Myogenic exosome miR-140-5p modulates skeletal muscle regeneration and injury repair by regulating muscle satellite cells.

Muscle satellite cells (SCs) play a crucial role in the regeneration and repair of skeletal muscle injuries. Previous studies have shown that myogenic exosomes can enhance satellite cell proliferation, while the expression of miR-140-5p is significantly reduced during the repair process of mouse skeletal muscle injuries induced by BaCl2. This study aims to investigate the potential of myogenic exosomes carrying miR-140-5p inhibitors to activate SCs and influence the regeneration of injured muscles. Myogenic progenitor cell exosomes (MPC-Exo) and contained miR-140-5p mimics/inhibitors myogenic exosomes (MPC-Exo140+ and MPC-Exo140-) were employed to treat SCs and use the model. The results demonstrate that miR-140-5p regulates SC proliferation by targeting Pax7. Upon the addition of MPC-Exo and MPC-Exo140-, Pax7 expression in SCs significantly increased, leading to the transition of the cell cycle from G1 to S phase and an enhancement in cell proliferation. Furthermore, the therapeutic effect of MPC-Exo140- was validated in animal model, where the expression of muscle growth-related genes substantially increased in the gastrocnemius muscle. Our research demonstrates that MPC-Exo140- can effectively activate dormant muscle satellite cells, initiating their proliferation and differentiation processes, ultimately leading to the formation of new skeletal muscle cells and promoting skeletal muscle repair and remodeling.

Exercise-inducible circulating extracellular vesicle irisin promotes browning and the thermogenic program in white adipose tissue.

AIM: Exercise can reduce body weight and promote white fat browning, but the underlying mechanisms remain largely unknown. This study investigated the role of fibronectin type III domain-containing protein 5 (FNDC5)/Irisin, a hormone released from exercising muscle, in the browning of white fat in circulating extracellular vesicles (EVs). METHODS: Mice were subjected to a 4 weeks of running table exercise, and fat browning was analyzed via histology, protein blotting and qPCR. Circulating EVs were extracted by ultrahigh-speed centrifugation, and ELISA was used to measure the irisin concentration in the circulating EVs. Circulating EVs that differentially expressed irisin were applied to adipocytes, and the effect of EV-irisin on adipocyte energy metabolism was analyzed by immunofluorescence, protein blotting, and cellular oxygen consumption rate analysis. RESULTS: During sustained exercise, the mice lost weight and developed fat browning. FNDC5 was induced, cleaved, and secreted into irisin, and irisin levels subsequently increased in the plasma during exercise. Interestingly, irisin was highly expressed in circulating EVs that effectively promoted adipose browning. Mechanistically, the circulating EV-irisin complex is transported intracellularly by the adipocyte membrane receptor integrin αV, which in turn activates the AMPK signaling pathway, which is dependent on mitochondrial uncoupling protein 1 to cause mitochondrial plasmonic leakage and promote heat production. After inhibition of the AMPK signaling pathway, the effects of the EV-irisin on promoting fat browning were minimal. CONCLUSION: Exercise leads to the accumulation of circulating EV-irisin, which enhances adipose energy metabolism and thermogenesis and promotes white fat browning in mice, leading to weight loss.

Natural Evolution of Porcine Epidemic Diarrhea Viruses Isolated from Maternally Immunized Piglets.

The porcine epidemic diarrhea virus (PEDV) can cause severe piglet diarrhea or death in some herds. Genetic recombination and mutation facilitate the continuous evolution of the virus (PEDV), posing a great challenge for the prevention and control of porcine epidemic diarrhea (PED). Disease materials of piglets with PEDV vaccination failure in some areas of Shanxi, Henan and Hebei provinces of China were collected and examined to understand the prevalence and evolutionary characteristics of PEDV in these areas. Forty-seven suspicious disease materials from different litters on different farms were tested by multiplex PCR and screened by hematoxylin-eosin staining and immunohistochemistry. PEDV showed a positivity rate of 42.6%, infecting the small and large intestine and mesenteric lymph node tissues. The isolated strains infected Vero, PK-15 and Marc-145 multihost cells and exhibited low viral titers in all three cell types, as indicated by their growth kinetic curves. Possible putative recombination events in the isolates were identified by RDP4.0 software. Sequencing and phylogenetic analysis showed that compared with the classical vaccine strain, PEDV SX6 contains new insertion and mutations in the S region and belongs to genotype GIIa. Meanwhile, ORF3 has the complete amino acid sequence with aa80 mutated wild strains, compared to vaccine strains CV777, AJ1102, AJ1102-R and LW/L. These results will contribute to the development of new PEDV vaccines based on prevalent wild strains for the prevention and control of PED in China.

MiR-222-3p suppresses C2C12 myoblast proliferation and differentiation via the inhibition of IRS-1/PI3K/Akt pathway.

Numerous studies have revealed the profound impact of microRNAs on regulating skeletal muscle development and regeneration. However, the biological function and regulation mechanism of miR-222-3p in skeletal muscle remains largely unknown. In this study, miR-222-3p was found to be abundantly expressed in the impaired skeletal muscles, indicating that it might have function in the development and regeneration process of the skeletal muscle. MiR-222-3p overexpression impeded C2C12 myoblast proliferation and myogenic differentiation, whereas inhibition of miR-222-3p got the opposite results. The dual-luciferase reporter assay showed that insulin receptor substrate-1 (IRS-1) was the target gene of miR-222-3p. We next found that knockdown of IRS-1 could obviously suppress C2C12 myoblast proliferation and differentiation. Additionally, miR-222-3p-induced repression of myoblast proliferation and differentiation was verified to be associated with a decrease in phosphoinositide 3-kinase (PI3K)-Akt signaling. Overall, we demonstrated that miR-222-3p inhibited C2C12 cells myogenesis via IRS-1/PI3K/Akt pathway. Therefore, miR-222-3p may be used as a therapeutic target for alleviating muscle loss caused by inherited and nonhereditary diseases.

CCE and EODF as two distinct non-shivering thermogenesis models inducing weight loss.

Increasing energy expenditure and reducing energy intake are considered two classical methods to induce weight loss. Weight loss through physical methods instead of drugs has been a popular research topic nowadays, but how these methods function in adipose and cause weight loss in body remains unclear. In this study, we set up chronic cold exposure (CCE) and every-other-day fasting (EODF) as two distinct models in long-term treatment to induce weight loss, recording their own characteristics in changes of body temperature and metabolism. We investigated the different types of non-shivering thermogenesis induced by CCE and EODF in white and brown adipose tissue through sympathetic nervous system (SNS), creatine-driven pathway, and fibroblast growth factor 21 (FGF21)-adiponectin axis. CCE and EODF could reduce body weight, lipid composition, increase insulin sensitivity, promote the browning of white fat, and increase the expression of endogenous FGF21 in adipose tissue. CCE stimulated the SNS and increased the thermogenic function of brown fat, and EODF increased the activity of protein kinase in white fat. In this study, we further explained the thermogenic mechanism function in adipose and metabolic benefits of the stable phenotype through physical treatments used for weight loss, providing more details for the literature on weight loss models. The influence on metabolism, non-shivering thermogenesis, endogenous FGF21, and ADPN changes in the long-term treatment of distinct methods (increasing energy expenditure and decreasing energy intake) to induce weight loss.

Bone marrow mesenchymal stem cell-derived exosomes reduce insulin resistance and obesity in mice via the PI3K/AKT signaling pathway.

Obesity is a common chronic metabolic disease that induces chronic systemic inflammation in the body, eventually leading to related complications such as insulin resistance (IR), type 2 diabetes mellitus, and metabolic syndromes such as cardiovascular disease. Exosomes transfer bioactive substances to neighboring or distal cells through autosomal, paracrine, or distant secretion, regulating the gene and protein expression levels of receptor cells. In this study, we investigated the effect of mouse bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) on high-fat diet obese mice and mature 3T3-L1 adipocyte models of IR. BMSC-Exo treatment of obese mice promoted their metabolic homeostasis, including reduction of obesity, inhibition of M1-type proinflammatory factor expression, and improvement of insulin sensitivity. In vitro analysis revealed that BMSC-Exos improved IR and lipid droplet accumulation in mature 3T3-L1 adipocytes treated with palmitate (PA). Mechanistically, BMSC-Exos cause increased glucose uptake and improved IR in high-fat chow-fed mice and PA-acting 3T3-L1 adipocytes by activating the phosphoinositide 3-kinases/protein kinase B (PI3K/AKT) signaling pathway and upregulating glucose transporter protein 4 (GLUT4) expression. This study offers a new perspective for the development of treatments for IR in obese and diabetic patients.

Expression analysis of irisin during different development stages of skeletal muscle in mice.

BACKGROUND: As a newly discovered muscle factor secreted by skeletal muscle cells, irisin is a polypeptide fragment formed from hydrolysis of fibronectin type Ⅲ domain-containing protein 5 (FNDC5). Irisin can promote beigeing of white adipose tissue (WAT) and regulate glucose and lipid metabolisms. However, the functions of irisin in skeletal muscle development remain largely unknown. In order to characterize the expression of irisin, this study investigated the expression of irisin precursor FNDC5 in myoblasts and skeletal muscles during different developmental stages of SPF mice. RESULTS: The Western blot, quantitative real-time PCR (qRT-PCR), and immunofluorescence assay results showed that FNDC5 was expressed in all the developmental stages of myoblasts and gastrocnemius, but its expression differed at different stages. FNDC5 protein exhibited the highest expression in gastrocnemius of sexually mature mice, followed by elderly mice and adolescent mice, and it displayed the lowest expression in pups. Additionally, FNDC5 protein was mainly expressed in cytoplasm, and it had the highest expression in primary myoblasts, followed by the myotubes with the lowest expression in C2C12 myogenic cells. CONCLUSIONS: Overall, FNDC5 was mainly expressed in cytoplasm and extracellular matrix with different expression levels at different developmental stages of skeletal muscle cells and tissues in mice. This study will provide new strategies for promoting skeletal muscle development and treating muscle- and metabolism-related disease by using irisin.

Myoblast-derived exosomes promote the repair and regeneration of injured skeletal muscle in mice.

When skeletal muscle is damaged, satellite cells (SCs) are activated to proliferate rapidly and fuse with the damaged muscle fibers to form new muscle fibers, thereby promoting muscle growth and remodeling and repair of trauma. Exosomes from differentiating human skeletal muscle cells trigger myogenesis of stem cells and provide biochemical cues for skeletal muscle regeneration. Therefore, we hypothesized that, when muscles are injured, myoblast-derived exosomes may regulate muscle repair and regeneration. Here, we investigated the underlying mechanism by applying C2C12-derived exosomes to injured mouse skeletal muscles. The expression levels of skeletal muscle regeneration factors paired box 7 and lipid-promoting factor peroxisome proliferator-activated receptor γ were upregulated, whereas the expression levels of fibrosis factors collagen-1 and α-smooth muscle actin decreased. The expression of proliferating cell nuclear antigen was elevated after applying C2C12-derived exosomes to SCs. Application of C2C12-derived exosomes to fibro-adipogenic progenitors resulted in an increase in peroxisome proliferator-activated receptor γ expression and adipogenesis capacity, whereas α-smooth muscle actin expression and fibrosis capacity decreased. Analysis of the transcriptome and proteome of SCs after treatment with exosomes showed the involvement of multiple biological processes, including proliferation and differentiation of SCs, muscle regeneration, skeletal muscle atrophy, and the inflammatory response after muscle injury. Hence, our data suggest that C2C12-derived exosomes can promote the regeneration of skeletal muscle fibers, accelerate the production of fat from damaged muscles, inhibit the fibrosis of damaged muscles, and accelerate injury repair, which is related to exosome-mediated regulation of the proliferation of SCs, differentiation of fibro-adipogenic progenitors, and modulation of SC mRNA expression and protein formation and decomposition.

Irisin promotes the browning of white adipocytes tissue by AMPKα1 signaling pathway.

The development of white adipose tissue (WAT) browning helps to protect animals from cold conditions and prevent obesity. AMPKα1 has been involved in the process of white adipocytes browning. Although Irisin plays a vital role in the browning of WAT, the detailed regulatory mechanism of Irisin inducing the browning of WAT remains unclear. Herein, we firstly investigated the potential roles of differentiation and Irisin in regulating the browning of 3T3-L1 cells. The results found that they could significantly increase the number of lipid droplets and upregulate the expression levels of UCP1, PGC-1α, PRDM16, and p-AMPKα1. Then we proved the effectiveness of the AMPKα1 signaling pathway in the process of Irisin inducing the browning of 3T3-L1 cells. Compared with si-NC, si-AMPKα1 not only decreased the number of Irisin-induced lipid droplets, but also attenuated the expression of Irisin-induced UCP1, PGC-1α, and PRDM16 protein and mRNA levels in 3T3-L1 cells. Furthermore, the results showed that Irisin increased the positive distribution of UCP1 and PGC-1α, and upregulated the expression of UCP1, PGC-1α, and PRDM16 at both protein and mRNA levels in WAT. Once siRNA treated mice, the facilitation of Irisin on UCP1 and PGC-1α in si-AMPKα1-injected mice was lower than that in si-NC-injected mice. Compared with si-NC, si-AMPKα1 significantly downregulated the expression of UCP1, PGC-1α, and PRDM16 in Irisin-injected mice. Taken together, our results demonstrate that Irisin activates the AMPKα1 pathway to promote the browning of WAT by upregulating the mRNA and protein levels of UCP1, PGC-1α, and PRDM16.

microRNA-140 Regulates PDGFRα and Is Involved in Adipocyte Differentiation.

In recent years, the studies of the role of microRNAs in adipogenesis and adipocyte development and the corresponding molecular mechanisms have received great attention. In this work, we investigated the function of miR-140 in the process of adipogenesis and the molecular pathways involved, and we found that adipogenic treatment promoted the miR-140-5p RNA level in preadipocytes. Over-expression of miR-140-5p in preadipocytes accelerated lipogenesis along with adipogenic differentiation by transcriptional modulation of adipogenesis-linked genes. Meanwhile, silencing endogenous miR-140-5p dampened adipogenesis. Platelet-derived growth factor receptor alpha (PDGFRα) was shown to be a miR-140-5p target gene. miR-140-5p over-expression in preadipocyte 3T3-L1 diminished PDGFRα expression, but silencing of miR-140-5p augmented it. In addition, over-expression of PDGFRα suppressed adipogenic differentiation and lipogenesis, while its knockdown enhanced these biological processes of preadipocyte 3T3-L1. Altogether, our current findings reveal that miR-140-5p induces lipogenesis and adipogenic differentiation in 3T3-L1 cells by targeting PDGFRα, therefore regulating adipogenesis. Our research provides molecular targets and a theoretical basis for the treatment of obesity-related metabolic diseases.

A novel strategy for optimal component formula of anti-PRRSV from natural compounds using tandem mass tag labeled proteomic analyses.

BACKGROUND: Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most important porcine viral diseases which have been threatening the pig industry in China. At present, most commercial vaccines fail to provide complete protection because of highly genetic diversity of PRRSV strains. This study aimed to optimize a component formula from traditional Chinese medicine(TCM)compounds with defined chemical characteristics and clear mechanism of action against PRRSV. METHODS: A total of 13 natural compounds were screened for the anti-PRRSV activity using porcine alveolar macrophages (PAMs). Three compounds with strong anti-PRRSV activity were selected to identify their potential protein targets by proteomic analysis. The optimal compound formula was determined by orthogonal design based on the results of proteomics. MTT assay was used to determine the maximum non-cytotoxic concentration (MNTC) of each compound using PAMs. QPCR and western blot were used to investigate the PRRSV N gene and protein expression, respectively. The Tandem Mass Tag (TMT) technique of relative quantitative proteomics was used to detect the differential protein expression of PAMs treated with PRRSV, matrine (MT), glycyrrhizic acid (GA) and tea saponin (TS), respectively. The three concentrations of these compounds with anti-PRRSV activity were used for orthogonal design. Four formulas with high safety were screened by MTT assay and their anti-PRRSV effects were evaluated. RESULTS: MT, GA and TS inhibited PRRSV replication in a dose-dependent manner. CCL8, IFIT3, IFIH1 and ISG15 were the top four proteins in expression level change in cells treated with MT, GA or TS. The relative expression of IFIT3, IFIH1, ISG15 and IFN-ß mRNAs were consistent with the results of proteomics. The component formula (0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS) showed synergistic anti-PRRSV effect. CONCLUSIONS: The component formula possessed anti-PRRSV activity in vitro, in which the optimal dosage on PAMs was 0.4 mg/mL MT + 0.25 mg/mL GA + 1.95 µg/mL TS. Compatibility of the formula was superposition of the same target with GA and TS, while different targets of MT. IFN-ß may be one of the targets of the component formula possessed anti-PRRSV activity.

Vitellogenin 2 promotes muscle development and stimulates the browning of white fat.

Eggs are rich in nutrients and contain a lot of protein. Although eggs have proved to accelerate the growth of C2C12 cells, the regulatory and mechanism of fertilized egg yolk extract (FEYE) on skeletal muscle development and fat metabolism remains unclearly. The mice were treated with FEYE by gavage for 24 d, we found that FEYE can inhibit the expression of skeletal muscle atrophy genes such as MSTN and Murf-1, and up-regulate the expression levels of MYOD, MYOG and Irisin. In addition, the treatment of FEYE induced UCP1 and PGC1α high expression in WAT, thereby causing WAT browning reaction. In order to confirm the composition of FEYE, we performed protein full spectrum identification (LC MS/MS) analysis and found the most enriched component is vitellogenin 2 (VTG2). Therefore, we added the recombinant protein VTG2 to C2C12 cells and found that VTG2 promoted the proliferation and differentiation of C2C12 cells. After that, we further proved that VTG2 inhibited the expression of MSTN and improved the expression of MYOD and Irisin. Finally, the dual luciferase test proved that VTG2 directly inhibited the transcriptional activity of MSTN. Our results conclude that FEYE inhibits the expression of MSTN in muscle tissues by delivering VTG2, thereby promoting skeletal muscle development, and can also promote the expression level of FNDC5 in serum. Then, FNDC5 acts on the fat through the serum, stimulating the browning reaction of white adipocytes. Therefore, VTG2 can be used to stop muscle consumption, improve skeletal muscle aging, and prevent obesity.

[Isolation, identification and prokaryotic expression of a bacteriocin-like substance from Bacillus licheniformis].

The ban on addition of antibiotics in animal feed in China has made the search for new antibiotics substitutes, e.g. bacteriocin, a hot topic in research. The present study successfully isolated an antibacterial substance producing strain of Bacillus sp. from alpaca feces by agar diffusion method, using Escherichia coli, Salmonella enterica, Staphylococcus aureus, Staphylococcus epidermidis, Micrococcus luteus and Listeria monocytogenes as indicator bacteria. The isolated strain was named as B. licheniformis SXAU06 based on colony morphology, Gram staining and 16S rRNA gene sequence. The antibacterial substance was isolated and purified through a series of procedures including (NH4)2SO4 precipitation, chloroform extraction, molecular interception and SDS-PAGE analysis. Bioinformatics analysis of the LC-MS/MS data indicated that the antibacterial substance was a bacteriocin-like substance (BLIS) with an approximate molecular weight of 14 kDa, and it was designated as BLIS_SXAU06. BLIS_SXAU06 exhibited high resistance to treatment of proteinase K, high temperature, high acidity and alkalinity. BLIS_SXAU06 was heterologously expressed in E. coli and the recombinant BLIS_SXAU06 exhibited effective antibacterial activity against S. aureus, S. epidermidis, M. luteus, and L. monocytogenes, showing potential to be investigated further.

Exosomal miRNA derived from keratinocytes regulates pigmentation in melanocytes.

BACKGROUND: Pigmentation is controlled by complex mechanisms. Evidence suggests that miRNAs can regulate pigmentation. However, the mechanism has not been fully elucidated. Objective In this study, we revealed a novel mechanism that regulates pigmentation involving exosomes, miRNAs and the crosstalk between keratinocytes and melanocytes. METHODS: The expression and localization of exosome specific marker TSG101 in keratinocytes and melanocytes; Changes of melanin content in melanocytes after co-culture of exosome and melanocytes; Expression changes of target gene TYR and its related genes and inhibitory effect of miR-330-5p on pigmentation were studied by using various molecular biological techniques. RESULTS: In this experiment, we used miR-330-5p in keratinocytes to verify the effect of keratinocyte derived exosome on melanocyte pigmentation. First, we found that keratinocytes secrete exosomes carrying miR-330-5p; moreover, greater miR-330-5p expression was found in exosomes derived from keratinocytes that overexpressed miR-330-5p. Second, we found that exosomes derived from keratinocytes with overexpression of miR-330-5p caused a significant increase in miR-330-5p in melanocytes. Finally, exosomes derived from keratinocytes that overexpressed miR-330-5p induced a significant decrease in the production of melanin and expression of TYR in melanocytes. Meanwhile, we overexpressed miR-330-5p in melanocytes, which also proved the inhibitory effect of miR-330-5p on pigmentation. CONCLUSION: These findings suggest that keratinocytes crosstalk with melanocytes in the epidermal melanin unit via exosomal miRNAs. These studies reveal an important role of exosomes in melanocyte pigmentation, which opens a new pathway of melanogenesis.

Effects of myostatin deficiency on PGC-1α and FNDC5 expression in three different murine muscle types.

Myostatin (MSTN) is a key negative regulator of muscle growth and development. Skeletal, cardiac, and smooth muscles were isolated from MSTN knockout (MSTN-∕-) and control mice to investigate the effect of knocking out MSTN on peroxisome proliferator-activated receptor 1 coactivator (PGC-1α)-III and fibronectin domain 5 (FNDC5) expression. Various molecular biology techniques were used to analyze the changes in PGC-1α-FNDC5 in different muscle types from MSTN-∕- mice. The expression levels of PGC-1α and FNDC5 in the skeletal, cardiac, and smooth muscles of MSTN-∕- mice differed from those in the skeletal, cardiac, and smooth muscles of normal mice. This study revealed that knocking out MSTN resulted in inconsistent PGC-1α and FNDC5 expression in specific muscles. It proved for the first time that MSTN deletion attenuated the expression of PGC-1α and FNDC5 in three different murine muscle types. MSTN deletion may have additional effects on the status ofFNDC5 expression. Further research, however, is needed to confirm this conclusion.

Let-7b regulates alpaca hair growth by downregulating ectodysplasin A.

Hypohidrotic ectodermal dysplasia (HED), also known as anhidrotic ectodermal dysplasia, is characterized by the clinical manifestations of less sweat or no sweat, sparse or no hair, tooth agenesis and/or abnormal tooth morphology. The characteristics of alpaca ear hair differ from the back hair. The ectodysplasin A (EDA) signaling pathway has a regulatory effect on skin development and hair growth. The aim of the present study was to study the effects of EDA on alpaca hair growth by examining the mRNA and protein expression levels of EDA in alpaca ear and back skin by reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. Results indicated that EDA expression was higher in the ear skin compared with the back skin. The expression levels of let­7b in the skin of healthy alpacas varies; the difference between let­7b expression levels of the ear and back have been reported to be >2­fold, suggesting a role for let­7b in the development of adult alpaca skin and hair follicles. A dual­luciferase reporter vector was constructed to verify the targeting relationship between microRNA let­7b and EDA, and the results revealed that EDA was a target gene of let­7b. Alpaca skin fibroblasts were transfected with a let­7b eukaryotic expression vector to investigate the regulatory relationship between let­7b and EDA. The expression of EDA was decreased in the transfected group; immunocytochemical results demonstrated that the EDA protein was abundantly expressed in the fibroblast cytoplasm. EDA protein expression was weaker in the transfected cells than in the untransfected cells. These results suggested that EDA may serve a role in alpaca hair growth and is probably a target gene of let­7b; let­7b downregulated EDA mRNA and protein expressions, which suggested that let­7b may regulate alpaca hair growth. These conclusions suggested that let­7b may be associated with HED.

FGF21 regulates melanogenesis in alpaca melanocytes via ERK1/2-mediated MITF downregulation.

Fibroblast growth factor 21 (FGF21) is known as a metabolic regulator to regulate the metabolism of glucose and lipids. However, the underlying mechanism of FGF21 on melanin synthesis remains unknown. Therefore, the current study investigates the effect of FGF21 on melanogenesis in alpaca melanocytes. We transfected the FGF21 into alpaca melanocytes, then detected the melanin contents, protein and mRNA levels of pigmentation-related genes in order to determine the melanogenesis-regulating pathway of FGF21. The results showed that FGF21 overexpression suppressed melanogenesis and decreased the expression of the major target genes termed microphthalmia-associated transcription factor (MITF) and its downstream genes, including tyrosinase (TYR) and tyrosinase-related protein 2 (TRP2). However FGF21 increased the expression of phospho-extracellular signal-regulated kinase (p-Erk1/2). In contrast, FGF21-siRNA, a small interference RNA mediating FGF21 silencing, abolished the inhibition of melanogenesis. Altogether, FGF21 may decrease melanogenesis in alpaca melanocytes via ERK activation and subsequent MITF downregulation, which is then followed by the suppression of melanogenic enzymes and melanin production.

MiR-137 affects melanin synthesis in mouse melanocyte by repressing the expression of c-Kit and Tyrp2 in SCF/c-Kit signaling pathway.

Previously, we created miR-137 overexpressing transgenic mice that produced lighten color phenotypes including gray mice phenotype. However, the miR-137 functional role in coat color regulation is still not well understood. In this study, the quantity of melanin granule and the relative expression of TYRP2 in gray miR-137 overexpression transgenic mouse skin were significantly lower than that in C57BL/6J black mouse skin. The mRNA and protein expression level of c-Kit and c-Kit downstream gene Tyrp2 in miR-137 expression plasmid-transfected melanocytes were significantly down-regulated comparing with that of the control melanocytes. In melanocytes, miR-137 overexpression could decrease the enhanced expression of c-Kit and Tyrp2 and the increased melanin production caused by UV treatment. The target relationship of miR-137 and c-Kit was identified by luciferase assay. The results suggest that miR-137 could inhibit melanogenesis in mouse skin melanocytes by repressing the expression of c-Kit and Tyrp2 in SCF/c-Kit signaling pathway.

Expression and tissue distribution of hepatocyte growth factor (HGF) and its receptor (c-Met) in alpacas (Vicugna pacos) skins associated with white and brown coat colors.

Hepatocyte growth factor (HGF)/c-Met signaling has been considered as a key pathway in both melanocyte development and melanogenesis. To understand better the expression patterns and tissue distribution characterization of HGF and its receptor c-Met in skin of white versus brown alpaca (Vicugna pacos), we detected the tissue distribution of HGF and c-Met using immunohistochemistry and analyzed the expression patterns by using Western blot and quantitative real time PCR (qPCR). Immunohistochemistry analysis demonstrated that HGF staining robustly increased in the dermal papilla and mesenchymal cells of white alpaca skin compared with that of brown. However, c-Met staining showed strongly positive result, particularly inhair matrix and root sheath in brown alpaca skin. Western blot and qPCR results suggested that HGF and c-Met were expressed at significantly high levels in white and brown alpaca skins, respectively, and protein and transcripts possessed the same expression pattern in white and brown alpaca skins. The results suggested that HGF/c-Met signaling functions in alpaca coat color formation offer essential theoretical basis for further exploration of the role of HGF/c-Met signaling in pigment formation.

MicroRNA-27a-3p Inhibits Melanogenesis in Mouse Skin Melanocytes by Targeting Wnt3a.

MicroRNAs (miRNAs) play an essential role in the regulation of almost all the biological processes, including melanogenesis. MiR-27a-3p is nearly six times higher in white alpaca skin compared to brown skin, which indicates that miR-27a-3p may be a candidate regulator for melanogenesis. Wnt3a plays an important role in promoting melanoblasts to differentiate into melanocytes and melanogenesis. To confirm the function of miR-27a-3p to melanogenesis in mammals, miR-27a-3p mimic, inhibitor and their negative control were transfected into mouse melanocytes. As a result, miR-27a-3p inhibits melanogenesis by repressing Wnt3a at post-transcriptional level. A significant decrease in Wnt3a luciferase activity was observed in 293T cells co-transfected with the matched luciferase reporter vector and pre-miR-27a. Furthermore, the presence of exogenous miR-27a-3p significantly decreased Wnt3a protein expression rather than mRNA and reduced ß-catenin mRNA levels in melanocytes. The over-expression of miR-27a-3p significantly increased the melanin content of melanocytes. However, miR-27a-3p inhibitor performs an opposite effect on melanogenesis. Wnt3a is one target of miR-27a-3p. MiR-27a-3p could inhibit Wnt3a protein amount by post-transcriptional regulation and melanogenesis in mouse melanocytes. Previous studies reported that Wnt3a promoted melanogenensis in mouse melanocytes. Thus, miR-27-3p inhibits melanogenesis by repressing Wnt3a protein expression.