FGF-2 mRNA Research Articles

Expression of fibroblast growth factor 10 and cognate receptors in the developing bovine ovary

In the mammalian ovary, FGF10 is expressed in oocytes and theca cells and is a candidate for paracrine signaling to the developing granulosa cells. To gain insight into the participation of FGF10 in the regulation of fetal folliculogenesis, we assessed mRNA expression patterns of FGF10 and its receptors, FGFR1B and FGFR2B, in relation to fetal follicle dynamics and localized FGF10 protein in bovine fetal ovaries at different ages. Primordial, primary, secondary, and antral follicles were first observed on Days 75, 90, 150, and 210 of gestation, respectively. The levels of GDF9 and BMP15 mRNA, markers for primordial and primary follicles, respectively, increased during fetal ovary development in a consistent manner with fetal follicle dynamics. CYP17A1 mRNA abundance increased from Day 60 to Day 75 and then from Day 120 to Day 150, coinciding with the appearance of secondary follicles. FGF10 mRNA abundance increased from Day 90, and this increase was temporally associated with increases in FGFR1B mRNA abundance and in the population of primary follicles. In contrast, FGFR2B mRNA expression was highest on Day 60 and decreased thereafter. FGF10 protein was localized to oogonia and oocytes and surrounding granulosa cells at all fetal ages. The present data suggest a role for FGF10 in the control of fetal folliculogenesis in cattle.

The effects of helium based atmospheric pressure cold plasma (APCP) on wound healing

Cold plasmas have been proposed for many applications in medicine, such as tissue disinfection and wound healing. Their antibacterial effects have been well demonstrated and depend mainly to the action of reactive oxygen species or ROS, but the molecular mechanisms promoting tissue regeneration in wound healing are not yet fully understood. We previously reported that the applications of 2-5 min of atmospheric pressure cold plasma (APCP) generated by helium ionization is effective in the inactivation bacteria without any visible changes in cells and tissues (1) and the aim of the presented study is to ascertain if the same doses of APCP are able to promote wound healing. At this purpose we tested the effects of APCP exposure on dermal fibroblasts viability obtained from 3 different donors by means of MTT and Tunnel test, in presence or absence of a potent antioxidant such as 5mM N-acetylcystein (NAC). To define the cellular response to APCP, we then analyzed by real-time PCR the expression changes of selected genes involved in oxidative stress-related response (OGG1, GPX1) and wound repair (IL1beta, IL6, TNFα, FGF2, TGFβ, SMAD3, α-SMA, collagen I) in the plasma treated cells cultures. Our results demonstrated that in all the three different fibroblasts cultures, 2 min APCP application did not cause any variation of viability 2 h after treatment and increased significantly after 24 h (p<0.05). In fibroblasts of two donors we also found at this time a significant increase of viability of cell exposed for 2 min to APCP when compared to the untreated control. Moreover, NAC pretreatment positively affected the viability of one cell preparations. Evaluation of apoptosis (Tunel test) on all fibroblast preparations treated with 2 min APCP and monitored after 24 h revealed that the number of apoptotic nuclei was comparable to those detected in untreated cells. RT-PCR analysis of cells two hours after APCP treatment revealed only a transient increasing in GPX1, IL6, FGF2 and TNFα mRNA levels. In conclusion, cell exposure of 2 min APCP, a dose that inactivates skin pathogens, maintain or increase their viability, but revealed a transitory alteration of the expression of some genes involved in oxidative stress and wound repair.

Sdc2 and Tbx16 regulate Fgf2-dependent epithelial cell morphogenesis in the ciliated organ of asymmetry

Heparan sulfate proteoglycans (HSPGs) control many cellular processes and have been implicated in the regulation of left-right (LR) development by as yet unknown mechanisms. Using lineage-targeted knockdowns, we found that the transmembrane HSPG Syndecan 2 (Sdc2) regulates LR patterning through cell-autonomous functions in the zebrafish ciliated organ of asymmetry, Kupffer's vesicle (KV), including regulation of cell proliferation and adhesion, cilia length and asymmetric fluid flow. Exploring downstream pathways, we found that the cell signaling ligand Fgf2 is exclusively expressed in KV cell lineages, and is dependent on Sdc2 and the transcription factor Tbx16. Strikingly, Fgf2 controls KV morphogenesis but not KV cilia length, and KV morphogenesis in sdc2 morphants can be rescued by expression of fgf2 mRNA. Through an Fgf2-independent pathway, Sdc2 and Tbx16 also control KV ciliogenesis. Our results uncover a novel Sdc2-Tbx16-Fgf2 pathway that regulates epithelial cell morphogenesis.

Molecular mechanism of congenital cataract induced by heat shock transcription factor 4 gene mutation in lop11 mouse

Background Mutation of the heat shock transcription factor 4 （HSF4） gene causes autosomal recessive hereditary cataract in lens opacity locus 11 （lopl I ） mouse, but the molecular mechanism of the pathogenesis has not been determined. Objective This study was to investigate the molecular mechanism of congenital cataract induced by HSF4 gene mutation in lopll mouse. Methods Twenty-four lopll mice and 24 wild type C57BL/6 mice were used in this study. The animals were sacrificed and the lenses were obtained on postnatal days 1,7 and 12. Regular pathological examination was carried out to evaluate the morphological changes of the lens and count the number of lens epithelial ceils （LECs） in the mice. The ctB-Crystallin （CRYAB） content in the lenses was detected in postnatal day 1 mice by Western blot. The expression of the fibroblast growth factor （FGF） mRNA in the lenses was assayed by quantitative PCR （q-PCR）. The data were compared between the lopl 1 mice and wild type C57BL/6 mice with independent sample t test. The use and care of the experimental animals complied with the ARVO statement. Results The morphology and array of LECs were uniform and regular in the wild type C57BL/6 mice, while proliferation of LECs and disorder of lens fibers were seen in the lopll mice on postnatal day 1. On postnatal day 7,vacuolar degeneration of the lens appeared in 7-day-old lopll mice. The numbers of LECs were （417±19）, （467±16） and （489 ±21 ） in lopll mice on the postnatal day 1,7 and 12, respectively, and those of wild type C57BL/6 mice were （ 378 ± 13 ） , （ 391 ±9 ） and （ 395 ±7 ） , respectively, showing statistically significant differences between them （1 day：t=6.696,P=0. O00;7 days：t=6.578,P=0.000;14 days：t=7.240,P=0.000）. The expression of CRYAB in the lenses was evidently weaker in the 1-day-old lopll mice compared with wild type C57BL/6 mice. The relative folds of expression of FGF-1, FGF-4, FGF-7 mRNA in the lenses were significantly higher in the lopll mice than those in the wild type C57BL/6 mice （2.04±0. 13 vs. 1. 037±0.06;2.0±0.08 vs. 0.97± 0.08;4.59±0.12 vs.1.0±0.04） （FGF-1 mRNA：t=14.000,P〈0.001;FGF-4 mRNA：t=15.510,P〈0.01;FGF-7 mRNA：t = 29.41 ,P〈0.01 ）. Conclusions HSF4 mutation leads to the abnormal development of the lens in lopl 1 mice by arresting the expression of cLB-Crystallin protein and increasing the expression of the FGF gene. Key words: Gene mutation; Heat shock transcription factor; Cataract/congenital; Crystallin; Fibroblast growth factor

Controlled release of rat adipose-derived stem cells from alginate microbeads

Cell-based therapies have potential for tissue regeneration but poor delivery methods lead to low viability or dispersal of cells from target sites, limiting clinical utility. Here, we developed a degradable and injectable hydrogel to deliver stem cells for bone regeneration. Alginate microbeads <200 μm are injectable, persist at implantation sites and contain viable cells, but do not readily degrade in-vivo. We hypothesized that controlled release of rat adipose-derived stem cells (ASCs) from alginate microbeads can be achieved by incorporating alginate-lyase in the hydrogel. Microbeads were formed using high electrostatic potential. Controlled degradation was achieved through direct combination of alginate-lyase and alginate at 4 °C. Results showed that microbead degradation and cell release depended on the alginate-lyase to alginate ratio. Viability of released cells ranged from 87% on day 2 to 71% on day 12. Monolayer cultures of released ASCs grown in osteogenic medium produced higher levels of osteocalcin and similar levels of other soluble factors as ASCs that were neither previously encapsulated nor exposed to alginate-lyase. Bmp2, Fgf2, and Vegfa mRNA in released cells were also increased. Thus, this delivery system allows for controlled release of viable cells and can modulate their downstream osteogenic factor production.

Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1α and STAT-3

Hypoxia-inducible factors (HIFs) and STAT-3 play essential roles in angiogenesis. HIF-1α and STAT-3 are clients of the heat shock protein 90 (HSP90). We hypothesized that ganetespib, a potent HSP90 inhibitor, would disrupt angiogenesis in colorectal cancer (CRC) through inhibition of HIF-1α and STAT-3. CRC cell lines (HCT116 and HT29) were used in all the experiments. Egg CAM and HUVEC assays revealed decreased angiogenesis in ganetespib treated cell lines. Ganetespib inhibited matrigel plug vascularization and tumor growth of xenografts. Significant inhibition of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, HIF-1α and STAT-3 expression was observed in both cell lines treated ganetespib. HIF-1α overexpression resulted in the increase VEGF and STAT-3 expression and this was inhibited by ganetespib. HIF-1α knockdown inhibited VEGF and STAT-3 expression. STAT-3 knockdown inhibited VEGF but not HIF-1α expression. HSP90, STAT-3 and VEGF expression was significantly higher in CRC compared to adjacent normal tissue. Significant downregulation of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, STAT-3 and HIF-1α mRNA was observed in the post ganetespib treatment tumor samples from patients with rectal cancer. These results collectively suggest that inhibition of HSP90 is a promising antiangiogenic strategy in CRC. HSP90 angiogenic effects are mediated through HIF-1α and STAT-3.

Paneth cells expand from newly created and preexisting cells during repair after doxorubicin-induced damage

Paneth cell numbers increase following intestinal damage, but mechanisms driving this process are not understood. We hypothesized that the increase in Paneth cell numbers is due to recruitment of cells from a preexisting pool of secretory progenitors. Mice were given a single injection of doxorubicin (Dox), and intestinal tissue was collected 0-168 h after treatment. Paneth, goblet, and intermediate cells were counted and evaluated for cell morphology. Quantitative RT-PCR was used to measure expression of various genes associated with Paneth cell allocation and maturation. Paneth cells were birth dated using incorporation of thymidine analogs given before or after Dox. Staining revealed "intermediate" cells, which were rarely observed in control crypts but increased significantly in number 96 and 120 h after Dox treatment. Birth dating of intermediate cells 5 days after Dox treatment revealed that 24% of these cells took up thymidine analog given prior to Dox treatment and 36% took up thymidine analog given after Dox treatment. Quantitative RT-PCR demonstrated a significant increase in Spdef, Atoh1, Sox9, EphB3, Mist, Wnt5a, FGF-9, and FGF-18 mRNAs and a significant decrease in Indian hedgehog mRNA. Expansion of the Paneth cell compartment after Dox treatment is due to generation of new cells and recruitment of cells from an existing pool. These cells express Paneth and goblet biomarkers and are found only during repair. Expansion of these cells correlates temporally with reduced Indian hedgehog and increased FGF and Wnt mRNA. These findings are significant, as they provide a first step in understanding mechanisms of Paneth cell expansion during mucosal repair.

Characterization of the expression profiles of adipogenesis-related factors, ZNF423, KLFs and FGF10, during preadipocyte differentiation and abdominal adipose tissue development in chickens

Adipogenesis is controlled by a complicated process involving certain transcriptional events. In chicken adipogenesis, peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of preadipocyte differentiation and abdominal fat accumulation. However, in a recent study in mammals, some novel factors related to regulation of adipogenesis, including preadipocyte differentiation, were identified in mammals. Therefore, in this study, we aimed to determine the expression profiles of these mammalian adipogenesis-related factors, such as zinc-finger protein 423 (ZNF423), Krüppel-like factor -2, -5, and -15 (KLF-2, -5, -15), and FGF10, in the chicken (Gallus gallus). Specifically, we analyzed their expression in primary preadipocyte differentiation in vitro and also analyzed their tissue distribution and their temporal expression in adipose tissue development in vivo. During chicken adipocyte differentiation, the gene expression of ZNF423, KLF-2, KLF-5 and FGF10 was found to rapidly decrease in the early stage of preadipocyte differentiation. Expression of ZNF423 then increased in the late stage of differentiation. KLF-15 expression increased in a time-dependent manner for 48 h. Protein expressions of these factors were reflected by Western blot analysis. High levels of aP2, PPARγ and FGF10 mRNA were found in adipose tissue. In addition, aP2, PPARγ and ZNF423 mRNA levels in the adipose tissue were elevated at days 10 and 20. These expression profiles of the adipogenesis-related factors in chicken are, in part, different from mammalian adipogenesis but this seems to reflect the differences in the regulation of adipogenesis and in adipose tissue functions between avians and mammals.

Forebrain and hindbrain development in zebrafish is sensitive to ethanol exposure involving agrin, Fgf, and sonic hedgehog function.

Ethanol is a teratogen that affects numerous developmental processes in the nervous system, which includes development and survival of GABAergic and glutamatergic neurons. Possible molecular mechanisms accounting for ethanol's effects on nervous system development include perturbed fibroblast growth factor (Fgf) and Sonic hedgehog (Shh) signaling. In zebrafish, forebrain GABAergic neuron development is dependent on Fgf19 and Shh signaling. The present study was conducted to test the hypothesis that ethanol affects GABAergic and glutamatergic neuron development by disrupting Fgf, Shh, and agrin function. Zebrafish embryos were exposed to varying concentrations of ethanol during a range of developmental stages, in the absence or presence of morpholino oligonucleotides (MOs) that disrupt agrin or Shh function. In situ hybridization was used to analyze glutamic acid decarboxylase (GAD1) gene expression, as well as markers of glutamatergic neurons. Acute ethanol exposure results in marked reduction in GAD1 gene expression in forebrain and hindbrain, and reduction of glutamatergic neuronal markers in hindbrain. Subthreshold ethanol exposure, combined with agrin or Shh MO treatment, produces a similar diminution in expression of markers for GABAergic and glutamatergic neurons. Consistent with the ethanol effects on Fgf and Shh pathways, Fgf19, Fgf8, or Shh mRNA overexpression rescues ethanol-induced decreases in GAD1 and Atonal1a gene expression. These studies demonstrate that GABAergic and glutamatergic neuron development in zebrafish forebrain or cerebellum is sensitive to ethanol exposure, and provides additional evidence that a signaling pathway involving agrin, Fgfs and Shh may be a critical target of ethanol exposure during zebrafish embryogenesis.

Does seminal plasma affect angiogenesis in the porcine oviduct?

In the present study we examined the effect of seminal plasma (SP) on angiogenesis in the porcine oviduct. Gene expressions of vascular endothelial growth factor (VEGF) and its two receptors (Flt-1: fms-like tyrosine kinase and Flk-1/KDR: fetal liver kinase-1/kinase insert domain-containing receptor) as well as fibroblast growth factors (FGF-1 and 2) and von Willenbrand factor (VWF) were determined in the oviduct of SP-treated and control (PBS-treated) gilts. Moreover, vascular density (VD) indicated by endothelial cell area immunolocalized by VWF staining, was assessed in the oviducts. Real-time PCR revealed significantly higher expression of FGF-2 and VWF on day 1 (p<0.05) after SP administration in comparison to control animals. In contrast, Flt-1 mRNA level on day 1 was lower in SP-treated gilts compared to controls (p<0.05). In the examined oviductal sections, VD did not differ between control and SP-treated animals. However, in SP-treated animals VD was higher on day 5 than on day 1 (p<0.05) or 3 (p<0.01). SP had no significant effect on VEGF, Flk-1/KDR and FGF-1 mRNA expression. In conclusion, our results suggest that SP affects the vascular network by changing the expression of factors contributing to angiogenesis.

Regulation of Fibroblast Growth Factor-2 Expression and Cell Cycle Progression by an Endogenous Antisense RNA

Basic fibroblast growth factor (FGF2) is a potent wide-spectrum mitogen whose overexpression is associated with immortalization and unregulated cell proliferation in many tumors. The FGF2 gene locus is bi-directionally transcribed to produce FGF2 mRNA from the “sense” strand and a cis-antisense RNA (NUDT6) from the NUDT6 gene on the “antisense” strand. The NUDT6 gene encodes a nudix motif protein of unknown function, while its mRNA has been implicated in the post-transcriptional regulation of FGF2 expression. FGF2 and NUDT6 are co-expressed in rat C6 glioma cells, and ectopic overexpression of NUDT6 suppresses cellular FGF2 accumulation and cell cycle progression. However, the role of the endogenous antisense RNA in regulation of FGF2 is unclear. In the present study, we employed siRNA-mediated gene knockdown to examine the role of the endogenous NUDT6 RNA in regulation of FGF2 expression and cell cycle progression. Knockdown of either FGF2 or NUDT6 mRNA was accompanied by a significant (>3 fold) increase in the complementary partner RNA. Similar reciprocal effects were observed at the protein level, indicating that these two transcripts are mutually regulatory. Remarkably, knockdown of either FGF2 or NUDT6 significantly reduced cell proliferation and inhibited S-phase re-entry following serum deprivation, implicating both FGF2 and NUDT6 in the regulation of cell transformation and cell cycle progression.

Dynamic modulation of basic Fibroblast Growth Factor (FGF-2) expression in the rat brain following repeated exposure to cocaine during adolescence

Our study stems from four related lines of evidence: (1) FGF-2 is expressed in the developing brain; (2) psychostimulants modulate FGF-2 expression; (3) stress alters FGF-2 expression; and (4) exogenous administration of FGF-2 long-lastingly alters cocaine acquisition of self-administration. This research aims to study the effects of adolescent cocaine exposure on FGF-2 mRNA levels and its influence on the response to stress. Rats were treated subcutaneously with saline or cocaine from postnatal day (PND) 28 to PND 42, a period that roughly approximates adolescence in humans. At PND 45 and PND 90, rats were exposed to an acute stress. Real-time PCRs were performed on total RNA extracted from the prefrontal cortex, hippocampus, nucleus accumbens and striatum. In the prefrontal cortex, repeated cocaine treatment during adolescence increased FGF-2 mRNA levels in PND 90 rats and altered its response to an acute stress in both PND 45 and PND 90 rats. In the hippocampus of PND 45 rats, we found an increase of FGF-2 mRNA levels following repeated cocaine administration. No changes in the trophic factor gene expression were found in the striatum and nucleus accumbens. Our data show that cocaine exposure during adolescence alters FGF-2 mRNA levels throughout life in rat prefrontal cortex and modulates its response to an adverse event. These results point to FGF-2 as a potential molecular target through which exposure to cocaine early in life may dynamically and persistently alter brain homeostasis.

Expression of fibroblast growth factor 4 in polydactyly of the thumb induced by cytarabine

Objective To observe the expression offibroblast growth factor4 (FGF-4) in polydactyly of the thumb induced by cytarabine and investigate the pathogenesis of polydactyly. Methods Sixty-six 2 to 3 months old Sprague Dawley rats were caged together daily from 6 pm to 8 am next morning by a male:female ratioof 1:2.The day that a vaginal plug was found was considered as embryonic day 0.5 ( E0.5).30 pregnant rats were assigned to the experimental group, while another 30 were assigned as controls.At day E10.5 intraperitoneal injection of cytarabine ( 100 mg/kg) was carried out in the experimental rats to induce polydactyly and syndactyly in the pupa.Normal saline was injected to the control rats.E10.5 to E12.5 embryos were removed from the pregnant female rats and limb buds were taken out and fast frozen.mRNA was extracted from the limb buds.The expression of FGF-4 mRNA was quantified using RT-PCR. Results In the experimental group FGF-4 expression in the limb buds was significantly higher and lasted much longer than the control group (P ＜0.01).Conclusion The rat embryo's polydactyly of the thumb can be induced by cytarabine.FGF-4 expression in the limb buds in the polydactyly embryo is increased. Key words: Cytarabine; Polysyndactyly; Rats; FGF-4

Neonatal fibroblast growth factor treatment enhances cocaine sensitization

Growth factors are critical in neurodevelopment and neuroplasticity, and recent studies point to their involvement in addiction. We previously reported increased levels of basic fibroblast growth factor (FGF2) in high novelty/drug-seeking rats (bred high responders, bHR) compared to low novelty/drug-seeking rats (bred low responders, bLRs). The present study asked whether an early life manipulation of the FGF system (a single FGF2 injection on postnatal day 2) can impact cocaine sensitization and associated neurobiological markers in adult bHR/bLR animals. Neonatal FGF2- and vehicle-treated bHR/bLR rats were sensitized to cocaine (7 daily injections, 15mg/kg/day, i.p.) in adulthood. Neonatal FGF2 markedly increased bLRs' typically low psychomotor sensitization to cocaine (day 7 locomotor response to cocaine), but had little effect on bHRs' cocaine sensitization. Gene expression studies examined dopaminergic molecules as well as FGF2 and the FGFR1 receptor in cocaine naïve animals, to investigate possible neurobiological alterations induced by neonatal FGF2 exposure that may influence behavioral response to cocaine. bLRs showed decreased tyrosine hydroxylase in the ventral tegmental area (VTA), decreased D1 and increased D2 receptor expression in the nucleus accumbens core, as well as decreased FGF2 in the VTA, substantia nigra, accumbens core, and caudate putamen compared to bHRs. Neonatal FGF2 selectively increased D1 receptor and FGF2 mRNA in the accumbens core of bLRs, which may contribute to their heightened cocaine sensitization. Our results suggest increased FGF2 in the mesodopaminergic circuit (as in baseline bHRs and neonatal FGF2-exposed bLRs vs. baseline bLRs) enhances an individual's susceptibility to cocaine sensitization and may increase vulnerability to drug seeking and addiction.

FGF signaling is involved in physiological adaptation to pressure overload in developing heart

Fibroblast growth factors (FGFs) play an important role during embryonic induction, growth and patterning. Exogenous FGF2 as well as pressure loading were shown to induce embryonic myocyte proliferation. Here we tested whether FGF2 signaling is involved in transmission of pressure overload to increased myocyte proliferation in vivo.Pressure overload was induced by conotruncus constriction in Stage 21 chick embryos. Anti‐bromodeoxyuridine labeling was used to assess proliferation at 24, 48 and 72 h. Western blotting and RT‐PCR to measure FGF2 protein and mRNA levels was performed on ventricular extracts at 48 h interval. Blood was collected at 48 h to assess the amount of FGF2 in serum by ELISA. Proliferation of ventricular myocytes was increased significantly at 48 h. Neither western blotting, nor immunohistochemistry revealed any changes in the amount of myocardial FGF2. ELISA showed a 93% increase of FGF2 in the serum. Increased amount of FGF2 mRNA in ventricular myocardium was confirmed by real time PCR.We conclude that FGF2 synthesis is increased in embryonic ventricular cardiomyocytes in response to increased stretch due to pressure overload. Increased stretch causes its release into serum, causing it to act in endocrine, rather then paracrine manner.Supported by Ministry of Education VZ 0021620806, ASCR AV0Z50450515 and AV0Z50110509 and GACR P302/11/1308.Grant Funding Source : P302/11/1308

781 UP-REGULATION OF FIBROBLAST GROWTH FACTOR 2 IN CASTRATION-INDUCED MOUSE PROSTATE STROMAL REMODELING LEAD TO IMBALANCE OF BASAL EPITHELIAL-STROMAL INTERACTIONS

You have accessJournal of UrologyProstate Cancer: Basic Research IV1 Apr 2012781 UP-REGULATION OF FIBROBLAST GROWTH FACTOR 2 IN CASTRATION-INDUCED MOUSE PROSTATE STROMAL REMODELING LEAD TO IMBALANCE OF BASAL EPITHELIAL-STROMAL INTERACTIONS Manabu Kato, Kenichiro Ishii, Yoichi Iwamoto, Yasuhide Hori, Yasushi Yamada, Kiminobu Arima, Taizo Shiraishi, and Yoshiki Sugimura Manabu KatoManabu Kato Mie, Japan More articles by this author , Kenichiro IshiiKenichiro Ishii Mie, Japan More articles by this author , Yoichi IwamotoYoichi Iwamoto Mie, Japan More articles by this author , Yasuhide HoriYasuhide Hori Mie, Japan More articles by this author , Yasushi YamadaYasushi Yamada Mie, Japan More articles by this author , Kiminobu ArimaKiminobu Arima Mie, Japan More articles by this author , Taizo ShiraishiTaizo Shiraishi Mie, Japan More articles by this author , and Yoshiki SugimuraYoshiki Sugimura Mie, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.869AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES In the tumor microenvironment of castration resistant prostate cancer (CRPC), paracrine support from the stroma surrounding prostate cancer (PCa) cells is considered to play a critical role in the promotion of tumorigenesis. Castration induces the stromal remodeling, e.g. regression of smooth muscle cells, increase of fibroblast, myofibroblast, and inflammatory cells that secrete growth factors (GFs). Although these changes might play important roles in the tumor progression, the castration-induced alteration of gene expression profile is not well elucidated. Thus, we analyzed the gene expression profile of stromal GFs in castrated mouse prostate and performed serum-free organ culture to investigate the direct effects of GFs on the prostate. METHODS Male 8 weeks C57BL/6 mice were castrated and killed at various time points (day 1, 3, 7, 28). The dorsolateral lobes were dissected out and prepared for further investigation. TaqMan® Gene Expression Assays for Acta2, Vim, Tnc, Emr1, Egf, Fgf2, Fgf7, Fgf10, Hgf, Igf1, Tgfa, and Tgfb were conducted. Explants of mouse prostate were treated with GFs or inhibitors for receptors of GFs in serum-free organ culture. RESULTS The ratio of p63-positive basal epithelial cells was significantly increased under castration in vivo or in the absence of androgen in vitro in a time dependent manner. Luminal epithelial cells shrunk but sustained without apoptosis after castration. mRNA expressions of Acta2, Egf, Fgf10, and Igf1 were down-regulated, while those of Vim, Emr1, Fgf2, Fgf7, Hgf, Tgfa, and Tgfb were up-regulated after castration. FGFR inhibitors (SU5402 and PD173074) but not EGFR inhibitor (AG1478) suppressed the increase in p63-positive basal epithelial cells in vitro. FGF2 but not FGF7 treatment increased in p63-positive basal epithelial cells in vitro. CONCLUSIONS We confirmed that the adult mouse prostate regression following castration-induced stromal remodeling including replacement of SMCs gradually by fibroblastic cells, along with infiltration of macrophage. Therefore, we suggest strongly that castration-induced stromal remodeling might induce the up-regulation of Fgf2, Fgf7, Hgf, and Tgfa mRNA. Furthermore, our investigations showed castration-induced stromal remodeling might lead to imbalance of basal epithelial-stromal interactions through up-regulations of FGF2. Our results warrant further investigations into the role of castration-induced stromal remodeling in the progression of CRPC under androgen-manipulated status. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e319 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Manabu Kato Mie, Japan More articles by this author Kenichiro Ishii Mie, Japan More articles by this author Yoichi Iwamoto Mie, Japan More articles by this author Yasuhide Hori Mie, Japan More articles by this author Yasushi Yamada Mie, Japan More articles by this author Kiminobu Arima Mie, Japan More articles by this author Taizo Shiraishi Mie, Japan More articles by this author Yoshiki Sugimura Mie, Japan More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

FGF10 inhibits dominant follicle growth and estradiol secretion in vivo in cattle

Fibroblast growth factors (FGFs) are involved in paracrine control of follicle development. It was previously demonstrated that FGF10 decreases estradiol (E(2)) secretion in granulosa cell culture and that theca cell FGF10 mRNA expression is decreased in healthy follicles from abattoir ovaries. The main objectives of this study were to evaluate FGF10 and FGFR2b mRNA expression during follicular development in vivo, to evaluate the effect of FGF10 on follicle growth using Bos taurus taurus cows as a model, and to gain more insight into the mechanisms through which FGF10 inhibits steroidogenesis. Messenger RNA encoding both FGF10 and FGFR2b (main FGF10 receptor) was significantly more expressed in subordinate follicles (SFs) than in dominant follicles (DFs). The intrafollicular injection of FGF10 into the largest growing follicle at 7-8 mm in diameter interrupted the DF growth in a dose-dependent manner (11±0.4, 8.3±1 and 5.9±0.3 mm for 0, 0.1, and 1 μg/ml FGF10, respectively, at 72 h after treatment; P<0.05). In a third experiment, follicles were obtained 24 h after FGF10 (1 μg/ml) or PBS treatment through ovariectomy. In theca cells, FGF10 treatment did not affect mRNA encoding steroidogenic enzymes, LHCGR and IGFBPs, but significantly upregulated FGF10 mRNA expression. The expression of CYP19A1 mRNA in granulosa cells was downregulated by FGF10 treatment, which was accompanied by a 50-fold decrease in E(2) production, and decreased cyclin D2 mRNA. These results have shown that FGF10 and its receptor FGFR2b are more expressed in SFs and provide solid in vivo evidence that FGF10 acts as an important regulator of follicular growth in cattle.

NOD2 Agonist Promotes the Production of Inflammatory Cytokines in VSMC in Synergy with TLR2 and TLR4 Agonists

Objective. To investigate the expression of NOD2 in human VSMCs, its role in the production of inflammatory cytokines in VSMC and the possible interaction of NOD2-mediated signaling pathway with those mediated by TLR2 and TLR4. Methods. Human coronary artery smooth muscle cells were stimulated with NOD2 agonist MDP alone or in combination with either TLR2 agonist PAM3 or TLR4 agonist LPSs. The mRNA expression of NOD2 and FGF-2 were measured by RT-PCR. The concentration of IL-8 and TNF-α in the culture supernatants was determined by ELISA. VSMC proliferation ability was analyzed by MTT assay. Results. MDP up regulated the expression of NOD2 mRNA in VSMC in a time-dependent manner, up regulated the expression of FGF-2 mRNA in VSMC, induced the production of IL-8 and TNF-α, and promoted the proliferation of VSMC. Additionally, MDP synergied with LPS and PAM3 to promote the proliferation of VSMC and induce the production of IL-8 and TNF-α. Conclusion. The activation of NOD2-mediated innate immune signaling pathway can increase the proliferation ability of VSMC and induce the production of inflammatory cytokines in VSMC. It is also shown a synergistic effect with TLR2- and TLR4-mediated signaling pathways in this process.

Core Binding Factor Beta Functions in the Maintenance of Stem Cells and Orchestrates Continuous Proliferation and Differentiation in Mouse Incisors

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.

Heterogenous induction of carcinoma-associated fibroblast-like differentiation in normal human prostatic fibroblasts by co-culturing with prostate cancer cells

In the tumor microenvironment, carcinoma-associated fibroblasts (CAFs) are considered to play a critical role in the promotion of tumorigenesis. However, the mechanisms that generate CAFs are not well elucidated. To understand how CAFs are generated during primary cancer progression, we investigated the biochemical characteristics of normal human prostate stromal cells (PrSC) co-cultured with human prostate cancer (PCa) cells in vitro. In primary cultures of human PCa-derived stromal cells (PCaSC-8 and PCaSC-9), expression of TNC, ACTA2, EGF, FGF7, and IGF1 mRNA was generally higher than PrSC but gene expression patterns were not uniform between PCaSC-8 and PCaSC-9 cells. Transforming growth factor β (TGFβ) and vascular endothelial growth factor (VEGF) protein levels in both PCaSC-8 and PCaSC-9 cells were generally higher than PrSC but levels of both secreted proteins were not same. When PrSCs were co-cultured with androgen-sensitive LNCaP cells or its sublines, androgen-low-sensitive E9 cells and androgen-insensitive AIDL cells, mRNA expression of IGF1 was significantly increased in all combinations. In contrast, expression of COL1A1, TNC, and ACTA2 mRNA was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Protein production of VEGF was significantly increased only in LNCaP + PrSC and E9 + PrSC co-cultures. Increase of TGFβ protein was observed only in E9 + PrSC co-cultures. These biochemical characteristics of PrSC were partially recapitulated in TGFβ-treated PrSC. We have demonstrated that normal fibroblasts co-cultured with cancer cells become activated and exhibit biochemical characteristics of CAFs in a heterogenous manner. Our results suggest that heterogenous induction of CAF-like differentiation might be strongly dependent on biochemical characteristics of adjacent cancer cells.