supplementary Fig. 2d, e). These data indicated that Wnt signal is necessary but not sufficient for the successful tail regeneration.

Low temperature increases activity of AMPKα during regeneration

AMPKα is an important energy-sensing factor that monitors variations in ATP concentration. Studies have shown that this process occurs through regulation of mTOR. Samples of the newly grown tail tissues were collected and analyzed by western blotting. The results revealed that the level of phosphorylated AMPKα (Thr172) was significantly higher in the geckos in the 10 °C group compared to that in the 28 °C group on days 3 and 7 (Fig. 6a, b). Moreover, the expression of total AMPKα in 10 °C group was higher than that in the 28 °C group on day 0, 3 and 7. The expression of AMPKα reached its peak on day 7 in the 10 °C group, and then declined over time (Fig. 6a, c). These data suggested that the higher activity of AMPK at early stage might suppress the initiation of regeneration.

**Fig. 6: Low temperature increased activity of AMPKα during regeneration.**

mTOR inhibition and AMPK activation reduce the proliferation of wound epithelium

To determine the necessity of mTOR activation for tail regeneration in geckos, RAD001, a small molecule inhibited the activation of mTOR, was administered by intraperitoneal injection. Similarly, the ATP sensing protein AMPK, a negative regulator of mTOR, was activated by the intraperitoneal injection of agonist A769662. Wound epithelium formation was analyzed on 7 days post-surgery. The results showed that the formation of wound epithelium was significantly blocked in both the mTOR inhibition and AMPKα activation groups (Fig. 7a, b). No individuals with regenerated tails were found in the mTOR inhibition or AMPKα activation groups (Fig. 7b). In addition, the results were further validated by morphological assay that also demonstrated the occurrence of only scar tissues in both the mTOR inhibition (RAD001) and AMPKα activation groups (A769662). Intact wound epithelium tissues were observed in the DMSO group (Fig. 7c). To confirm the effects of the inhibitor and agonist, Western Blotting analysis was performed to determine the expression of mTOR and AMPK. The results showed that the ratios of p-AMPKα/AMPKα were significantly increased in the RAD001- (Fig. 7d, e) and A769662-treated groups (Fig. 7d, e). Total AMPKα was also increased in the RAD001- and A769662-treated groups (Fig. 7d, g). Phosphorylated mTOR/mTOR was significantly decreased in the RAD001-treated group (Fig. 7d, f) and the A769662-treated group (Fig. 7d, f). To investigate cell proliferation in the wound epithelium, BrdU incorporation was analyzed in the geckos. The results showed that the number of BrdU positive cells was significantly decreased in RAD001- and A769662-treated group (Fig. 7h, upper panel, Fig. 7i). WE6 was used to shape the site of wound epithelium (Fig. 7h, lower panel). To confirm the effect of mTOR inhibition on the formation of wound epithelium, another mTOR inhibitor, Torin1, was administrated as well, the treatment obviously inhibited tail regeneration (Supplementary Fig. 3a, b). The treatment also blunted the level of phosphorylated mTOR (Supplementary Fig. 3c, d). The BrdU positive cells was significantly decreased in Torin1-treated group (Supplementary Fig. 3f, upper panel, Fig. 3e). WE6 was used to shape the site of wound epithelium (Supplementary Fig. 3f, lower panel). Taken together, inhibition of mTOR and activation of AMPK may hinder the proliferation of wound epithelium.

**Fig. 7: AMPK activation reduced the proliferation of wound epithelium.**

Discussion

Energy supplements maintain cellular activities and influence cell cycle and growth⁹. Energy metabolism is regulated by many signaling pathways and affected by the surrounding temperature, especially in the heterothermic animals⁶. Thus, heterothermic animals could be applied as a model to investigate the biological process that involved in metabolic regulation. In this study, we focused on the role of Akt/mTOR activation in energy deficiency situations during the tail regeneration of geckos. Our results revealed that the injury induced Wnt signal and the energy sensing AMPK pathway coordinately modulated the activity of mTOR and affected epithelium proliferation.

As an upstream signaling molecule, Wnt is strongly implicated in cell proliferation, differentiation, and regeneration process in response to injury^32,33,34. Our results revealed that Wnt pathway promoted wound epithelium formation and Wnt inhibition significantly decreased Akt activation. Furthermore, we addressed whether Wnt signal regulating Akt activity. Previous studies for the cross-talk of Wnt and Akt/mTOR pathway was described as inhibition of GSK3-β by Akt, which could lead to stable β-catenin and Wnt activation^35,36. Our data revealed an opposite regulatory pathway from Wnt to Akt, and this effect might be mediated by other growth factors indirectly. Therefore, we proposed a possible link between injury induced Wnt signal and metabolic related pathway. It might be essential for the appendage regeneration when a robust proliferation is required to initiate and sustain the regeneration process.

AMPK, an upstream regulator of mTOR, plays an important role in regulating the energy balance in cells during the regeneration process. Several studies have suggested that the AMPK/mTOR axis maintains the energy status and thus coordinates cellular activities^9,15,32. To confront harsh winter conditions and food deficiency, geckos survive by hibernating and thus minimizing their energy expenditure. How can geckos maintain a lower metabolic level, a potential approach is to sustain a high activity of AMPK when confronting low temperature. In this study, we found that the expression of total AMPK was higher at low temperature than that at high temperature at the early stages of regeneration in geckos. Moreover, the robust expression of phosphorylated AMPK following surgery provided evidence for the retardation of wound epithelium at low temperature in geckos. Then another interesting question is that how the activity of AMPK is maintained or even increased in low temperature. Usually, as an enzyme, the activity is positively correlated with the temperature. How the structure of AMPK is orchestrated upon low temperature is an amazing topic in future study.

In summary, as illustrated in Fig. 8, we demonstrated that low temperature and AMPK activation led to a downregulation of mTOR activity. The Wnt pathway is evoked by the injury signal and sequentially boosts the Akt activity. The AMPK and Wnt pathway exhibitesd opposing effects on the activity of mTOR, which is involved in regulating proliferation of the wound epithelium.

**Fig. 8: Schematic representation of working model.**

Methods

Gekko japonicus breeding and modeling

Adult G. japonicus with intact tails were grouped into control and experimental groups after health evaluation by veterinarians. The number of males and females were equal in each group. The average body weight was 4.4 ± 0.495 g. All geckos had free access to mealworms and sterilized water during the entire experiment. The culture incubator was set at 28 °C, 17 °C or 10 °C based on the test protocol. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee of the Nantong University (Anima protocol: S20190405-402).

Tail autotomy was exerted following the protocols issued previously by our lab members²⁴. Concisely, caudectomy was performed by locating a nylon slipknot at the 6th tail segment and then pulling gently to simulate the stimulation of autotomy in the natural environment. The successful regeneration was determined by the featured viewing as scab shedding and exposing of pink epithelium at ~7 day. The delayed scab shedding indicated deficiency of the wounding epithelium formation, which always led to failure of regeneration. The subsequent regeneration was evaluated by the length of regenerated tails that described in our previous study²⁴, mainly detected by morphology observation, pathological investigation and molecular markers indication that detailed below.

ATP extraction and evaluation

Liver and tail tissues near the 6th caudal vertebrae were collected and weighed, after which ATP lysis buffer (100 μL/10 mg sample, S0026, Beyotime Biotechnology, Nantong, China) was added into the tubes and the tissues were minced. The samples were incubated on ice for 5 min and then centrifuged at 12,000 × g at 4 °C. The supernatant (50 μL) was transferred into an opaque 96-well plate, which was pre-loaded with 100 μL ATP detection buffer. The absorbance of the samples was measured with an illuminometer (Synergy TM 2, BioTek, Waldbronn, Germany).

Detection of metabolic parameters

The animals were maintained at either 28 °C or 10 °C for 1 week. Blood was drained from the hearts and the serum was separated by centrifugation at 12,000 × g for 10 min at 4 °C. The GLU, CHOL, TG, LDLC, HDLC, UA, and CR levels were determined using Siemens ADVIA® 2400 (Erlangen, Germany) based on the manufacturer’s instructions.

Drug administration

The tails were amputated at the 6th caudal vertebrae to set up the regeneration models. Akt inhibitor MK-2206 (5 mg/kg body weight (BW) S1078, Selleck, Houston, TX) was administered after autotomy by i.p. injection every other day. BrdU solvent (20 μL of 20 mM solution, B5002, Sigma, Darmstadt, Germany) was administered on the last 2 days before sample collection. The samples were collected on the 7th day or other time point indicated. For the treatment of mTOR inhibitor (RAD001, Torin1) and AMPK activator (A769662, Selleck, Houston, TX), the gecko models were administered RAD001 (10 mg/kg BW, S1120, Selleck, Houston, TX), Torin1 (20 mg/kg BW, S2827, Selleck, Houston, TX) and A769662 (30 mg/kg BW, S2697, Selleck, Houston, TX) individually or together by i.p. injection post autotomy every other day. For the treatment of Wnt agonist, CHIR98014 was administered (5 mg/kg BW, S2745, Selleck, Houston, TX) by i.p. injection post autotomy every other day. The BrdU was administered as mentioned above. For treatment with the Wnt inhibitor, IWP-4 (2 mg/kg BW, SML1114, Sigma, Darmstadt, Germany) or XAV939 solution (40 mg/kg BW, S1180, Selleck, Houston, TX) was administered after amputation by i.p. injection every other day.

Cell culture and drug treatment

Geckos with well-regenerated blastema at day 12 post autotomy were prepared. The anesthetized ones were sterilized with 70% EtOH and the blastema tissues were dissected out and digested in 0.25% trypsin-EDTA buffer at 30 °C for 10 min. Then cells were harvested and resuspended in 3 mL DMEM/F12 (10-092-CV, Thermo Fisher Scientific) containing 10% FBS, 2 mM glutamine (C0212, Beyotime, Nantong, China) and Penicillin-Streptomycin-Amphotericin B solution (100X, C0224, Beyotime, Nantong, China). Cells then were plated in 8-cm² cell-culture dish under 30 °C and 5% CO2. The cells of passage 2 were gathered by conventional trypsin dislocation and allocated into a 6-well plate with 3 × 10⁵ cells per well for 12 h. The Wnt/β-catenin signaling agonist SKL2001 (40 μM, S8320, Selleck, Houston, TX) or CHIR98014 (8 μM, S2745, Selleck, Houston, TX) were applied into culture medium for additional 24 h before assays. The Akt antagonist MK-2206 (8 μM, S1078, Selleck, Houston, TX) were administrated as above.

RNA isolation and quantitative real-time PCR

Cells were collected and total RNA was serparated by TRIzol (Vazyme, Nanjing, China). RNA pellets were qualified and quantified to be further reverse-transcribed with HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China), and qRT-PCR was performed using a TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara, Beijing, China) following the supplier’s instructions. Primers were synthesized by Invitrogen company (Shanghai, China). Sequences of the primer were listed below: ef1α sense: 5'- GATGGAAAGTGACCCGCA-3', ef1α antisense: 5'-GAGGAAGACGCAGAGGTTTG-3', FGF7 sense: 5'-AGCCATGAACAAGTCTGGGA-3', FGF7 antisense: 5'-GCACCGTTCTGTTTCAATGC-3', FGF8 sense: 5'-AGAGCAACGGCAAAGG-3', FGF8 antisense: 5'-TGCTGGCGAGTCTTGG-3', FGF10 sense: 5'-GGCCACTAACTCTTCCTCGT-3', FGF10 antisense: 5'-CTTTCCCGTTCGGCTCAATC-3', FGF20 sense: 5'-TGGCAGTGGGTTTAGTCAG-3', FGF20 antisense: 5'-ACATTGGAGGAGTAAGTGTTGT-3'. All measurements were taken out at least in triplicates and data were normalized to endogenous ef1α expression. Gene expression was set as the fold change compared with the threshold cycle (Ct) and the relative expression levels were calculated by 2^-ΔΔCt method.

Immunohistochemistry and immunofluorescence assay

The geckos were anesthetized by freezing on ice and perfused with pre-cooled 4% paraformaldehyde (PFA) solution. The blastema (5 mm before injury site) was sampled and subjected to 4% PFA fixation for 4 h and then washed with 0.1 M phosphate buffered saline (PBS) three times. The samples were then dehydrated in sequential concentrations of sucrose solution (5%, 10%, 20%, and 30%). The 12 μM sections were prepared from the OCT embedded blastema. For hematoxylin and eosin (H&E) staining, the sections were washed with PBS and double distilled water (DDW), stained with hematoxylin, eosin, and dehydrated by sequential concentration of ethanol solution. Sections were immersed in xylene three times and mounted with resin. Images were captured using an Olympus DP71 microscope (Tokyo, Japan) for further analysis.

For BrdU immunofluorescence detection, sections were warmed at 37 °C and rinsed with PBS. Sections were soaked in 1.5 M HCl for 30 min in a 37 °C water bath. After neutralization in 0.02 mM sodium tetraborate solution for 10 min at room temperature (RT) and PBS washes, the sections were then incubated with normal goat serum for 2 h at 37 °C. The extra blocking solution was drained and the sections incubated with anti-BrdU primary antibody overnight at 4 °C. The secondary antibody was incubated with the PBS-washed specimen for 2 h at RT. The sections were incubated with Hoechst 33342 antibody for 15 min at RT. Slides were mounted and observed by Zeiss M6 microscopy, and images were analyzed using Image Pro Plus. The fields of interested budding center were recorded and then merged for further BrdU positive cells counting. Data were collected from 5 gecko’s wound healing center area.

For immunofluorescence assay of cultured blastema cells, the samples were fixed with 4% paraformaldehyde for 20 min at 25 °C, then washed by PBS three times and blocked with 10 mg/mL bovine serum albumin, 0.1% Triton X-100 at 37 °C for 2 h. The cells were then incubated at 4 °C overnight with β-catenin antibody (1:200, Cat. ab32572, RRID: AB_725966, Abcam). After rewarmed at 25 °C for 30 min, cells were rinsed with PBS three times, and incubated with the corresponding Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:400, Jackson ImmunoResearch, West Grove, PA, USA, Cat.111-545-003, RRID: AB_2338046) for 2 h at 25 °C. After counterstaining with DAPI (1:2500, Cat.D9542, Merck, Germany), the cells were washed with PBS and mounted to reduce photo bleaching. Fluorescence images were obtained using a Zeiss Axio Imager M2 microscope (Jena, Germany).

Western blotting

Tail tissues (3 mm) before the injury site were excised and frozen in liquid nitrogen and stored at -80 °C. Protein samples were extracted and adjusted to identical concentration. Protein mixtures were loaded onto different percentage SDS-polyacrylamide gels (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Primary antibodies used were as follows: Akt (1:1000, 4685, Cell Signaling Technology (CST)), p-Akt Ser473 (1:1000, 4060, CST), p-Akt Thr308 (1:1000, 13038 S, CST), AMPKα (1:1000, 5832, CST), p-AMPKα Thr172 (1:1000, 2535, CST), mTOR (1:1000, 2983, CST), p-mTOR (1:1000, 2976, CST, 2976), p-p70 S6K Thr389 (1:1000, 9205, CST), p70 S6K (1:1000, 2708, CST), LEF1 (1:1000, ab85052, Abcam), β-catenin (1:1000, ab32572, Abcam), WE6 (1:5, WE6-s, Developmental Studies Hybridoma Bank) and β-actin (1:2000, ab8826, Abcam). β-actin was used as an internal control.

Statistics and reproducibility

All data are presented as mean ± SD values. Replicates are biologically representing independent experimental subject. Experiments were duplicated at least three times and sample size was qualified according to methodology and was specialized in figure legends. Two groups of data were compared using homogeneity and t-tests, and multiple groups of data were analyzed by One-way (one factor comparation) or Two-way (two factor comparation) ANOVA followed by Bonferroni post-hoc test using SPSS Statistics 22.0 (SPSS Inc., Chicago, IL, USA). Chi-square test was adopted in the analysis for regeneration rate. p < 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

All data are contained within the manuscript and Supplementary Data files. The source data for graphs are provided in the Supplementary Data 1. Uncropped immunoblots are provided in the Supplementary Fig. 4.

References

Nacu, E., Gromberg, E., Oliveira, C. R., Drechsel, D. & Tanaka, E. M. FGF8 and SHH substitute for anterior-posterior tissue interactions to induce limb regeneration. Nature 533, 407–410 (2016).
Article CAS PubMed Google Scholar
Sánchez-Iranzo, H. et al. Tbx5a lineage tracing shows cardiomyocyte plasticity during zebrafish heart regeneration. Nat. Commun. 9, 428 (2018).
Article PubMed PubMed Central Google Scholar
Alibardi, L. & Toni, M. Wound keratins in the regenerating epidermis of lizard suggest that the wound reaction is similar in the tail and limb. J. Exp. Zool. A Comp. Exp. Biol. 303, 845–860 (2005).
Article PubMed Google Scholar
Liu, Y. et al. Gekko japonicus genome reveals evolution of adhesive toe pads and tail regeneration. Nat. Commun. 6, 10033 (2015).
Article CAS PubMed Google Scholar
Delorme, S. L., Lungu, I. M. & Vickaryous, M. K. Scar-free wound healing and regeneration following tail loss in the leopard gecko, Eublepharis macularius. Anat. Rec. 295, 1575–1595 (2012).
Article CAS Google Scholar
Milsom, W. K. et al. Seasonal changes in daily metabolic patterns of tegu lizards (Tupinambis merianae) placed in the cold (17 degrees C) and dark. Physiol. Biochem. Zool. PBZ 81, 165–175 (2008).
Article CAS PubMed Google Scholar
Fulco, M. et al. Glucose restriction inhibits skeletal myoblast differentiation by activating SIRT1 through AMPK-mediated regulation of Nampt. Develop. Cell 14, 661–673 (2008).
Article CAS Google Scholar
García-Rodríguez, J. L. et al. SIRT1 controls liver regeneration by regulating bile acid metabolism through farnesoid X receptor and mammalian target of rapamycin signaling. Hepatology 59, 1972–1983 (2014).
Article PubMed Google Scholar
Mo, J. S. et al. Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway. Nat. Cell Biol. 17, 500–510 (2015).
Article CAS PubMed PubMed Central Google Scholar
Igarashi, M. & Guarente, L. mTORC1 and SIRT1 cooperate to foster expansion of gut adult stem. Cells Calor. Restriction. Cell 166, 436–450 (2016).
CAS Google Scholar
Loeffler, J., Duda, G. N., Sass, F. A. & Dienelt, A. The metabolic microenvironment steers bone tissue regeneration. Trends Endocrinol. Metab. TEM 29, 99–110 (2018).
Article CAS PubMed Google Scholar
Miao, L. et al. mTORC1 is necessary but mTORC2 and GSK3β are inhibitory for AKT3-induced axon regeneration in the central nervous system. eLife 5, e14908 (2016).
Article PubMed PubMed Central Google Scholar
Zhang, P. et al. mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration. Biochem. Biophys. Res. Commun. 463, 102–108 (2015).
Article CAS PubMed PubMed Central Google Scholar
Wu, C. C. et al. mTORC1-mediated inhibition of 4EBP1 is essential for hedgehog signaling-driven translation and medulloblastoma. Develop. Cell 43, 673–688.e675 (2017).
Article CAS Google Scholar
Giovannini, L. & Bianchi, S. Role of nutraceutical SIRT1 modulators in AMPK and mTOR pathway: evidence of a synergistic effect. Nutrition 34, 82–96 (2017).
Article CAS PubMed Google Scholar
Peacock, H. M., Gilbert, E. A. & Vickaryous, M. K. Scar-free cutaneous wound healing in the leopard gecko, Eublepharis macularius. J. Anat. 227, 596–610 (2015).
Article CAS PubMed PubMed Central Google Scholar
Christensen, R. N. & Tassava, R. A. Apical epithelial cap morphology and fibronectin gene expression in regenerating axolotl limbs. Develop. Dyn. 217, 216–224 (2000).
Article CAS Google Scholar
Saunders, J. W. Jr. The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. 1948. J. Exp. Zool. 282, 628–668 (1998).
Article PubMed Google Scholar
Wehner, D. et al. Wnt/β-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin. Cell Rep. 6, 467–481 (2014).
Article CAS PubMed Google Scholar
Stoick-Cooper, C. L. et al. Distinct Wnt signaling pathways have opposing roles in appendage regeneration. Development. 134, 479–489 (2007).
Article CAS PubMed Google Scholar
Schunk, S. J., Floege, J., Fliser, D. & Speer, T. WNT-β-catenin signalling—a versatile player in kidney injury and repair. Nat. Rev. Nephrol. 17, 172–184 (2021).
Article CAS PubMed Google Scholar
Mehta, A. S. & Singh, A. Insights into regeneration tool box: an animal model approach. Develop. Biol. 453, 111–129 (2019).
Article CAS PubMed Google Scholar
Kim, D. I. et al. Spring and summer microhabitat use by Schlegel’s Japanese gecko, Gekko japonicus (Reptilia: Squamata: Gekkonidae), in urban areas. Anim. Cells Syst. 23, 64–70 (2019).
Article Google Scholar
Xu, M. et al. PGE2 facilitates tail regeneration via activation of Wnt signaling in Gekko japonicus. J. Mol. Histol. 50, 551–562 (2019).
Article CAS PubMed Google Scholar
Manning, B. D. & Toker, A. AKT/PKB signaling: navigating the network. Cell 169, 381–405 (2017).
Article CAS PubMed PubMed Central Google Scholar
Bockaert, J. & Marin, P. mTOR in brain physiology and pathologies. Physiol. Rev. 95, 1157–1187 (2015).
Article CAS PubMed Google Scholar
Bao, F. et al. Akt scaffold proteins: the key to controlling specificity of Akt signaling. Am. J. Physiol. Cell Physiol. 321, C429–C442 (2021).
Article CAS PubMed Google Scholar
Copp, J., Manning, G. & Hunter, T. TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2. Cancer Res. 69, 1821–1827 (2009).
Article CAS PubMed PubMed Central Google Scholar
Dufner, A. & Thomas, G. Ribosomal S6 kinase signaling and the control of translation. Exp. Cell Res. 253, 100–109 (1999).
Article CAS PubMed Google Scholar
Hornberger, T. A., Sukhija, K. B., Wang, X. R. & Chien, S. mTOR is the rapamycin-sensitive kinase that confers mechanically-induced phosphorylation of the hydrophobic motif site Thr(389) in p70(S6k). FEBS Lett. 581, 4562–4566 (2007).
Article CAS PubMed PubMed Central Google Scholar
Hirai, H. et al. MK-2206, an allosteric Akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo. Mol. Cancer Ther. 9, 1956–1967 (2010).
Article CAS PubMed Google Scholar
Aros, C. J. et al. Distinct spatiotemporally dynamic Wnt-secreting niches regulate proximal airway regeneration and aging. Cell Stem Cell 27, 413–429.e414 (2020).
Article CAS PubMed PubMed Central Google Scholar
Sun, X., Peng, X., Cao, Y., Zhou, Y. & Sun, Y. ADNP promotes neural differentiation by modulating Wnt/β-catenin signaling. Nat. Commun. 11, 2984 (2020).
Article CAS PubMed PubMed Central Google Scholar
Jung, Y.S., et al. TMEM9-v-ATPase activates Wnt/β-Catenin signaling Via APC lysosomal degradation for liver regeneration and tumorigenesis. Hepatology 73, 776–794 (2020).
Korkaya, H. et al. Regulation of mammary stem/progenitor cells by PTEN/Akt/beta-catenin signaling. PLoS Biol. 7, e1000121 (2009).
Article PubMed PubMed Central Google Scholar
Eiraku, N. et al. BMP9 directly induces rapid GSK3-β phosphorylation in a Wnt-independent manner through class I PI3K-Akt axis in osteoblasts. FASEB J. 33, 12124–12134 (2019).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

This work was supported by National Natural Science Foundation of China (Grant No. 31970412, 31472004 and 32100380); Natural science foundation of Jiangsu province (Grant No. BK20191446); the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Author information

These authors contributed equally: Qinghua Wang, Zuming Mao.

Authors and Affiliations

Key Laboratory of Neuroregeneration of Jiangsu Province and Ministry of Education, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, 226001, China
Qinghua Wang, Zuming Mao, Zhuang Liu, Man Xu, Shuai Huang, Yin Wang, Yanran Xu, Mei Liu & Yan Liu
Comparative Medicine Research Institution, Nantong University, Nantong, 226001, China
Qinghua Wang
Affiliated Nantong Hospital 3 of Nantong University, Nantong University, Nantong, 226001, China
Longju Qi

Authors

Qinghua Wang
View author publications
You can also search for this author in PubMed Google Scholar
Zuming Mao
View author publications
You can also search for this author in PubMed Google Scholar
Zhuang Liu
View author publications
You can also search for this author in PubMed Google Scholar
Man Xu
View author publications
You can also search for this author in PubMed Google Scholar
Shuai Huang
View author publications
You can also search for this author in PubMed Google Scholar
Yin Wang
View author publications
You can also search for this author in PubMed Google Scholar
Yanran Xu
View author publications
You can also search for this author in PubMed Google Scholar
Longju Qi
View author publications
You can also search for this author in PubMed Google Scholar
Mei Liu
View author publications
You can also search for this author in PubMed Google Scholar
Yan Liu
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Y.L., M.L., and Q.W. conceptualized the project. Y.L., M.L., Q.W., Z.M., and M.X. designed experiments. Q.W., Z.M., M.X., S.H., Y.W., Y.X., and L.Q. performed experiments and analyses. Q.W., M.L., and Y.L. wrote the paper and all authors were involved in the editing process. Y.L. and M.L. acquired funding.

Corresponding author

Correspondence to Yan Liu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Jesmond Dalli and Zhijuan Qiu.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary data 1

Reporting Summary

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Wang, Q., Mao, Z., Liu, Z. et al. Akt/mTOR integrate energy metabolism with Wnt signal to influence wound epithelium growth in Gekko Japonicus. Commun Biol 5, 1018 (2022). https://doi.org/10.1038/s42003-022-04004-5

Download citation

Received: 01 April 2022
Accepted: 15 September 2022
Published: 27 September 2022
DOI: https://doi.org/10.1038/s42003-022-04004-5

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.