Insights into the molecular characteristics of embryonic cranial neural crest cells and their derived mesenchymal cell pools.

Neural crest cells (NCCs) are central to vertebrate embryonic development, giving rise to diverse cell types with unique migratory and differentiation capacities. This study examines the molecular characteristics of cranial neural crest cell (CNCC)-derived mesenchymal cells, specifically those from teeth which in deer show continuous but limited growth, and antlers, which exhibit remarkable regenerative capabilities. Here, through single-cell RNA sequencing analysis, we uncover shared gene expression profiles between adult antlerogenic and dental mesenchymal cells, indicating common developmental pathways. We identify a striking resemblance in transcriptomic features between antlerogenic progenitor cells and dental pulp mesenchymal cells. Comparative analysis of CNCC-derived and non-CNCC-derived mesenchymal cell pools across species reveals core signature genes associated with CNCCs and their derivatives, delineating essential connections between CNCCs and CNCC-derived adult mesenchymal pools. Furthermore, whole-genome DNA methylation analysis unveils hypomethylation of CNCC derivate signature genes in regenerative antlerogenic periosteum, implying a role in maintaining multipotency. These findings offer crucial insights into the developmental biology and regenerative potential of CNCC-derived mesenchymal cells, laying a foundation for innovative therapeutic strategies in tissue regeneration.

Conditioned Media from Deer Antler Stem Cells Effectively Alleviate Type 1 Diabetes Mellitus Possibly via Inhibiting the NF-κB Signaling Pathway.

BACKGROUND: Type 1 diabetes mellitus (T1D) represents a severe threat to human health. Persistent hyperglycemia and dyslipidemia can lead to damaged liver function, while effective interventions for these complications are currently lacking. Deer antler stem cells (AnSCs), a novel type of adult stem cells, significantly reduced liver injury, which was speculated to be achieved through the paracrine pathway. METHODS: In this study, AnSC-conditioned medium (AnSC-CM) was used to treat C57BL/6 mice with T1D symptoms induced by streptozotocin (STZ). The therapeutic effects of AnSC-CM on T1D were evaluated, and the underlying mechanism was investigated. RESULTS: It was shown that AnSC-CM alleviated the T1D symptom: decreased body weight, increased blood glucose levels and islet lesions, and reduced insulin secretion. Moreover, AnSC-CM treatment improved liver function and mitigated liver injury in T1D mice. Impressively, the therapeutic effects of AnSC-CM on T1D were better than those of bone marrow mesenchymal stem cell-CM (BMSC-CM). The mechanistic study revealed that AnSC-CM significantly downregulated the NF-κB signaling pathway in both pancreatic and liver tissues. CONCLUSIONS: Therapeutic effects of AnSC-CM on STZ-induced T1D and liver injury may be achieved through targeting the NF-κB signaling pathway.

A population of stem cells with strong regenerative potential discovered in deer antlers.

The annual regrowth of deer antlers provides a valuable model for studying organ regeneration in mammals. We describe a single-cell atlas of antler regrowth. The earliest-stage antler initiators were mesenchymal cells that express the paired related homeobox 1 gene (PRRX1+ mesenchymal cells). We also identified a population of "antler blastema progenitor cells" (ABPCs) that developed from the PRRX1+ mesenchymal cells and directed the antler regeneration process. Cross-species comparisons identified ABPCs in several mammalian blastema. In vivo and in vitro ABPCs displayed strong self-renewal ability and could generate osteochondral lineage cells. Last, we observed a spatially well-structured pattern of cellular and gene expression in antler growth center during the peak growth stage, revealing the cellular mechanisms involved in rapid antler elongation.

Stem cells drive antler regeneration.

Understanding the rapid growth of deer antlers could have applications in medicine.

Antlerogenic stem cells extract accelerate chronic wound healing: a preliminary study.

BACKGROUND: Chronic wounds constitute a significant medical and social problem. Chronic wound treatment may be supported by various techniques, such as negative pressure therapy, phototherapy or stem cells therapy, yet most of those supporting therapies need more evidence to be used for standard wound care. Current study covers the use of sonicated Antlerogenic Stem Cells (ASC) extract on chronic wounds. METHODS: Study was performed on 20 dermatological patients with venous leg ulcers, divided into two groups - treated with and without ASC extract respectively. The area and circumference of the wounds during the follow-up visits were measured on the wound imprint. Dynamics of wound healing was determined and compared between control and study group; statistics includes changes in absolute values (wound area, circumference), as well as relative (percentage of wound decrease, circumference/area ratio) and their change in time. For the purpose of Ki-67 immunohistochemical staining, sections were sampled from the wound edge at distinct check-points during therapy. Results of both groups were compared with Student test or Mann-Whitney test, depending on results distribution. RESULTS: Besides Ki-67 expression, all tested wound healing parameters (including relative and absolute wound decrease and changes in circumference/area ratio) were statistically significant more favorable in experimental group. CONCLUSION: ASC extract significantly supported standard chronic wound treatment. Due to small population of study the results should be considered preliminary, yet promising for further research.

Effects and mechanism of lncRNA-27785.1 that regulates TGF-ß1 of Sika deer on antler cell proliferation.

Transforming growth factor (TGF-ß) plays an important role in the development of deer antlers. The purpose of this study was to investigate the role of long noncoding RNA in the transcriptional regulation of TGF-ß1 and its relationship with the proliferation and differentiation of antler chondrocytes. High-throughput sequencing was used to screen lncRNAs related to TGF-ß1. Next, the overexpression plasmid and interference sequence of target lncRNA27785.1 were constructed and transfected into chondrocytes. We found that lncRNA27785.1 inhibited the proliferation and migration of chondrocytes and delayed the transition of cells from G1 to S phase. qRT-PCR and Western blot analysis indicated that the overexpression of lncRNA27785.1 may downregulate mRNA and protein expression of TGF-BR2, Smad3, pSmad3, and Smad4. Our findings highlight lncRNA27785.1 as an inhibitor of chondrocytes proliferation and differentiation by negatively regulating the TGF-ß/Smad signaling pathway; this implicates an important regulatory role for long noncoding RNA in the regeneration of antler.

PTN-PTPRZ signalling is involved in deer antler stem cell regulation during tissue regeneration.

A growing deer antler contains a stem cell niche that can drive endochondral bone regeneration at up to 2 cm/day. Pleiotrophin (PTN), as a multifunctional growth factor, is found highly expressed at the messenger RNA level within the active antler stem cell tissues. This study aims to map the expression patterns of PTN protein and its receptors in a growing antler and investigate the effects of PTN on antler stem cells in vitro. Immunohistochemistry was employed to localise PTN/midkine (MDK) and their functional receptors, protein tyrosine phosphatase receptor type Z (PTPRZ), anaplastic lymphoma kinase (ALK), NOTCH2, and integrin αV ß3, on serial slides of the antler growth centre. PTN was found to be the dominantly expressed growth factor in the PTN/MDK family. High expression of PTPRZ and ALK co-localised with PTN was found suggesting a potential interaction. The high levels of PTN and PTPRZ reflected the antler stem cell activation status during the regenerative process. When antler stem cells were cultured in vitro under the normoxic condition, no PTN protein was detected and exogenous PTN did not induce differentiation or proliferation but rather stem cell maintenance. Collectively, the antler stem cell niche appears to upregulate PTN and PTPRZ in vivo, and PTN-PTPRZ signalling may be involved in regulating antler stem cell behaviour during rapid antler regeneration.

Transplanted Antler Stem Cells Stimulated Regenerative Healing of Radiation-induced Cutaneous Wounds in Rats.

Radiation-induced cutaneous injury is the main side effect of radiotherapy. The injury is difficult to cure and the pathogenesis is complex. Mesenchymal stem cells (MSCs) serve as a promising candidate for cell-based therapy for the treatment of cutaneous wounds. The aim of the present study was to investigate whether antler stem cells (AnSCs) have better therapeutic effects on radiation-induced cutaneous injury than currently available ones. In this study, a rat model of cutaneous wound injury from Sr-90 radiation was used. AnSCs (1 × 106/500 µl) were injected through the tail vein on the first day of irradiation. Our results showed that compared to the control group, AnSC-treated rats exhibited a delayed onset (14 days versus 7 days), shorter recovery time (51 days versus 84 days), faster healing rate (100% versus 70% on day 71), and higher healing quality with more cutaneous appendages regenerated (21:10:7/per given area compared to those of rat and human MSCs, respectively). More importantly, AnSCs promoted much higher quality of healing compared to other types of stem cells, with negligible scar formation. AnSC lineage tracing results showed that the injected-dye-stained AnSCs were substantially engrafted in the wound healing tissue, indicating that the therapeutic effects of AnSCs on wound healing at least partially through direct participation in the wound healing. Expression profiling of the wound-healing-related genes in the healing tissue of AnSC group more resembled a fetal wound healing. Revealing the mechanism underlying this higher quality of wound healing by using AnSC treatment would help to devise more effective cell-based therapeutics for radiation-induced wound healing in clinics.

Influence of antlerogenic stem cells on the healing of lesions in the corneal epithelium and corneal stroma in rabbits.

The aim of the study was to compare the effects of corneal healing in case of application of stem cells in various forms, in relation to the antibiotic-assisted procedures. Rabbits were divided into 4 groups in the first stage of the experiment. Group 0 (negative control group) was not subjected to any actions, which would cause damage to the cornea. The remaining three groups had their cornea damaged. Group 1 (positive control group) - no drugs were administered during the experiment. Rabbits in group 2 were administered with ointment containing stem cells to the lesion, while group 3 - with ofloxacinum. The stem cells were administered during the first five days, twice a day, onto the corneal surface. The further course of the experiment consisted of observing the rate of healing of the injured cornea and assessment of its transparency, size of lesion, hyperaemia, eyelid spasm and outflow from the conjunctival sac after 5, 10 and 20 days.In the second stage the animals were euthanised after clinical examination on the twentieth day of the experiment, in order to analyse the corneal reparative processes on the same day. The studies revealed that the application of antlerogenic stem cells had a positive effect on the healing process of corneal defects. The application thereof not only shortened the healing time, but also weakened or arrested the development of side effects. The results have demonstrated that the epithelial proliferation in each group was different. The longest was maintained in the group with stem cells, the shortest - in the group with chemotherapeutics. The use of antlerogenic stem cells had a positive effect on the healing process of corneal lesions. The use of stem cells helped to maintain high transparency of the cornea.

Screening and functional identification of lncRNAs in antler mesenchymal and cartilage tissues using high-throughput sequencing.

Long non-coding RNA (lncRNA) is a transcription product of the mammalian genome that regulates the development and growth in the body. The present study aimed to analyze the expression dynamics of lncRNA in sika antler mesenchymal and cartilage tissues by high-throughput sequencing. Bioinformatics was applied to predict differentially expressed lncRNAs and target genes and screen lncRNAs and mRNAs related to osteogenic differentiation, cell proliferation, and migration. Finally, the expression of the lncRNAs and target genes were analyzed by qRT-PCR. The results showed that compared to the cartilage tissue, the transcription levels of lncRNA and mRNA, 1212 lncRNAs and 518 mRNAs, in mesenchymal tissue were altered significantly. Thus, a complex interaction network was constructed, and the lncRNA-mRNA interaction network correlation related to osteogenic differentiation, cell proliferation, and migration was analyzed. Among these, the 26 lncRNAs and potential target genes were verified by qRT-PCR, and the results of qRT-PCR were consistent with high-throughput sequencing results. These data indicated that lncRNA promotes the differentiation of deer antler mesenchymal tissue into cartilage tissue by regulating the related osteogenic factors, cell proliferation, and migration-related genes and accelerating the process of deer antler regeneration and development.

Antler stem cells as a novel stem cell source for reducing liver fibrosis.

Liver fibrosis results from collagen fiber deposition. Antler stem cells (ASCs) naturally in vivo differentiate into cartilage, which is only made of Col II in collagen component; whereas liver fibrosis is caused by over-abundance of Col I and III. In addition, ASCs can effectively promote regenerative wound healing in which tissue contains very few collagen fibers (Col I). In this study, we investigate the therapeutic effects of ASCs in a rat model of CCl4-induced liver fibrosis. Rats were treated with ASCs for 4 weeks in vivo, then biochemical and histopathological analyses were performed. Furthermore, we established cell co-culture systems of hepatic stellate cells (HSCs) and ASCs and of M1 macrophages and ASCs in vitro. Mesenchymal stem cells (MSCs) were used as a positive control. The results showed that ASC transplantation alleviated liver fibrosis effectively as evidenced by reduced collagen accumulation, decreased fatty degeneration, increased hepatocyte regeneration, decreased inflammation and significantly enhanced liver function; moreover, ASCs decreased the expression of pro-fibrogenic factors including TGF-ß and α-SMA. Additionally, our study showed that ASCs inhibit HSC activation and proliferation by controlling the expression of MMPs, TIMP1, TGF-ß, α-SMA and COL1A2 involved in these processes. Our results suggested that ASCs alleviate liver fibrosis effectively and inhibit HSC activation. Thus, ASCs may serve as a novel stem cell source for the treatment of liver fibrosis in the clinic.

S100A4: a novel partner for heat shock protein 47 in antler stem cells and insight into the calcium ion-induced conformational changes.

S100A4 is a multiple-function protein highly expressed in tumor or stem cells. We found S100A4 was a novel protein partner for heat shock protein 47 (HSP47) in deer antlerogenic periosteum cells (AP cells), indicating that S100A4 could bind with HSP47. S100A4 had both calcium-dependent and calcium-independent patterns (labeled as SCd and SCi, respectively) to execute different biological activities. Homology models of HSP47, SCd and SCi were constructed. HSP47:collagen model, HSP47:collagen I-V, HSP47:SCd and HSP47:SCi complexes were built using ZDOCK software. Together with free SCd and SCi, 200 ns molecular dynamic (MD) simulations were performed to analyze binding free energies and SCi/SCd conformational changes. The energetic results showed that SCi had the strongest affinity to HSP47, and followed by collagens. SCd had little interaction with HSP47. Decomposition energy results showed that collagen model interacted with HSP47 mainly though neutral amino acids. When SCi bound with HSP47, the majority of mediated amino acids were charged. These results indicated that SCi could compete with collagen on the binding site of HSP47. Root mean square fluctuation (RMSF) values and cross-correlation matrices of principal component analysis (PCA) were calculated to evaluate the SCi/SCd structural variation during MD simulation. Both HSP47 and Ca2+ could stabilize the conformation of SCi/SCd. The loops interacting with Ca2+s and linking the two EF-hand motifs were impacted particularly. The relative moving directions of α-helices in EF-hands were distinct by the binding effect of HSP47 and Ca2+. We found that SCi may regulate the differentiation of AP cells by disturbing the interaction between HSP47 and collagen. Communicated by Ramaswamy H. Sarma.

Identification of interactive molecules between antler stem cells and dermal papilla cells using an in vitro co-culture system.

Deer antlers are the only mammalian organs capable of complete renewal. Antler renewal is a stem cell-based [antler stem cells (ASCs)] process. Maintenance and activation of the ASCs require them to be located in a specialized microenvironment (niche), and to interact with the cells resident in the niche. Based on previous experiments we found that niche of the ASCs is provided by the closely associated enveloping skin, which currently was known includes dermal papilla cells (DPCs) and epidermal cells. Antler generation/regeneration are triggered by the interactions between ASCs and the niche. In the present study, we established an in vitro co-culture system in which ASCs and DPCs, were cultured together to mimic the in vivo state. A MLEFF strategy was adopted to identify the interactive molecules from the co-culture system. In total, 128 molecules were identified and over 60% belonged to exosomes. Important biological processes that were activated by these molecules included osteoblast differentiation, angiogenesis, and the PI3K-AKT signaling pathway. In so doing, we have significantly simplified the process for identifying interactive molecules, which may be the key signals for triggering antler formation/renewal. Further study of these molecules will help us to gain insights into the mechanism of mammalian organ regeneration.

Antler stem cell-conditioned medium stimulates regenerative wound healing in rats.

BACKGROUND: When the deer antler is cast, it leaves a cutaneous wound that can achieve scarless healing due to the presence of antler stem cells (ASCs). This provides an opportunity to study regenerative wound healing. METHODS: In this study, we investigated the therapeutic effects and mechanism of antler stem cell-conditioned medium (ASC-CM) on cutaneous wound healing in rats. In vitro, we investigated the effects of the ASC-CM on proliferation of HUVEC and NIH-3T3 cell lines. In vivo, we evaluated the effects of ASC-CM on cutaneous wound healing using full-thickness skin punch-cut wounds in rats. RESULTS: The results showed that ASC-CM significantly stimulated proliferation of the HUVEC and NIH-3T3 cells in vitro. In vivo, completion of healing of the rat wounds treated with ASC-CM was on day 16 (± 3 days), 9 days (± 2 days) earlier than the control group (DMEM); the area of the wounds treated with ASC-CM was significantly smaller (p < 0.05) than the two control groups. Further molecular characterization showed that the ratios of Col3A1/Col1A2, TGF-ß3/TGF-ß1, MMP1/TIMP1, and MMP3/TIMP1 significantly increased (p < 0.01) in the healed tissue in the ASC-CM group. CONCLUSIONS: In conclusion, ASC-CM effectively accelerated the wound closure rate and enhanced the quality of healing, which might be through transforming wound dermal fibroblasts into the fetal counterparts. Therefore, the ASC-CM may have potential to be developed as a novel cell-free therapeutic for scarless wound healing.

Deer antler stem cells are a novel type of cells that sustain full regeneration of a mammalian organ-deer antler.

Deer antlers are extraordinary mammalian organs that can fully regenerate annually. Antler renewal is a stem cell-based epimorphic process and antler stem (AS) cells can initiate de novo generation of antlers in postnatal mammals. However, although being called stem cells, the AS cells have not been characterized at molecular level based on the stem cell criteria. Comprehensive characterization of the AS cells would undoubtedly help to decipher the mechanism underlying the full regeneration of deer antlers, the only case of stem cell-based epimorphic regeneration in mammals. In the present study, three types of AS cells (antlerogenic periosteal cells APCs, for initial pedicle and first antler formation; pedicle periosteal cells PPC, for annual antler regeneration; and reserve mesenchyme cells RMCs, for rapid antler growth), were isolated for comprehensive molecular characterization. A horn-growth-related gene, RXFP2, was found to be expressed only in AS cells lineages but not in the facial periosteal cells (FPCs, locates geographically in the vicinity of the APCs or PPCs), suggesting the RXFP2 might be a specific marker for the AS cell lineage in deer. Our results demonstrated that AS cells expressed classic MSC markers including surface markers CD73, CD90, CD105 and Stro-1. They also expressed some of the markers including Tert, Nestin, S100A4, nucleostemin and C-Myc, suggesting that they have some attributes of the ESCs. Microinjection of male APC into deer blastocysts resulted in one female foetus (110 days gestation) recovered with obvious pedicle primordia with both male and female genotype detected in the ovary. In conclusion, the AS cells should be defined as MSCs but with partial attributes of ESCs.

DNA methylation and mRNA expression of COL6A3 in antler mesenchyme of female and male reindeer.

BACKGROUD: Reindeer is the only deer species that both male and female produce antlers, which provides a particularly interesting case in studying the differences between antlers of the two sexes. Alpha 3(VI) Collagen Gene (COL6A3), forms a microfibrillar network associated with the structural integrity and biomechanical properties, has been found to be one of the differentially expressed genes in antler mesenchyme of female and male reindeer. OBJECTIVE AND METHODS: The promoter sequence of reindeer COL6A3 gene was obtained using the cloning technology and analyzed by the bioinformatics methods. Bisulfite sequencing PCR (BSP) was used to detect the methylation status of the COL6A3 promoter in reindeer antler mesenchyme. Real-time quantitative PCR was used to detect COL6A3 expression in the antler mesenchyme of female and male reindeer. RESULTS: Sequence analysis revealed that the reindeer COL6A3 partial promoter sequence was 983 bp including the possible promoter region at + 105 bp to + 155 bp. Homology and phylogenetic analysis indicated that the COL6A3 promoter of reindeer had the closest genetic distance with Bos taurus, Capra hircus and Ovis aries. BSP results indicated that the methylation level of COL6A3 promoter in the female reindeer antler mesenchyme was significantly higher than in the male. Correlating with increased methylation status, we also found that COL6A3 mRNA expression in female reindeer antler mesenchyme was significantly lower than in the male. CONCLUSION: The higher methylation level of the COL6A3 gene in female reindeer antler mesenchyme coincides with decreased COL6A3 mRNA expression, thereby affecting the transposon silencing mechanism and possibly contributing to apparent differences of antlers in female and male reindeer.

Single-cell transcriptome provides novel insights into antler stem cells, a cell type capable of mammalian organ regeneration.

Antler regeneration, a stem cell-based epimorphic process, has a potential as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler development is located in the antlerogenic periosteum (AP). However, whether this ASC pool is homogenous or heterogeneous has not been fully evaluated. In this study, we produced a comprehensive transcriptome dataset at the single-cell level for the ASCs based on the 10× Genomics platform (scRNA-seq). A total of 4565 ASCs were sequenced and classified into a large cell cluster, indicating that the ASC resident in the AP are likely to be a homogeneous population. The scRNA-seq data revealed that tumor-related genes were highly expressed in these homogeneous ASCs, i.e., TIMP1, TMSB10, LGALS1, FTH1, VIM, LOC110126017, and S100A4. Results of screening for stem cell markers suggest that the ASCs may be considered as a special type of stem cell between embryonic (CD9) and adult (CD29, CD90, NPM1, and VIM) stem cells. Our results provide the first comprehensive transcriptome analysis at the single-cell level for the ASCs and identified only one major cell type resident in the AP and some key stem cell genes, which may hold the key to why antlers, the unique mammalian organ, can fully regenerate once lost.

Identifying deer antler uhrf1 proliferation and s100a10 mineralization genes using comparative RNA-seq.

BACKGROUND: Deer antlers are bony structures that re-grow at very high rates, making them an attractive model for studying rapid bone regeneration. METHODS: To identify the genes that are involved in this fast pace of bone growth, an in vitro RNA-seq model that paralleled the sharp differences in bone growth between deer antlers and humans was established. Subsequently, RNA-seq (> 60 million reads per library) was used to compare transcriptomic profiles. Uniquely expressed deer antler proliferation as well as mineralization genes were identified via a combination of differential gene expression and subtraction analysis. Thereafter, the physiological relevance as well as contributions of these identified genes were determined by immunofluorescence, gene overexpression, and gene knockdown studies. RESULTS: Cell characterization studies showed that in vitro-cultured deer antler-derived reserve mesenchyme (RM) cells exhibited high osteogenic capabilities and cell surface markers similar to in vivo counterparts. Under identical culture conditions, deer antler RM cells proliferated faster (8.6-11.7-fold increase in cell numbers) and exhibited increased osteogenic differentiation (17.4-fold increase in calcium mineralization) compared to human mesenchymal stem cells (hMSCs), paralleling in vivo conditions. Comparative RNA-seq identified 40 and 91 previously unknown and uniquely expressed fallow deer (FD) proliferation and mineralization genes, respectively, including uhrf1 and s100a10. Immunofluorescence studies showed that uhrf1 and s100a10 were expressed in regenerating deer antlers while gene overexpression and gene knockdown studies demonstrated the proliferation contributions of uhrf1 and mineralization capabilities of s100a10. CONCLUSION: Using a simple, in vitro comparative RNA-seq approach, novel genes pertinent to fast bony antler regeneration were identified and their proliferative/osteogenic function was verified via gene overexpression, knockdown, and immunostaining. This combinatorial approach may be applicable to discover unique gene contributions between any two organisms for a given phenomenon-of-interest.

Differential effects of the PI3K/AKT pathway on antler stem cells for generation and regeneration of antlers in vitro.

Understanding the role of the PI3K/AKT pathway in regulating basic antler stem cell parameters and angiogenesis may provide an insight into the mechanisms underlying mammalian appendage development. The present study took multiple approaches in vitro to investigate the effects of the PI3K/AKT pathway on antler stem cells. By addition of LY294002, proliferation rate of the antlerogenic periosteum (AP) cells was decreased significantly (p<0.01), while the proliferation rate of the pedicle periosteum (PP) cells decreased to a lesser extent; the cytoskeleton of the AP cells was essentially collapsed; and the PP cells significantly shrunken. By addition of LY294002 or KU-0063794, formation of networking tubular structures from HUVECs in the AP or PP cell conditioned medium was significantly inhibited; whereas, expression level of VEGF-B mRNA in the AP or PP cells was decreased by the former, and increased by the latter significantly. Therefore, the results suggest that the PI3K/AKT pathway is involved in proliferation and differentiation of the AP and the PP cells, and plays a more important role in the former than in the latter.

WNT4 acts downstream of BMP2 to mediate the regulation of ATRA signaling on RUNX1 expression: Implications for terminal differentiation of antler chondrocytes.

Although ATRA is involved in regulating the proliferation and differentiation of chondrocytes, its underlying mechanism remains unknown. Here we showed that ATRA could stimulate the proliferation of antler chondrocytes and expression of COL X and MMP13 which were two well-known markers for hypertrophic chondrocytes. Silencing of CRABP2 prevented the induction of ATRA on chondrocyte terminal differentiation, while overexpression of CRABP2 exhibited the opposite effects. CYP26A1 and CYP26B1 weakened the sensitivity of antler chondrocytes to ATRA. Further analysis evidenced that ATRA might induce chondrocyte terminal differentiation and modulate the expression of BMP2, WNT4, and RUNX1 through RARα/RXRα. Knockdown of BMP2 enhanced the induction of ATRA on the expression of COL X and MMP13, whereas overexpression of BMP2 abrogated this effectiveness. WNT4 might mediate the effects of ATRA and BMP2 on chondrocyte terminal differentiation. Dysregulation of BMP2 impaired the regulation of ATRA on WNT4 expression. Administration of ATRA to antler chondrocytes transfected with RUNX1 siRNA failed to induce the differentiation. Conversely, rRUNX1 strengthened the stimulation of ATRA on the expression of COL X and MMP13. Simultaneously, RUNX1 was a downstream effector of BMP2 and WNT4 in chondrocyte terminal differentiation. Moreover, WNT4 might play an important role in the crosstalk between BMP2 and RUNX1. Attenuation of BMP2 or WNT4 enhanced the interaction between ATRA and RUNX1, while constitutive expression of BMP2 or WNT4 reversed the regulation of ATRA on RUNX1. Collectively, WNT4 may act downstream of BMP2 to mediate the effects of ATRA on the terminal differentiation of antler chondrocytes through targeting RUNX1.