Butyrate inhibits the retinoic acid-induced differentiation of F9 teratocarcinoma stem cells

F9 mouse teratocarcinoma stem cells differentiate into parietal endoderm cells in the presence of retinoic acid, dibutyryl cyclic AMP, and theophylline (RACT). When F9 cells are exposed to 2-5 mM sodium butyrate plus RACT, they fail to differentiate. Differentiation is assessed by induction of laminin and collagen IV mRNA, the synthesis of laminin, collagen IV and plasminogen activator proteins, and alterations in cell morphology. Butyrate inhibits differentiation only when added within 8 hr after retinoic acid addition. Thus an early event in retinoid action on F9 cells is butyrate-sensitive. The population doubling time and cell cycle distribution of F9 cells are not altered within the first 24 hr after butyrate addition, suggesting that butyrate does not inhibit differentiation by inhibition of growth or normal cycling. However, butyrate does inhibit histone deacetylation in F9 cells, and this could be the mechanism by which butyrate inhibits differentiation.     

Expression of fibroblast growth factor by F9 teratocarcinoma cells as a function of differentiation

F9 teratocarcinoma stem cells treated with retinoic acid (RA) and dibutyryl cAMP (but2 cAMP) differentiate into embryonic parietal endoderm. Using heparin-affinity chromatography, endothelial cell proliferation assays, immunoprecipitation, and Western analysis with antibodies specific for acidic and basic fibroblast growth factors (FGFs), we detected biologically active FGF in F9 cells only after differentiation. A bovine basic FGF cDNA probe hybridized to 2.2-kb mRNAs in both F9 stem and parietal endoderm cells and to a 3.8-kb mRNA in F9 stem cells. A genomic DNA probe for acidic FGF hybridized to a 5.8-6.0-kb mRNA in both F9 stem and parietal endoderm cells, and to a 6.0-6.3-kb mRNA only in parietal endoderm cells. Although these FGF mRNAs were present in the stem cells, we could find no evidence that F9 stem cells synthesized FGFs, whereas differentiated F9 cells synthesized both acidic and basic FGF-like proteins. We conclude that biologically active factors with properties characteristic of acidic and basic FGF are expressed by F9 parietal endoderm cells after differentiation. Differentiating embryonic parietal endoderm thus may serve as a source of FGF molecules in the developing blastocyst, where these factors appear to play a central role in subsequent embryogenesis.     

Studies on the relation of DNA synthesis to retinoic acid-induced differentiation of F9 teratocarcinoma cells

Inhibition of DNA synthesis in F9 embryonal carcinoma cells with high thymidine induces differentiation similar to that induced with retinoic acid (RA). The presence of differentiated cells is evident after 15 h of treatment with 2 mM thymidine, during which period DNA synthesis is inhibited 99%. The addition of RA during the period of high thymidine treatment does not increase the amount of differentiation seen at the end of the 15-h treatment, but does increase the amount seen after thymidine is removed. The inhibition of proliferation by low serum concentration does not induce differentiation in the absence of RA. In partially synchronized cultures of F9 cells, the addition of RA alters the pattern of DNA replication during the first third of S phase. If RA is present during this part of S phase, differentiation is evident both morphologically and biochemically during the following cell cycle. Addition of RA during the second half of S phase does not lead to obvious differentiation until after the next cell cycle. These results suggest that particular events during the early replication period of F9 cells are targets for RA action in induction of differentiation of F9 cells.     

Retinoic acid-induced differentiation of F9 embryonal carcinoma cells

The ability of retinoic acid (RA) to induce differentiation in embryonal carcinoma (EC) cells was examined by growing mouse F9 cells in a medium containing 1 μM RA. The altered properties of the cells became apparent after a lag period of approx. 24 h and were fully expressed after 5 days. The RA-induced phenotype was characterized by changes in cell morphology, slowing of the rate of cell multiplication, reduced DNA and protein synthesis, altered pattern of polypeptide synthesis and changes in cell surface components. The slowing of cell multiplication and general reduction in the rate of protein synthesis was paralleled by changes in the relative rates at which different polypeptides were synthesized. Two-dimensional gel electrophoretic analysis of [³⁵S]methioninelabelled cell proteins showed an altered relative synthesis of at least fifty polypeptides. The relative rate of synthesis of two components of the cytoskeleton identified as vimentin and tropomyosin were shown to increase.     

Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) expression during induced luteolysis in the bovine corpus luteum

Angiogenic factors, like vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), and their receptors, are strongly regulated during the development of bovine corpus luteum (CL). The aim of this study was to investigate real-time changes of these factors in luteal tissue of cows (n = 4-5 per group) in the mid-luteal phase (day 8-12) after intramuscular injection of the PGF2alpha-analog Cloprostenol. Before (control) and 2, 4, 12, 48, and 64 hr after prostaglandin (PG) injection, CL were collected by transvaginal ovariectomy. RT-PCR for VEGF, VEGF-receptor type 1 (VEGF-R1), VEGF-R2, acidic FGF (FGF-1), basic FGF (FGF-2), and FGF-receptor (FGF-R) was performed. Additionally, the protein concentration for VEGF was determined. The mRNA expression of VEGF and its two receptors (VEGF-R1 and -R2) was significantly downregulated during structural luteolysis (after 12 hr). VEGF protein concentration already significantly declined 2 hr after PGF2alpha. Surprisingly FGF-1 and FGF-2 were significantly and maximally upregulated during functional luteolysis (until 12 hr). Furthermore, FGF-R mRNA was significantly upregulated at 2 hr after PGF2alpha, when compared with the control group. During structural luteolysis, the expression of FGFs and their receptors was not significantly different from control, except FGF-2 mRNA, which was downregulated at 64 hr. We conclude that the cessation of VEGF-support for the CL plays a role during structural luteolysis, whereas FGFs seem to have a major impact on functional luteolysis. The possible role of these growth factors could be a transient counter-regulation of luteolysis, but also an involvement in preventing inflammatory reactions during luteal regression.     

Co-purification of proteases with basic fibroblast growth factor (FGF)

Acidic and basic fibroblast growth factors (FGFs) are proteins of 16-18 kDa. Other forms of 25-30 kDa related to this growth factor family have recently been described. All these components bind tightly to heparin-Sepharose, a property that allows the purification of several FGF-related proteins. During the purification of acidic and basic FGFs from bovine pituitary glands, we detected the presence of 28-30 kDa components that are immunoreactive against anti-basic FGF antisera. However, microsequencing analysis revealed that the 28-30 kDa components are lysosomal proteases that co-elute with basic FGF from heparin-Sepharose columns. The involvement of these proteases in the etiology of microheterogenous forms of FGFs and/or release of FGFs from the extracellular matrix is discussed.     

Expression and function of fibroblast growth factor (FGF) 9 in hepatic stellate cells and its role in toxic liver injury

Hepatic injury and regeneration of the liver are associated with activation of hepatic stellate cells (HSC). Fibroblast growth factors (FGFs) and their receptors are important regulators of repair in various tissues. HSC express FGFR3IIIc as well as FGFGR4 and different spliced FGFR1IIIc and FGFR2IIIc isoforms which differ in the presence or absence of the acid box and of the first Ig-like domain. Expression of FGF9, known to be capable to activate the HSC FGFR2/3-isoforms, was increased in HSC in liver slice cultures after exposition to carbon tetrachloride, as an acute liver injury model. FGF9 significantly stimulated 3-H thymidine incorporation of hepatocytes, but failed to induce DNA synthesis in HSC despite the fact that FGF9 induced a sustained activation of extracellular signal-related kinases (ERK) 1/2. FGF9 induced an increased phosphorylation of Tyr436 of the fibroblast growth factor receptor substrate (FRS) 2, while phosphorylation of Tyr196 which is required for efficient Grb2 recruitment remained unchanged. Our findings suggest that HSC FGF9 provide a paracrine mitogenic signal to hepatocytes during acute liver injury, while the autocrine FGF9 signaling appears to be not sufficient to induce cell proliferation.     

Stimulation of aromatase activity in immature porcine Leydig cells by fibroblast growth factor (FGF)

The effects of fibroblast growth factor (FGF) on testicular aromatase activity has been studied using primary cultures of porcine Leydig cells. After culture for 3 days in the absence or presence of FGF, the ability of the cells to produce estrogen was examined in a 4h-test period in which either (a) hCG (10(-9) M) or (b) androstenedione (3 x 10(-6) M) was added to the medium. FGF produced a 3- to 20-fold increase in estrogen formation from endogenous or exogenous substrate during the test period, in spite of a marked decrease (approximately equal to 60%) in [125I]-hCG binding and no significant change in testosterone concentration. Stimulation of estrogen secretion by FGF was dose-(ED50 approximately equal to 2 ng/ml) and time-dependent, the first and maximal effects were observed after 12h and 48h, respectively. Preliminary tests with several other factors (insulin, EGF, TGF-beta, FSH and hCG) showed that hCG alone directly stimulated aromatase activity. From these findings a role is suggested for FGF as a paracrine/autocrine agent in the control of estrogen secretion by Leydig cells.     

Rapid and transient decrease of N-myc expression in retinoic acid-induced differentiation of OTF9 teratocarcinoma stem cells.

The level of expression of N-myc in mouse teratocarcinoma stem cells is very high. Previous studies have shown that N-myc expression significantly decreases when the stem cells are subjected to long-term induction for differentiation by retinoic acid (RA). We found that in a stem cell line, OTF9, a steep yet transient decrease of N-myc expression takes place much earlier, immediately after induction by RA. To examine whether this decrease is responsible for differentiation, we constructed a gene, miwNmyc, to express N-myc cDNA constitutively and transformed OTF9 cells with this gene construct. Transformants under the constitutive expression of miwNmyc differentiated normally, as judged by morphological changes and by modulation of c-myc, Hox1.1, and laminin B1 expression. Therefore, transient decrease of N-myc expression may be the consequence of RA-induced differentiation, even though it occurs very early in the process. Alternatively, in addition to N-myc decrease, there may be redundant mechanisms which lead to OTF9 differentiation after induction by RA, so that suppression of N-myc decrease is bypassed by at least one other mechanism.     

Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo

Although it has been clearly established that basic fibroblast growth factor (FGF) is a potent mitogen for chondrocytes in vitro, there is little evidence that it can stimulate this cell type in vivo. In an effort to address this problem, we examined the effect of an intraarticular administration of basic FGF. Alzet osmotic pumps delivering the mitogen to the site of injury promotes the healing of intra-chondrial lesions by stimulating chondrocyte proliferation and the formation of extracellular matrix. The observation that chronic infusions of basic FGF can elicit a repair response at the site of injury suggests that this growth factor may have therapeutic applications that extend beyond its capacity to induce neovascularization. The results also suggest that one of the ways that the perichondrium mediates cartilage repair may be by the local production of FGF-like mitogens.     

Comparative proteomic analysis of differentiation of mouse F9 embryonic carcinoma cells induced by retinoic acid

The multipotent mouse F9 embryonic carcinoma cell is an ideal model system to investigate the mechanism of retinoic acid (RA) in cell differentiation and cell growth control and the biochemical basis of early embryonic development. We reported here a proteomics approach to study protein expression changes during the differentiation of F9 cells into the visceral endoderm. F9 cells were incubated with or without RA at 0, 24, 48, and 72 h. Total proteins extracted were separated by two‐dimensional electrophoresis (2‐DE) and the protein patterns on the gels were comparatively analyzed by computer. Approximately 1,100 protein spots were detected in the F9 proteome, within the pH 3–10 range. Fourteen protein spots which the levels of expression were found to be altered dramatically during the F9 cells differentiating, and were identified by MALDI‐TOF MS or ESI‐MS/MS. These proteins included metabolism enzymes, HSP60s, RAN, hnRNP K, FUBP1, VDAC1, STI1, and prohibitin. These proteins are involved in cellar metabolism, gene expression regulation, stress response, and apoptosis, respectively. The data from proteomic analyze are consistent with the result obtained from Western blot analysis. This study increases our understanding of the proteomics changes during F9 cells differentiation induced by RA. J. Cell. Biochem. 113: 1811–1819, 2012. © 2012 Wiley Periodicals, Inc.