Growth Factor Fgf Research Articles

FGF-2 suppresses expression of nephronectin via JNK and PI3K pathways.

Nephronectin (Npnt), an extracellular matrix protein, is a ligand for integrin α8β1 and is involved in the development of various organs, such as the kidneys, bones, liver, and muscles. Previously, we found that Npnt expression was inhibited by various cytokines including transforming growth factor‐β (Tgf‐β) and oncostatin M (Osm). Fibroblast growth factor (Fgf)‐2, otherwise known as basic Fgf, also plays important roles in skeletal development and postnatal osteogenesis. In this study, Npnt expression was found to be suppressed by Fgf‐2 in MC3T3‐E1 cells, an osteoblast‐like cell line, in a dose‐ and time‐dependent manners. Furthermore, Fgf‐2‐mediated Npnt mRNA suppression was shown to involve the Jun N‐terminal kinase (JNK) and phosphoinositide‐3 kinase (PI3K) pathways. Together, our results suggest that FGF‐2 suppresses Npnt gene expression via JNK and PI3K pathways.

Optimizing a three dimensional in vitro intervertebral disc environment for the co-culture of human nucleus pulposus cells and mesenchymal stromal cells

Purpose: Current clinical trials have attempted the injection of mesenchymal stromal cells (MSCs) into degenerated intervertebral discs (IVDs) to restore height and function. However, retrieval of tissue for intensive characterization of the mechanisms behind any apparent regenerative effect is very limited. In the present research study, a 3D culture system has been developed to act as an ex vivo model for the intervertebral disc that mimics the native environment physically and chemically. The development of a cellularized nucleus pulposus (NP) analogue will help to unravel the cell-cell signaling between MSCs and NP cells in an in vitro setting, and enable identification of specific immunomodulatory targets that are regulated by MSCs. Methods: Alginate co-culture construct creation: Alginate constructs were created in two steps; beads were created by external gelation followed by bead encapsulation within an internally gelling cylindrical geometry. Alginate droplets extruded through a needle into a solution of 102 mM CaCl2 were left to crosslink for 10 min. The beads were rinsed in PBS and added in a 1:1 volumetric ratio to a solution of alginate with 30 mM CaCO3. Glucono-del-lactone (GDL, 60 mM) was added; the combination was mixed to create 3 mm diameter × 3 mm height cylindrical constructs with a central bead (Figure A, B). The addition of cells in UV-sterilized alginate requires little modification to the gelation protocol; cells at greater than 2 × 106 cells/mL concentration are added before crosslinking. Stimulative and degenerative media formulation: Human NP cells (passage 3, ScienCell), were seeded at a concentration of 5000 cells/cm2 into poly-l-lysine coated 96 well plates. Base media (DMEM-lg, 10% FBS, 1% ABAM) was combined with variations of pH, osmolarity and growth factors (n = 6) to replicate healthy or degenerative conditions. Cells were cultured for 14 days in 5% O2 to determine ideal conditions for MSC:NPC co-culture. Cells were assayed and imaged at various timepoints over the 14 day period. Media reserved from the wells was analyzed for ECM and inflammatory factor content via ELISA. Results: Three dimensional constructs of varying concentrations were optimized; overall, 1.2% alginate beads encased in 2% alginate showed 93% bead retention rate (Figure 1A–C). Encapsulated hNPCs showed no migration between the two constructs over 14 days in culture and full bead separation was achieved over the full time scale, reinforcing the construct as a separatable 3D co-culture method (Figure 1C, D). The addition of the degenerative growth factors TNFα and IL-1β as well as the adjustment of media pH to degenerative levels (pH 6.8) caused the hNPCs to decrease in size and proliferate significantly higher than control levels. Additions of any combination enhanced proliferation over base media control; brightfield images show an appearance change as well, which may indicate phenotype shift. Stimulative factors such as increased osmolarity with the growth factor FGF-2 caused a reduction in proliferation and a drastic morphology change irrespective of altered pH levels, whereas the addition of stimulative factor TGF-β3 under high osmolarity conditions showed a moderate increase in proliferation over control levels. Conclusions: The results of this study indicate that the optimized alginate 3D co-culture construct is a feasible method to co-culture encapsulated cells while enabling complete separation for ease of analysis. The addition of degenerative media or stimulative media, when optimized, can further enhance the ability to accurately mimic in vivoenvironments to replicate MSC-NPC interactions in a clinical setting. Future results include ELISA and immunofluorescence for Collagen I, Collagen II, Aggrecan, and Interferon gamma secretion. Further studies include the co-culture of MSCs and hNPCs and the corresponding analysis of the secreted inflammasome in both the optimized degenerative and stimulative ex vivo NP environment.

Growth factors FGF8 and FGF2 and their receptor FGFR1, transcriptional factors Msx-1 and MSX-2, and apoptotic factors p19 and RIP5 participate in the early human limb development

The expression pattern of fibroblast growth factors FGF8 and FGF2 and their receptor FGFR1, transcription factors MSX-1 and MSX-2, as well as cell proliferation (Ki-67) and cell death associated caspase-3, p19 and RIP5 factors were analyzed in histological sections of eight 4th-9th-weeks developing human limbs by immunohistochemistry and semi-thin sectioning. Increasing expression of all analyzed factors (except FGF8) characterized both the multilayered human apical ectodermal ridge (AER), sub-ridge mesenchyme (progress zone) and chondrocytes in developing human limbs. While cytoplasmic co-expression of MSX-1 and MSX-2 was observed in both limb epithelium and mesenchyme, p19 displayed strong cytoplasmic expression in non-proliferating cells. Nuclear expression of Ki-67 proliferating cells, and partly of MSX-1 and MSX-2 was detected in the whole limb primordium. Strong expression of factors p19 and RIP5, both in the AER and mesenchyme of human developing limbs indicates their possible involvement in control of cell senescence and cell death. In contrast to animal studies, expression of FGFR1 in the surface ectoderm and p19 in the whole limb primordium might reflect interspecies differences in limb morphology. Expression of FGF2 and downstream RIP5 gene, and transcription factors Msx-1 and MSX-2 did not show human-specific changes in expression pattern. Based on their spatio-temporal expression during human limb development, our study indicates role of FGFs and Msx genes in stimulation of cell proliferation, limb outgrowth, digit elongation and separation, and additionally MSX-2 in control of vasculogenesis. The cascade of orchestrated gene expressions, including the analyzed developmental factors, jointly contribute to the complex human limb development.

Short-term methionine deprivation improves metabolic health via sexually dimorphic, mTORC1-independent mechanisms.

Obesity and diabetes are major challenges to global health, and there is an urgent need for interventions that promote weight loss. Dietary restriction of methionine promotes leanness and improves metabolic health in mice and humans. However, poor long-term adherence to this diet limits its translational potential. In this study, we develop a short-term methionine deprivation (MD) regimen that preferentially reduces fat mass, restoring normal body weight and glycemic control to diet-induced obese mice of both sexes. The benefits of MD do not accrue from calorie restriction, but instead result from increased energy expenditure. MD promotes increased energy expenditure in a sex-specific manner, inducing the fibroblast growth factor (Fgf)-21-uncoupling protein (Ucp)-1 axis only in males. Methionine is an agonist of the protein kinase mechanistic target of rapamycin complex (mTORC)-1, which has been proposed to play a key role in the metabolic response to amino acid-restricted diets. In our study, we used a mouse model of constitutive hepatic mTORC1 activity and demonstrate that suppression of hepatic mTORC1 signaling is not required for the metabolic effects of MD. Our study sheds new light on the mechanisms by which dietary methionine regulates metabolic health and demonstrates the translational potential of MD for the treatment of obesity and type 2 diabetes.-Yu, D., Yang, S. E., Miller, B. R., Wisinski, J. A., Sherman, D. S., Brinkman, J. A., Tomasiewicz, J. L., Cummings, N. E., Kimple, M. E., Cryns, V. L., Lamming, D. W. Short-term methionine deprivation improves metabolic health via sexually dimorphic, mTORC1-independent mechanisms.

Characterization of New Human Glioblastoma Cell Lines

Glioblastomas are tumors of neuroectodermal origin with a high degree of diversity. Tumor cells isolated from the surgical material of patients with glioblastomas are heterogeneous populations varying in morphology, phenotype, and genetic characteristics. This paper presents a description of two new glioblastoma cell lines, R1 and T2, isolated from tumor tissue of patients in 2010. We investigated their morphological and cytochemical cell characteristics and the expression of neuronal, mesenchymal, and endothelial markers, as well as the activity of genes encoding a number of growth factors, extracellular matrix proteins, and intracellular proteins typical for cells of mesenchymal origin. R1 and T2 cell lines are morphologically different. T2 cell line was characterized by the presence of multinuclear cells. In terms of the expression of β-tubulin III, MGMT, and p53 protein, R1 cell line was more heterogeneous than T2. R1 and T2 glioblastoma cell lines also differed in the presence and ratio of cell populations with mesenchymal, neuronal, and endothelial markers. Thus, neuronal markers CD133/2 and CD56 were detected only on R1 cells. Both lines were characterized by high activity of growth factor genes TGFβ1, VEGF, and FGF2(b), lower activity of EGF, and high expression of THBS1 and αSMA genes. However, the activity of most of the genes under study in R1 cells was higher than in T2 cells. The greatest difference lay in the expression of HGF, FAP, and TNC. Comparison of two new glioblastoma cell lines, R1 and T2, with the continuously cultivated lines А172 and T98G showed that R1 line had remarkable similarity with A172, while T2 glioblastoma resembled T98G cells. Apparently, the differences between R1 and T2 cell lines are determined by the properties of the initial tumors rather than the time of the cell cultivation.

Hyperinnervation improves Xenopus laevis limb regeneration

Xenopus laevis (an anuran amphibian) shows limb regeneration ability between that of urodele amphibians and that of amniotes. Xenopus frogs can initiate limb regeneration but fail to form patterned limbs. Regenerated limbs mainly consist of cone-shaped cartilage without any joints or branches. These pattern defects are thought to be caused by loss of proper expressions of patterning-related genes. This study shows that hyperinnervation surgery resulted in the induction of a branching regenerate. The hyperinnervated blastema allows the identification and functional analysis of the molecules controlling this patterning of limb regeneration. This paper focuses on the nerve affects to improve Xenopus limb patterning ability during regeneration. The nerve molecules, which regulate limb patterning, were also investigated. Blastemas grown in a hyperinnervated forelimb upregulate limb patterning-related genes (shh, lmx1b, and hoxa13). Nerves projecting their axons to limbs express some growth factors (bmp7, fgf2, fgf8, and shh). Inputs of these factors to a blastema upregulated some limb patterning-related genes and resulted in changes in the cartilage patterns in the regenerates. These results indicate that additional nerve factors enhance Xenopus limb patterning-related gene expressions and limb regeneration ability, and that bmp, fgf, and shh are candidate nerve substitute factors.

180 Increasing GfrA1-Positive Spermatogonial Stem Cell Population of Goat

Spermatogonial stem cells (SSC) form the basis of spermatogenesis and continuous fertility in male. Their meagre population in the testis is a hindrance in the in vitro study of biological activity of these cells. The objective of the present study was to isolate and characterise goat SSC and increase their number during in vitro culture by different methods. Two goat testes (3 to 4 months of age) were collected from the slaughterhouse and transported to the laboratory. The testes were washed and seminiferous tubules were collected and minced in the laminar flow hood. The seminiferous tubules were washed twice with PBS to remove spermatozoa and subjected to double enzymatic digestion (collagenase, 1 mg mL−1, hyaluronidase, 1 mg mL−1, trypsin, 0.05%, and DNaseI, 10 µg mL−1 for 45 min and second digestion with same set of enzymes except trypsin for 30 min). The isolated cells were filtered sequentially through nylon mesh filters of pore size 70 and 40 µm. The cells were plated on DSA-lectin coated dishes for 4 h and the unattached cells were cultured on a Sertoli cell feeder layer prepared by treating with mitomycin-C for 3 h. The cells were cultured in DMEM/F-12 supplemented with human recombinant growth factors (glial cell-derived neurotrpic factor, 10 ng mL−1, fibroblast growth factor FGF, 10 ng mL−1, epidermal growth factor, 20 ng mL−1), 10% fetal bovine serum, and antibiotics. The expression of pluripotency markers (Oct4, Nanog, Sox2) and SSC-specific markers (Thy1, GfrA1, and Uchl1) in the SSC colonies was determined by RT-PCR and immunostaining, after in vitro culture of 3 weeks. The SSC population was enhanced by differential plating, Percoll density gradient (on Day 1) and SSC passaging (by passaging SSC colonies on Day 20). The cells were tagged with GfrA1 antibody and their population was tested by flow cytometry. The SSC colonies started appearing after 7 days and continued to grow in size and number until 3 weeks. The SSC colonies were positive for the pluripotency markers Oct4, Nanog, and Sox2 by RT-PCR and immunostaining. The SSC were also positive for the SSC-specific markers Thy1, GfrA1, and Uchl1 by RT-PCR and immunostaining. Flow cytometry showed that the GfrA1-positive population in the SSC enriched by the differential plating was 11.23%, Percoll density gradient was 23.57%, and by passaging of SSC colonies, after picking and trypsinising with 0.05% trypsin, was 91.23%. In vitro culture of the SSC enriched by these methods also revealed that the number of SSC colonies appearing in the cells enriched by passaging was higher than the other methods. From the results of present study, we conclude that SSC are positive for markers of pluripotency and SSC-specific markers. The SSC population can be enhanced to a very high level following SSC passaging, which is an inexpensive method and does not require expensive instruments like fluorescence- or magnetic-activated cell sorting.

Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart.

Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

Plasticity of Human Meniscus Fibrochondrocytes: A Study on Effects of Mitotic Divisions and Oxygen Tension

Meniscus fibrochondrocytes (MFCs) may be the optimal cell source to repair non-healing meniscus injuries using tissue engineering strategies. In this study, we investigated the effects of mitotic divisions and oxygen tension on the plasticity of adult human MFCs. Our assessment techniques included gene expression, biochemical, histological, and immunofluorescence assays. MFCs were expanded in monolayer culture with combined growth factors TGFβ1 and FGF-2 (T1F2) under normoxia (21% O2). Trilineage (adipogenesis, chondrogenesis and osteogenesis) differentiation was performed under both normoxic (21% O2) and hypoxic (3% O2) conditions. The data demonstrated that MFCs with a mean total population doubling of 10 can undergo adipogenesis and chondrogenesis. This capability was enhanced under hypoxic conditions. The MFCs did not undergo osteogenesis. In conclusion, our findings suggest that extensively expanded human MFCs have the capacity to generate tissues with the functional matrix characteristics of avascular meniscus. To this end, expanded MFCs may be an ideal cell source for engineering functional constructs for the replacement or repair of avascular meniscus.

Adipose-derived mesenchymal stromal cells prevented rat vocal fold scarring.

This study aimed to reveal the effects of adipose-derived mesenchymal stromal cells (ASCs) on prevention of vocal fold scarring by investigating how the immediate ASCs transplantation into the injured rat vocal fold affect the levels of gene transcription and translation. Prospective animal experiments with controls. ASCs harvested from green fluorescent protein transgenic rat (ASCs group) or saline (sham group) were injected into the thyroarytenoid muscle of Sprague-Dawley rats immediately after stripping the vocal fold. For histological examinations, larynges were extirpated at 3, 14, and 56 days after the injection. Quantitative real-time polymerase chain reaction (PCR) analyses were performed at 3 and 14 days after the injection. Transplanted ASCs were detected only in larynges at day 3. At days 14 and 56, histological examination showed significantly higher amounts of hyaluronic acid and lower deposition of collagen in the ASCs group compared to the sham group. Real-time PCR revealed that the ASCs group showed low expression of procollagen (Col)1a1, Col1a3, matrix metalloproteinase (Mmp)1 and Mmp8 in each time points. The ASCs group showed high expression of fibroblast growth factor (Fgf)2 and Hepatocyte growth factor (Hgf) compared to the sham group at day 14. ASCs increased expressions of Fgf2 and Hgf, and suppressed excessive collagen deposition during vocal fold wound healing. Given the fact that ASCs survived no more than 14 days, ASCs were thought to induce upregulations of growth factors' genes in surrounding cells. These results suggested that ASCs have potential to prevent vocal fold scarring. NA. Laryngoscope, 128:E33-E40, 2018.

Tumor-associated B-cells induce tumor heterogeneity and therapy resistance

In melanoma, therapies with inhibitors to oncogenic BRAFV600E are highly effective but responses are often short-lived due to the emergence of drug-resistant tumor subpopulations. We describe here a mechanism of acquired drug resistance through the tumor microenvironment, which is mediated by human tumor-associated B cells. Human melanoma cells constitutively produce the growth factor FGF-2, which activates tumor-infiltrating B cells to produce the growth factor IGF-1. B-cell-derived IGF-1 is critical for resistance of melanomas to BRAF and MEK inhibitors due to emergence of heterogeneous subpopulations and activation of FGFR-3. Consistently, resistance of melanomas to BRAF and/or MEK inhibitors is associated with increased CD20 and IGF-1 transcript levels in tumors and IGF-1 expression in tumor-associated B cells. Furthermore, first clinical data from a pilot trial in therapy-resistant metastatic melanoma patients show anti-tumor activity through B-cell depletion by anti-CD20 antibody. Our findings establish a mechanism of acquired therapy resistance through tumor-associated B cells with important clinical implications.

Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish.

The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.

ID: 1032 Differentiation Potential and Characteristics of Mesenchymal Stem Cells Isolated from Human Umbilical Cord membrane to Hepatocyte-like Cells

Recent studies indicated that Mesenchymal stem cell has become a potential objective for therapy. In this study, umbilical cord cells were isolated and analyzed the expression of mesenchymal stem cells specific markers then they were differentiated into hepatocyte-like cells by DMSO and Gene transfection. Umbilical cord mesenchymal stem cell (MSC) was isolated by explant culture in media DMEM/F12, complementing with growth factors EGF, FGF and IST. After that, they were exposured to DMSO with three concentrations: 0.01%, 0.1%, 1% and another group was transfection with HNF4α by Lipofectamin LX plus. The cells were analyzed at 1, 2, 3, and 4 weeks after treatment. The cells isolation was shown the positive with markers CD73, CD34, CD86, CD90, CD105, eras, Oct 1, GATA, and negative with markers HNF4α, Alb and G6P. In group 0,1% DMSO treatment, after 3 weeks the cells were positive with markers HNF4α but it was also negative with markers Alb and G6P. In the transfection group, the cell expresses HNF4α at three weeks after treatment. Although our results exposure that the umbilical cord mesenchymal stem cells expressed hepatic specific marker after DMSO induced and DNA treatment. So it will be necessary to optimize research conditional and investigate the hepatic functions of these cells in a longer in vitro culture.

Ectodysplasin target gene Fgf20 regulates mammary bud growth and ductal invasion and branching during puberty

Mammary gland development begins with the appearance of epithelial placodes that invaginate, sprout, and branch to form small arborized trees by birth. The second phase of ductal growth and branching is driven by the highly invasive structures called terminal end buds (TEBs) that form at ductal tips at the onset of puberty. Ectodysplasin (Eda), a tumor necrosis factor-like ligand, is essential for the development of skin appendages including the breast. In mice, Eda regulates mammary placode formation and branching morphogenesis, but the underlying molecular mechanisms are poorly understood. Fibroblast growth factor (Fgf) receptors have a recognized role in mammary ductal development and stem cell maintenance, but the ligands involved are ill-defined. Here we report that Fgf20 is expressed in embryonic mammary glands and is regulated by the Eda pathway. Fgf20 deficiency does not impede mammary gland induction, but compromises mammary bud growth, as well as TEB formation, ductal outgrowth and branching during puberty. We further show that loss of Fgf20 delays formation of Eda-induced supernumerary mammary buds and normalizes the embryonic and postnatal hyperbranching phenotype of Eda overexpressing mice. These findings identify a hitherto unknown function for Fgf20 in mammary budding and branching morphogenesis.

Renal findings in patients with Mulibrey nanism

Mulibrey nanism (MUL) is a rare inherited disease caused by genetic defects affecting peroxisomal TRIM37 protein. MUL affects multiple organs, leading to growth retardation and early onset type 2 diabetes. We aimed to characterize the structure and function of kidneys and the urinary tract in a large cohort of Finnish MUL patients. Ultrasound, magnetic resonance imaging (MRI), and autopsy findings of the kidneys and urinary tract from 101 MUL patients were retrospectively analyzed. Renal function was examined using blood and urine biochemistry. Kidney pathology was assessed by histology and immunohistochemistry from biopsy and autopsy samples. Structural anomalies of the kidneys and urinary tract were found in 13 % of MUL patients and renal tumors and macroscopic cystic lesions in 14 % and 43 % respectively. Overall, kidney histology was well preserved, but glomerular cysts with a wide Bowman's space were observed in most samples (87 %). Also, prominent and abundant blood vessels with thick walls were typically seen. Expression of endothelial cell markers and angiogenic growth factors PDGF-B and FGF1 (but not VEGF-A) was significantly increased in MUL kidneys. Markers of fibrosis and epithelial-mesenchymal transformation, α-SMA, and vimentin were moderately up-regulated. Despite radiological and histological changes, most MUL patients (age 0.2-51 years) had normal kidney function. However, 9 out of 36 patients (25 %) had hypertension and 6 out of 26 (23 %) had mildly decreased glomerular filtration. Genetic defects in the TRIM37 gene lead to an increased risk for kidney anomalies, renal tumors, and solitary cysts in addition to glomerular cystic lesions, but not to progressive deterioration of renal function.

127 - From heart development to cardiac regeneration: role of the Fibroblast Growth Factor FGF10

127 - From heart development to cardiac regeneration: role of the Fibroblast Growth Factor FGF10

Activation of GR but not PXR by dexamethasone attenuated acetaminophen hepatotoxicities via Fgf21 induction

Glucocorticoid receptor (GR) signaling is indispensable for cell growth and development, and plays important roles in drug metabolism. Fibroblast growth factor (Fgf) 21, an important regulator of glucose, lipid, and energy metabolism, plays a cytoprotective role by attenuating toxicities induced by chemicals such as dioxins, acetaminophen (APAP), and alcohols. The present study investigates the impact of dexamethasone (DEX)-activated GR on Fgf21 expression and how it affects the progression of APAP-induced hepatotoxicity. Our results showed that DEX dose/concentration- and time-dependently increased Fgf21 mRNA and protein expression in mouse liver as well as cultured mouse and human hepatoma cells. By using PXR-null mouse model, we demonstrated that DEX induced Fgf21 expression by a PXR-independent mechanism. In cultured mouse and human hepatoma cells, inhibition of GR signaling, by RU486 (Mifepristone) or GR silencing using GR-specific siRNA, attenuated DEX-induced Fgf21 expression. In addition, DEX increased luciferase reporter activity driven by the 3.0-kb mouse and human Fgf21/FGF21 gene promoter. Further, ChIP-qPCR assays demonstrated that DEX increased the binding of GR to the specific cis-regulatory elements located in the 3.0-kb mouse and human Fgf21/FGF21 gene promoter. Pretreatment of 2mg/kg DEX ameliorated APAP-induced liver injury in wild-type but not Fgf21-null mice. In conclusion, via GR activation, DEX induced Fgf21 expression in mouse liver and human hepatoma cells.

From nucleus pulposus mesenchymal stem cells towards neural differentiation: an interesting prospect

Regenerative medicine arouses great interest for the treatment of many neurological diseases. Since nucleus pulposus of the invertebral discs is a postembryonic vestige of the notochord, it has been hypothesized that mesenchymal stem cells (MSCs) isolated from nucleus pulposus (NP-MSCs) can more easily differentiate into neurons. In this study, MSCs from nucleus pulposus were successfully isolated and characterized. Then, neural differentiation was induced by using a medium consisted of DMEM/F12 supplemented with B27 and the growth factors FGF and EGF for 10 days. Immunocytochemistry, molecular studies, SEM and TEM microscopy analyses were performed. NP-MSCs exhibited the typical features of MSCs, revealing spindle-shape morphology, specific immunophenotype attributable to MSCs and the ability to differentiate in osteogenic and chondrogenic lineages. After neurodifferentiation induction, compared to NP-MSCs in only DMEM/F12, proliferation rate decreased and cells changed morphology acquiring an increased number of the so-called neural-like extensions. Neural progenitor marker NESTIN and mature neuronal marker ENOLASE-2 were up-regulated, while GFAP was not detected. Moreover, cells after differentiation were small rounded and fusiform, with tendency to organize in clumps; they had elongated extrusions containing oriented cytoskeletal elements, classifiable as microtubules and intermediate filaments, as visualized by SEM and TEM microscopy. Dense vesicles similar to lipid droplet were also observed. NP-MSCs in differentiation medium were able to form neurospheres. In conclusion, even if more analysis have to be done and the way to treat neurodegenerative disease with regenerative medicine is still long, NP-MSCs represent a promising resource.

Antiangiogenic platinum through glycan targeting.

Heparan sulfate is identified as a ligand receptor for polynuclear platinum anti-cancer agents through sulfate cluster binding. We present a new biological role for platinum and coordination compounds and a new target for metal-based drugs while presenting a new chemotype for heparanase and growth factor inhibition through modulation (metalloshielding) of their interactions. Masking of extracellular (ECM)-resident heparan sulfate (HS) through metalloshielding results in very effective inhibition of physiologically critical HS functions including enzyme (heparanase, HPSE) and protein growth factor recognition. The interaction of the highly cationic polynuclear platinum complexes (PPCs) with the highly sulfated pentasaccharide Fondaparinux (FPX, in this case as a model HS-like substrate) results in inhibition of its cleavage by the HS-related enzyme heparanase. Binding of the fibroblast growth factor FGF-2 to HS is also inhibited with consequences for downstream signalling events as measured by a reduction in accumulation of phospho-S6 ribosomal protein in human colon tumor HCT-116 cells. The end-point of inhibition of HPSE activity and growth factor growth factor signaling is the prevention of cell invasion and angiogenesis. Finally these events culminate in inhibition of HCT-116 cell invasion at sub-cytotoxic concentrations and the process of angiogenesis. A competition assay shows that Fondaparinux can sequester the 8+ TriplatinNC from bound DNA, emphasising the strength of PPC-HS interactions. Altering the profile of platinum agents from cytotoxic to anti-metastatic has profound implications for future directions in the development of platinum-based chemotherapeutics.

P38 (Mapk14/11) occupies a regulatory node governing entry into primitive endoderm differentiation during preimplantation mouse embryo development.

During mouse preimplantation embryo development, the classically described second cell-fate decision involves the specification and segregation, in blastocyst inner cell mass (ICM), of primitive endoderm (PrE) from pluripotent epiblast (EPI). The active role of fibroblast growth factor (Fgf) signalling during PrE differentiation, particularly in the context of Erk1/2 pathway activation, is well described. However, we report that p38 family mitogen-activated protein kinases (namely p38α/Mapk14 and p38β/Mapk11; referred to as p38-Mapk14/11) also participate in PrE formation. Specifically, functional p38-Mapk14/11 are required, during early-blastocyst maturation, to assist uncommitted ICM cells, expressing both EPI and earlier PrE markers, to fully commit to PrE differentiation. Moreover, functional activation of p38-Mapk14/11 is, as reported for Erk1/2, under the control of Fgf-receptor signalling, plus active Tak1 kinase (involved in non-canonical bone morphogenetic protein (Bmp)-receptor-mediated PrE differentiation). However, we demonstrate that the critical window of p38-Mapk14/11 activation precedes the E3.75 timepoint (defined by the initiation of the classical ‘salt and pepper’ expression pattern of mutually exclusive EPI and PrE markers), whereas appropriate lineage maturation is still achievable when Erk1/2 activity (via Mek1/2 inhibition) is limited to a period after E3.75. We propose that active p38-Mapk14/11 act as enablers, and Erk1/2 as drivers, of PrE differentiation during ICM lineage specification and segregation.