Fibroblast growth factor-4 enhances proliferation of mouse embryonic stem cells via activation of c-Jun signaling.

Fibroblast growth factor-4 (FGF4) is expressed in embryonic stages and in adult tissues, where it plays critical roles in modulating multiple cellular functions. However, the exact roles of FGF4 on proliferation and differentiation of embryonic stem cells (ESCs) are not completely understood. Exogenous addition of FGF4 stimulated proliferation of mouse ESCs (mESCs), as proven by the increases in DNA synthesis and cell cycle regulatory protein induction. These increases were almost completely inhibited by pre-treating cells with anti-FGF4 antibody. FGF4 also activated c-Jun N-terminal kinase (JNK) and extracellular-signal regulated kinase (ERK) signaling, but not p38 kinase. Blockage of JNK signaling by SP600125 or by transfection with its specific siRNA significantly inhibited FGF4-stimulated cell proliferation through the suppression of c-Jun induction and activator protein-1 (AP-1) activity. However, ERK or p38 kinase inhibitor did not affect FGF4-stimulated proliferation in mESCs. FGF4 suppressed osteogenic differentiation of mESCs by inhibiting expression of transcription factors involved in bone formation. Further, exogenous FGF4 addition stimulated proliferation of human periodontal ligament stem cells (hPDLSCs) and bone marrow mesenchymal stem cells (BMMSCs) via activation of ERK signaling. FGF4 also augmented mineralization of hPDLSCs, but not of BMMSCs. Collectively, it is suggested that FGF4 triggers proliferation of stem cells by activating MAPK-mediated signaling, while it affects differently osteogenic differentiation according to the origins of stem cells

Protective effects of recombinant staphylococcal enterotoxin type C mutant vaccine against experimental bovine infection by a strain of Staphylococcus aureus isolated from subclinical mastitis in dairy cattle

Staphylococcus aureus is one of the main etiological agents of bovine mastitis; however, antibiotics that are effective against bovine strains of S. aureus are not currently available. Staphylococcal enterotoxin type C (SEC), a superantigen, is the enterotoxin most frequently expressed by bovine strains of S. aureus and one of immunogenic determinants. The purpose of this study was to evaluate the protective effectiveness of recombinant SEC mutant vaccine (MastaVac™) against experimentally induced bovine infection. Three representative SEC secreting strains were selected from 9 candidate isolates that showed various intensities of pathogenicity on mice and inoculated into 5 lactating dairy cattle at a concentration of 50–5.0 × 108 CFU per quarter. The optimal experimental bovine subclinical mastitis model was produced by inoculation with 50 CFU of S. aureus 409 per quarter, a level which was not lethal to mice. After the experimental model was determined, other 3 cattle were intramuscularly administered three doses of vaccine at day 0, at 2 wks and at 6 wks. Nine quarters of 3 vaccinated cattle and 8 quarters of 3 control cattle were then challenged with S. aureus 409. An SEC-specific ELISA test conducted at 4 wks post-immunization confirmed the presence of a high antibody titer against SEC in all vaccinated cattle. The somatic cell counts from the vaccinated group remained relatively low, whereas those of control group increased significantly after challenge with S. aureus. After challenge, S. aureus was not isolated from any cattle in the vaccinated group, whereas it was isolated from 75% of the cattle in the control group. These results indicate that recombinant SEC mutant vaccine had a protective effect against S. aureus intramammary infection in lactating cattle.This study was funded by LG life Sciences, Ltd. as well as the National Veterinary Research & Quarantine Service. Drs. Hye Cheong Koo and Yong Ho Park were supported by the Korean Research Foundation Grant (KRF-2006-005-J02903 and KRF-2007-331-E00254), Research Institute of Veterinary Science, Department of Veterinary Microbiology, College of Veterinary Medicine, and the BK21 Program for Veterinary Science, Seoul National University. The authors thank Gum Chan Jang from NVRQS and Sook Shin from Seoul National University for their technical assistance

FGF4 stimulates proliferation and mineralization of hPDLSCs.

<p>(A) hPDLSCs at passage 3 were labeled with the indicated primary antibodies and then analyzed by flow cytometer. The cells were incubated for 48 h with 50 ng/ml FGF4 and/or 100 ng/ml (B), or with each of MAPK inhibitors (C) in 96-multiwell plates. The cells were processed for cell proliferation assay. **p<0.01 vs. GM alone. ^#p<0.05 vs. FGF4 treatment alone. (D) hPDLSCs were incubated in the presence of DAG with and without 50 ng/ml FGF4 and 100 ng/ml anti-FGF4 for 14 days and then were processed for Alizarin red staining. (E) Absorbance of the dye was determined at 560 nm. hPDLSCs were also processed for analysis of ALP activity (F) and mRNA expression (G) after 5 days of differentiation. *p<0.05 vs. DAG alone. F4, FGF4; GM, growth medium; NC, non-significant.</p

FGF4 increases proliferation of mESCs.

<p>mESCs were incubated in the presence of 0 to 200 ng/ml FGF4 in 96-multiwell plates or with the indicated concentration of FGF4 in 6-well culture plates for 48 h and then further processed for BrdU incorporation assay (A) and hematoxylin staining (B), respectively. Experimental results shown in (A) represent the mean ± SD from three separate experiments. **p<0.01 indicates a significant difference between experiments and untreated control cells.</p

Effects of anti-FGF4 antibody on FGF4-induced cell proliferation and protein expression associated with cell cycle progression.

<p>mESCs cultured in 6-well culture plates were incubated in the presence of 50 ng/ml FGF4 and/or 100 ng/ml anti-FGF4 antibody and then proliferation rates were analyzed using ³H-TdR incorporation (A, left panel) and Cell Counting Kit-8 (A, right panel) 48 h after treatment. *p<0.05, **p<0.01, and ***p<0.001 indicate significant differences between experiments. (B) Expression patterns of cell cycle regulatory proteins 24 h after treatment were analyzed by Western blotting. (C) Data from Western blot analysis were quantified by densitometry after normalizing bands to α-tubulin levels. **p<0.01 and ***p<0.001 vs. untreated cells. ^##p<0.01 and ^###p<0.001 vs. cells treated with FGF4 only. (D) mESCs cultured under the same conditions as (C) were also processed for analysis of PCNA expression by flow cytometry. a-F4, anti-FGF4 antibody.</p