In Utero Cell Treatment of Hemophilia A Mice via Human Amniotic Fluid Mesenchymal Stromal Cell Engraftment.

Hemophilia is a genetic disorder linked to the sex chromosomes, resulting in impaired blood clotting due to insufficient intrinsic coagulation factors. There are approximately one million individuals worldwide with hemophilia, with hemophilia A being the most prevalent form. The current treatment for hemophilia A involves the administration of clotting factor VIII (FVIII) through regular and costly injections, which only provide temporary relief and pose inconveniences to patients. In utero transplantation (IUT) is an innovative method for addressing genetic disorders, taking advantage of the underdeveloped immune system of the fetus. This allows mesenchymal stromal cells to play a role in fetal development and potentially correct genetic abnormalities. The objective of this study was to assess the potential recovery of coagulation disorders in FVIII knockout hemophilia A mice through the administration of human amniotic fluid mesenchymal stromal cells (hAFMSCs) via IUT at the D14.5 fetal stage. The findings revealed that the transplanted human cells exhibited fusion with the recipient liver, with a ratio of approximately one human cell per 10,000 mouse cells and produced human FVIII protein in the livers of IUT-treated mice. Hemophilia A pups born to IUT recipients demonstrated substantial improvement in their coagulation issues from birth throughout the growth period of up to 12 weeks of age. Moreover, FVIII activity reached its peak at 6 weeks of age, while the levels of FVIII inhibitors remained relatively low during the 12-week testing period in mice with hemophilia. In conclusion, the results indicated that prenatal intrahepatic therapy using hAFMSCs has the potential to improve clotting issues in FVIII knockout mice, suggesting it as a potential clinical treatment for individuals with hemophilia A.

miR-196a provides antioxidative neuroprotection via USP15/Nrf2 regulation in Huntington's disease.

Huntington's disease (HD) is a devastating neurodegenerative disorder characterized by the accumulation of mutant Huntingtin protein (mHTT) and oxidative stress-induced neuronal damage. Based on previous reports, microRNA-196a (miR-196a) has emerged as a potential therapeutic target due to its neuroprotective effects in various neurodegenerative diseases. However, whether miR-196a functions through antioxidative effects is still unknown. In this study, we demonstrated that HD models, both in vitro and in vivo, exhibit elevated levels of reactive oxygen species (ROS) and increased neuronal death, and miR-196a mitigates ROS levels and reduces cell death in HD cells. Moreover, we elucidated that miR-196a facilitates the translocation of nuclear factor erythroid 2 (Nrf2) into the nucleus, enhancing the transcription of antioxidant genes, including heme oxygenase-1 (HO-1). We further identified ubiquitin-specific peptidase 15 (USP15), a direct target of miR-196a related to the Nrf2 pathway, and USP15 exacerbates mHTT aggregate formation while partially counteracting miR-196a-induced reductions in mHTT levels. Taken together, these findings shed light on the multifaceted role of miR-196a in HD, highlighting its potential as a therapeutic avenue for ameliorating oxidative stress and neurodegeneration in this debilitating disease.

Lactoferrin Ameliorates Ovalbumin-Induced Asthma in Mice through Reducing Dendritic-Cell-Derived Th2 Cell Responses.

Asthma is a chronic respiratory disease with symptoms such as expiratory airflow narrowing and airway hyperresponsiveness (AHR). Millions of people suffer from asthma and are at risk of life-threatening conditions. Lactoferrin (LF) is a glycoprotein with multiple physiological functions, including antioxidant, anti-inflammatory, antimicrobial, and antitumoral activities. LF has been shown to function in immunoregulatory activities in ovalbumin (OVA)-induced delayed type hypersensitivity (DTH) in mice. Hence, the purpose of this study was to investigate the roles of LF in AHR and the functions of dendritic cells (DCs) and Th2-related responses in asthma. Twenty 8-week-old male BALB/c mice were divided into normal control (NC), ovalbumin (OVA)-sensitized, and OVA-sensitized with low dose of LF (100 mg/kg) or high dose of LF (300 mg/kg) treatment groups. The mice were challenged by intranasal instillation with 5% OVA on the 21st to 27th day after the start of the sensitization period. The AHR, cytokines in bronchoalveolar lavage fluid, and pulmonary histology of each mouse were measured. Serum OVA-specific IgE and IgG1 and OVA-specific splenocyte responses were further detected. The results showed that LF exhibited protective effects in ameliorating AHR, as well as lung inflammation and damage, in reducing the expression of Th2 cytokines and the secretion of allergen-specific antibodies, in influencing the functions of DCs, and in decreasing the level of Th2 immune responses in a BALB/c mouse model of OVA-induced allergic asthma. Importantly, we demonstrated that LF has practical application in reducing DC-induced Th2 cell responses in asthma. In conclusion, LF exhibits anti-inflammation and immunoregulation activities in OVA-induced allergic asthma. These results suggest that LF may act as a supplement to prevent asthma-induced lung injury and provide an additional agent for reducing asthma severity.

miR-196a enhances polymerization of neuronal microfilaments through suppressing IMP3 and upregulating IGF2 in Huntington's disease.

Huntington's disease (HD) is one of the inheritable neurodegenerative diseases, and these diseases share several similar pathological characteristics, such as abnormal neuronal morphology. miR-196a is a potential target to provide neuroprotective functions, and has been reported to enhance polymerization of neuronal microtubules in HD. While microtubules and microfilaments are two important components of the neuronal cytoskeleton, whether miR-196a improves neuronal microfilaments is still unknown. Here, we identify insulin-like growth factor 2 mRNA binding protein 3 (IMP3), and show that miR-196a directly suppresses IMP3 to increase neurite outgrowth in neurons. In addition, IMP3 disturbs neurite outgrowth in vitro and in vivo, and worsens the microfilament polymerization. Moreover, insulin-like growth factor-II (IGF2) is identified as the downstream target of IMP3, and miR-196a downregulates IMP3 to upregulate IGF2, which increases microfilamental filopodia numbers and activates Cdc42 to increase neurite outgrowth. Besides, miR-196a increases neurite outgrowth through IGF2 in different HD models. Finally, higher expression of IMP3 and lower expression IGF2 are observed in HD transgenic mice and patients, and increase the formation of aggregates in the HD cell model. Taken together, miR-196a enhances polymerization of neuronal microfilaments through suppressing IMP3 and upregulating IGF2 in HD, supporting the neuroprotective functions of miR-196a through neuronal cytoskeleton in HD.

A new mouse model of ankle instability induced by multiple mechanical sprains with controlled inversion angle and speed.

Ankle sprain occurs by a sudden and extreme inversion and plantarflexion at the ankle joint to cause ligamentous injuries. A portion of ankle sprain patients experience recurrent ankle sprains and develop chronic ankle instability (CAI). The present CAI animal models are single events with severe ligamentous injury using surgical transection of ligaments or manually overextending the ankle. Purpose: To simulate the mechanical and recurrent sprain injuries in CAI patients, we established a new ankle instability model with multiple ankle injuries using a self-designed machine to sprain the ankle with a controlled inversion angle and speed. Methods: Male C57BL/6J mice were used and respectively subjected to a sham operation, calcaneofibular ligament (CFL) transection, and mechanical ankle sprains. Three mechanical sprains were performed on the 13th and 185th day after the initial mechanical ankle sprain. Results: The first mechanical sprain and CFL transection induced ankle injury as indicated by an average of a 62% decrease in ankle pressure pain threshold and a 114% increase in the ankle thickness compared with the contralateral untreated ankle. The second and third mechanical sprains induced recurrent ankle injuries. The foot slips during beam tests were increased after mechanical ankle sprains but not after CFL transection, indicating the induction of motor balance deficits. Multiple mechanical ankle sprains induced significant gait changes in longer duration of stance (an average of 194% increase), swing (134%), and step cycle (147%) compared with CFL transection or sham operation, and slower walking speed (78% reduction) and shorter step distance (91%) after the third sprain. Conclusion: These results elucidate that multiple mechanical sprains, which induce recurrent ankle injuries, balance deficits, and gait changes, are a good model for investigating the mechanisms of CAI induced by recurrent sprain injuries.

Cruciform DNA Structures Act as Legible Templates for Accelerating Homologous Recombination in Transgenic Animals.

Inverted repeat (IR) DNA sequences compose cruciform structures. Some genetic disorders are the result of genome inversion or translocation by cruciform DNA structures. The present study examined whether exogenous DNA integration into the chromosomes of transgenic animals was related to cruciform DNA structures. Large imperfect cruciform structures were frequently predicted around predestinated transgene integration sites in host genomes of microinjection-based transgenic (Tg) animals (αLA-LPH Tg goat, Akr1A1eGFP/eGFP Tg mouse, and NFκB-Luc Tg mouse) or CRISPR/Cas9 gene-editing (GE) animals (αLA-AP1 GE mouse). Transgene cassettes were imperfectly matched with their predestinated sequences. According to the analyzed data, we proposed a putative model in which the flexible cruciform DNA structures acted as a legible template for DNA integration into linear DNAs or double-strand break (DSB) alleles. To demonstrate this model, artificial inverted repeat knock-in (KI) reporter plasmids were created to analyze the KI rate using the CRISPR/Cas9 system in NIH3T3 cells. Notably, the KI rate of the 5' homologous arm inverted repeat donor plasmid (5'IR) with the ROSA gRNA group (31.5%) was significantly higher than the knock-in reporter donor plasmid (KIR) with the ROSA gRNA group (21.3%, p < 0.05). However, the KI rate of the 3' inverted terminal repeat/inverted repeat donor plasmid (3'ITRIR) group was not different from the KIR group (23.0% vs. 22.0%). These results demonstrated that the legibility of the sequence with the cruciform DNA existing in the transgene promoted homologous recombination (HR) with a higher KI rate. Our findings suggest that flexible cruciform DNAs folded by IR sequences improve the legibility and accelerate DNA 3'-overhang integration into the host genome via homologous recombination machinery.

Cerebral Aß deposition in an Aß-precursor protein-transgenic rhesus monkey.

With the ultimate goal of developing a more representative animal model of Alzheimer's disease (AD), two female amyloid-ß-(Aß) precursor protein-transgenic (APPtg) rhesus monkeys were generated by lentiviral transduction of the APP gene into rhesus oocytes, followed by in vitro fertilization and embryo transfer. The APP-transgene included the AD-associated Swedish K670N/M671L and Indiana V717F mutations (APPSWE/IND) regulated by the human polyubiquitin-C promoter. Overexpression of APP was confirmed in lymphocytes and brain tissue. Upon sacrifice at 10 years of age, one of the monkeys had developed Aß plaques and cerebral Aß-amyloid angiopathy in the occipital, parietal, and caudal temporal neocortices. The induction of Aß deposition more than a decade prior to its usual emergence in the rhesus monkey supports the feasibility of creating a transgenic nonhuman primate model for mechanistic analyses and preclinical testing of treatments for Alzheimer's disease and cerebrovascular amyloidosis.

FGF9 promotes cell proliferation and tumorigenesis in TM3 mouse Leydig progenitor cells.

Fibroblast growth factor 9 (FGF9) modulates cell proliferation, differentiation and motility for development and tissue repair in normal cells. Growing evidence shows that abnormal activation of FGF9 signaling is associated with tumor malignancy. We have previously reported that FGF9 increases MA-10 mouse Leydig tumor cell proliferation, in vitro, and tumor growth, in vivo. Also, FGF9 promotes the tumor growth and liver metastasis of mouse Lewis lung cancer cells, in vivo. However, the effects of FGF9 in the early stage of tumorigenesis remains elusive. In this study, TM3 mouse Leydig progenitor cells, that are not tumorigenic in immunocompromised mice, were used as a model cell line to investigate the role of FGF9 in tumorigenesis. The results demonstrated that FGF9 significantly induced cell proliferation and activated the MAPK, PI3K and PLCγ signaling pathways in TM3 cells. The percentage of the cell number in G1 phase was reduced and that in S and G2/M phases was increased after FGF9 stimulation in TM3 cells. Cyclin D1, cyclin A1, CDK2, CDK1, and p21 expressions and the phosphorylation level of Rb were all induced in FGF9-treated TM3 cells. In addition, FGF9 increased the expression of FGF receptor 1-4 in TM3 cells, suggesting the positive feedback loop between FGF9 and FGFRs. Furthermore, in the allograft mouse model, FGF9 promoted the tumorigenesis of TM3 cells characterized by higher expression of tumor markers, such as tumor necrosis factor alpha (TNFα) and α-fetoprotein (AFP), in the subcutaneously inoculated TM3 cell tissue. Conclusively, FGF9 induced cell cycle to increase cell proliferation of TM3 cells through FAK, MAPK, PI3K/Akt and PLCγ signaling pathways, in vitro, and promoted the tumorigenesis of TM3 cell allograft tissue, in vivo, which is a potential marker for tumor as well as a target for cancer therapeutic strategies.

Suppression of Dendritic Cell Maturation by Kefir Peptides Alleviates Collagen-Induced Arthritis in Mice.

Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases.

The Role of Autophagy in Anti-Cancer and Health Promoting Effects of Cordycepin.

Cordycepin is an adenosine derivative isolated from Cordyceps sinensis, which has been used as an herbal complementary and alternative medicine with various biological activities. The general anti-cancer mechanisms of cordycepin are regulated by the adenosine A3 receptor, epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPKs), and glycogen synthase kinase (GSK)-3ß, leading to cell cycle arrest or apoptosis. Notably, cordycepin also induces autophagy to trigger cell death, inhibits tumor metastasis, and modulates the immune system. Since the dysregulation of autophagy is associated with cancers and neuron, immune, and kidney diseases, cordycepin is considered an alternative treatment because of the involvement of cordycepin in autophagic signaling. However, the profound mechanism of autophagy induction by cordycepin has never been reviewed in detail. Therefore, in this article, we reviewed the anti-cancer and health-promoting effects of cordycepin in the neurons, kidneys, and the immune system through diverse mechanisms, including autophagy induction. We also suggest that formulation changes for cordycepin could enhance its bioactivity and bioavailability and lower its toxicity for future applications. A comprehensive understanding of the autophagy mechanism would provide novel mechanistic insight into the anti-cancer and health-promoting effects of cordycepin.

The regulatory roles of microRNAs toward pathogenesis and treatments in Huntington's disease.

Huntington's disease (HD) is one of neurodegenerative diseases, and is defined as a monogenetic disease due to the mutation of Huntingtin gene. This disease affects several cellular functions in neurons, and further influences motor and cognitive ability, leading to the suffering of devastating symptoms in HD patients. MicroRNA (miRNA) is a non-coding RNA, and is responsible for gene regulation at post-transcriptional levels in cells. Since one miRNA targets to several downstream genes, it may regulate different pathways simultaneously. As a result, it raises a potential therapy for different diseases using miRNAs, especially for inherited diseases. In this review, we will not only introduce the update information of HD and miRNA, but also discuss the development of potential miRNA-based therapy in HD. With the understanding toward the progression of miRNA studies in HD, we anticipate it may provide an insight to treat this devastating disease, even applying to other genetic diseases.

FGF9/FGFR1 promotes cell proliferation, epithelial-mesenchymal transition, M2 macrophage infiltration and liver metastasis of lung cancer.

Fibroblast growth factors 9 (FGF9) modulates cell proliferation, differentiation and motility for development and repair in normal cells. Abnormal activation of FGF9 signaling is associated with tumor progression in many cancers. Also, FGF9 may be an unfavorable prognostic indicator for non-small cell lung cancer patients. However, the effects and mechanisms of FGF9 in lung cancer remain elusive. In this study, we investigated the FGF9-induced effects and signal activation profiles in mouse Lewis lung carcinoma (LLC) in vitro and in vivo. Our results demonstrated that FGF9 significantly induced cell proliferation and epithelial-to-mesenchymal transition (EMT) phenomena (migration and invasion) in LLC cells. Mechanism-wise, FGF9 interacted with FGFR1 and activated FAK, AKT, and ERK/MAPK signal pathways, induced the expression of EMT key proteins (N-cadherin, vimentin, snail, MMP2, MMP3 and MMP13), and reduced the expression of E-cadherin. Moreover, in the allograft mouse model, intratumor injection of FGF9 to LLC-tumor bearing C57BL/6 mice enhanced LLC tumor growth which were the results of increased Ki67 expression and decreased cleaved caspase-3 expression compared to control groups. Furthermore, we have a novel finding that FGF9 promoted liver metastasis of subcutaneous inoculated LLC tumor with angiogenesis, EMT and M2-macrophage infiltration in the tumor microenvironment. In conclusion, FGF9 activated FAK, AKT, and ERK signaling through FGFR1 with induction of EMT to stimulate LLC tumorigenesis and hepatic metastasis. This novel FGF9/LLC allograft animal model may therefore be useful to study the mechanism of liver metastasis which is the worst prognostic factor for lung cancer patients with distant organ metastasis.

STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos.

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (-1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (-1369~+28 nt), Δ2-pGzmg (-939~+28 nt), Δ3-pGzmg (-711~+28 nt) and Δ4-pGzmg (-417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the -417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Fibroblast Growth Factor 9 Stimulates Neuronal Length Through NF-kB Signaling in Striatal Cell Huntington's Disease Models.

Proper development of neuronal cells is important for brain functions, and impairment of neuronal development may lead to neuronal disorders, implying that improvement in neuronal development may be a therapeutic direction for these diseases. Huntington's disease (HD) is a neurodegenerative disease characterized by impairment of neuronal structures, ultimately leading to neuronal death and dysfunctions of the central nervous system. Based on previous studies, fibroblast growth factor 9 (FGF9) may provide neuroprotective functions in HD, and FGFs may enhance neuronal development and neurite outgrowth. However, whether FGF9 can provide neuronal protective functions through improvement of neuronal morphology in HD is still unclear. Here, we study the effects of FGF9 on neuronal length in HD and attempt to understand the related working mechanisms. Taking advantage of striatal cell lines from HD knock-in mice, we found that FGF9 increases total neuronal length and upregulates several structural and synaptic proteins under HD conditions. In addition, activation of nuclear factor kappa B (NF-kB) signaling by FGF9 was observed to be significant in HD cells, and blockage of NF-kB leads to suppression of these structural and synaptic proteins induced by FGF9, suggesting the involvement of NF-kB signaling in these effects of FGF9. Taken these results together, FGF9 may enhance total neuronal length through upregulation of NF-kB signaling, and this mechanism could serve as an important mechanism for neuroprotective functions of FGF9 in HD.

FGF9 induces neurite outgrowth upon ERK signaling in knock-in striatal Huntington's disease cells.

AIMS: Huntington's disease (HD) is a neurodegenerative disease that causes deficits in neurite outgrowth, which suggests that enhancement of neurite outgrowth is a potential direction by which to improve HD. Our previous publications showed that fibroblast growth factor 9 (FGF9) provides anti-apoptosis and anti-oxidative functions in striatal cell models of HD through the extracellular signal-regulated kinases (ERK) pathway, and FGF9 also stimulates cytoskeletons to enhance neurite outgrowth via nuclear factor kappa B (NF-kB) signaling. In this study, we further demonstrate the importance of the ERK pathway for the neurite outgrowth induced by FGF9 in HD striatal models. MATERIALS AND METHODS: FGF9 was treated with ERK (U0126) or NF-kB (BAY11-7082) inhibitors in STHdhQ7/Q7 and STHdhQ111/Q111 striatal knock-in cell lines to examine neurite outgrowth, cytoskeletal markers, and synaptic proteins via immunofluorescence staining and Western blotting. NF-kB activity was analyzed by NF-kB promoter reporter assay. KEY FINDINGS: Here, we show that suppression of ERK signaling significantly inhibits FGF9-induced neurite outgrowth, cytoskeletal markers, and synaptic proteins in HD striatal cells. In addition, we also show suppression of ERK signaling significantly decreases FGF9-induced NF-kB activation, whereas suppression of NF-kB does not decrease FGF9-induced ERK signaling. These results suggest that FGF9 activates ERK signaling first, stimulates NF-kB upregulation, and then enhances neurite outgrowth in HD striatal cells. SIGNIFICANCE: We elucidate the more detailed mechanisms of neurite outgrowth enhanced by FGF9 in these HD striatal cells. This study may provide insights into targeting neurite outgrowth for HD therapy.

Anti-Cancer Effect of Cordycepin on FGF9-Induced Testicular Tumorigenesis.

Cordycepin, a bioactive constituent from the fungus Cordyceps sinensis, could inhibit cancer cell proliferation and promote cell death via induction of cell cycle arrest, apoptosis and autophagy. Our novel finding from microarray analysis of cordycepin-treated MA-10 mouse Leydig tumor cells is that cordycepin down-regulated the mRNA levels of FGF9, FGF18, FGFR2 and FGFR3 genes in MA-10 cells. Meanwhile, the IPA-MAP pathway prediction result showed that cordycepin inhibited MA-10 cell proliferation by suppressing FGFs/FGFRs pathways. The in vitro study further revealed that cordycepin decreased FGF9-induced MA-10 cell proliferation by inhibiting the expressions of p-ERK1/2, p-Rb and E2F1, and subsequently reducing the expressions of cyclins and CDKs. In addition, a mouse allograft model was performed by intratumoral injection of FGF9 and/or intraperitoneal injection of cordycepin to MA-10-tumor bearing C57BL/6J mice. Results showed that FGF9-induced tumor growth in cordycepin-treated mice was significantly smaller than that in a PBS-treated control group. Furthermore, cordycepin decreased FGF9-induced FGFR1-4 protein expressions in vitro and in vivo. In summary, cordycepin inhibited FGF9-induced testicular tumor growth by suppressing the ERK1/2, Rb/E2F1, cell cycle pathways, and the expressions of FGFR1-4 proteins, suggesting that cordycepin can be used as a novel anticancer drug for testicular cancers.

FGF9 is a downstream target of SRY and sufficient to determine male sex fate in ex vivo XX gonad culture.

Fibroblast growth factor 9 (FGF9) is an autocrine/paracrine growth factor that plays critical roles in embryonic and organ developments and is involved in diverse physiological events. Loss of function of FGF9 exhibits male-to-female sex reversal in the transgenic mouse model and gain of FGF9 copy number was found in human 46, XX sex reversal patient with disorders of sex development. These results suggested that FGF9 plays a vital role in male sex development. Nevertheless, how FGF9/Fgf9 expression is regulated during testis determination remains unclear. In this study, we demonstrated that human and mouse SRY bind to -833 to -821 of human FGF9 and -1010 to -998 of mouse Fgf9, respectively, and control FGF9/Fgf9 mRNA expression. Interestingly, we showed that mouse SRY cooperates with SF1 to regulate Fgf9 expression, whereas human SRY-mediated FGF9 expression is SF1 independent. Furthermore, using an ex vivo gonadal culture system, we showed that FGF9 expression is sufficient to switch cell fate from female to male sex development in 12-16 tail somite XX mouse gonads. Taken together, our findings provide evidence to support the SRY-dependent, fate-determining role of FGF9 in male sex development.

CDK4 and CDK5 Inhibition Have Comparable Mild Hypothermia Effects in Preventing Drp1-Dependent Mitochondrial Fission and Neuron Death Induced by MPP.

Mild hypothermia has promising effects in the treatment of acute brain insults and also affects cell cycle progression. Mitochondrial dynamics, fusion and fission, are changed along with the cell cycle and disrupted in neurodegenerative diseases, including Parkinson's disease (PD). However, the effects of hypothermia on aberrant mitochondrial dynamics in PD remain unknown. We hypothesized that mild hypothermia protects neurons by regulating cell cycle-dependent protein expression and mitochondrial dynamics in a 1-methyl-4-phenylpyridinium (MPP+)-induced cell model of PD. We found that the hypothermia treatment at 32 °C prevented MPP+-induced neuron death; however, 32 °C treatment itself also reduced cell viability. This reduction was associated with cell cycle arrest and downregulation of cyclin-dependent kinase 4 (CDK4) in proliferating human SK-N-SH neuroblastoma cells but upregulation in well-differentiated primary rat cortical neurons. In both types of neurons, hypothermia upregulated p27 (an endogenous inhibitor of CDKs) and p35 (CDK5 activator) protein expression. Treatment with hypothermia, or a selective CDK4 inhibitor, or roscovitine (CDK5 inhibitor) prevented MPP+-induced mitochondrial fission, upregulation of mitochondrial fission protein dynamin-related protein 1 (Drp1), and neuron death. In addition, overexpression of dominant negative mutant Drp1K38A improved MPP+-induced mitochondrial fission while overexpression of wild-type Drp1 blunted the prevention of mitochondrial fission by hypothermia as well as CDK4 inhibitor and roscovitine. These results elucidate that hypothermia may inhibit CDK4 and CDK5 activation by upregulating p27 and p35 expression to prevent Drp1-dependent mitochondrial fission and neuron loss after MPP+ treatment. CDK4 and CDK5 inhibition imitates the neuroprotective functions of hypothermia as a potential therapy for PD.

FGF9 induces functional differentiation to Schwann cells from human adipose derived stem cells.

Rationale: The formation of adipose-derived stem cells (ASCs) into spheres on a chitosan-coated microenvironment promoted ASCs differentiation into a mixed population of neural lineage-like cells (NLCs), but the underline mechanism is still unknown. Since the fibroblast growth factor 9 (FGF9) and fibroblast growth factor receptors (FGFRs) play as key regulators of neural cell fate during embryo development and stem cell differentiation, the current study aims to reveal the interplay of FGF9 and FGFRs for promoting peripheral nerve regeneration. Methods: Different concentration of FGF9 peptide (10, 25, 50, 100 ng/mL) were added during NLCs induction (FGF9-NLCs). The FGFR expressions and potential signaling were studied by gene and protein expressions as well as knocking down by specific FGFR siRNA or commercial inhibitors. FGF9-NLCs were fluorescent labeled and applied into a nerve conduit upon the injured sciatic nerves of experimental rats. Results: The FGFR2 and FGFR4 were significantly increased during NLCs induction. The FGF9 treated FGF9-NLCs spheres became smaller and changed into Schwann cells (SCs) which expressed S100ß and GFAP. The specific silencing of FGFR2 diminished FGF9-induced Akt phosphorylation and inhibited the differentiation of SCs. Transplanted FGF9-NLCs participated in myelin sheath formation, enhanced axonal regrowth and promoted innervated muscle regeneration. The knockdown of FGFR2 in FGF9-NLCs led to the abolishment of nerve regeneration. Conclusions: Our data therefore demonstrate the importance of FGF9 in the determination of SC fate via the FGF9-FGFR2-Akt pathway and reveal the therapeutic benefit of FGF9-NLCs.