Biochemical Society Transactions (1999) 27 111
DIFFERENTIAL ACTIVATION OF MAP& BY MEMBERS OF THE FGF FAMILY. Mehta. PB.. Robson, CN., Neal, DE, and Leung, HY. Department of Surgical and Reproductive Sciences, University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK. (
[email protected]) Members of the fibroblast growth factor (FGF) family function as both paracrine and autocrine agents to regulate proliferation, differentiation, cellular motility and angiogenesis. We have previously demonstrated FGF overexpression, namely FGF-I, -2, -7 and -8,in resected prostate tumours. As FGFs have a wide range of functional capabilities, we hypothesised that different FGFs have unique signalling pathways to account for various functional consequences. Here we report characterisation of differential activation of members of the MAPK family by FGFs. Applying exogenous FGFs on DU145 prostate cells (FGFR positive), we performed comprehensive studies on the signalling activities to include STATS 1.3 and 5 as well as the MAPKS [Erk (p42/44), JNK (p46/54) and ~381. Serum-starved DU145 cells were stimulated for fifteen minutes with FGFs at the corresponding ED50 doses [FGFl(38.1 pM with 10 pdml heparin), FGF-2 (31.25 pM) or FGF-7 ( I nM)]. STAT activities were evaluated using mobility shift assays; Erk and JNK activities were assayed using phosphospecific antibodies on western blotting and p38 activity with an in vitro kinase assay with myelin basic protein as substrate. FGF-I and -2 strongly activated Erkl/2 and p38 while FGF-7 induced p38 alone, but more potently than FGF-I and -2. Assays for JNK and STAT activities showed no significant FGF-induced activation. Transfection assays in COS-7 cells were. performed using Elk-I-FLAG and GALCMEF2A plasmids (substrates for Erk and p38 respectively) and confirmed similar patterns of FGF-induced MAPK activations and their downstream activities. Co-incubation with FGF-7 did not influence epidermal growth factor or FGF-I activated Erk. This work provides the first insight into FGF-induced differential MAPK activations to account for the diversity of FGFfunctions.
-
113 Clenbuterol a n d denervation upregulate IGF-I1 a n d H I 9 mRNA expression in rat skeletal muscle. A.A. Sneddon, M. I. Delday, Steven, J. and C. A. Maltin.
The Rowett Research Institute, Greenbum Road, Bucksburn, Aberdeen. AB2l 9SB. Scotland U.K. The 02-adrenoceptor agonist clenbuterol specifically promotes hypertrophy of skeletal muscle in a wide variety of animals The anabolic effect of this drug is manifest in both growing (innervated) as well as atrophic (eg. denervated) muscle, Although such hypertrophy has been shown to be achieved through an initial increase in the rate of protein synthesis in addition to a later, more prolonged decrease in the rate of protein degradation, the precise signal transduction pathway(s) involved are unknown. In view of the known hypertrophic effects of IGF-I1 on muscle, we examined the expression of the IGF-I1 gene, as well as the associated H19 gene, in muscle treated with clenbuterol. Transcript levels of both IGF-I1 and H19 were upregulated with clenbuterol at a time which correlates with the clenbuterol associated increase in the rate of protein synthesis. The expression of both HI9 and IGF-I1 transcripts were also upregulated in denervated muscle, where clenbuterol induced an additional expression ofthese genes. These findings implicate both IGF-I1 and H19 as playing potentially important roles in the response of differentiated muscle to anabolic stimuli as well as in the amelioration of denervation-induced muscle atrophy.
114
Diurnal variation in S-oxidation of S-caboxymethyl-L-cysteine metabolism in Man
G.B. Steventon
112 THE HISTONE ACETYLTRANSFERASETIP60 IS A
CO-ACTIVATOR PROTEIN OF THE HUMAN ANDROGEN RECEPTOR PROTEIN. Gauehan. L., Brady, M.E., Cook, S., Neal, D.E. and Robson, C.N.
Department of Pharmacy, King's College London, London SW3 6LX.
School of Surgical and Reproductive Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK
The metabolism of Scarboxymethyl-LGysteine(SCMC) is complex and he identity of the major urinary metabolite of SCMC has been under considerable debate over the last 20 years with two schools of thought;l.That SCMC and SCMC S-oxide are the major drug related compounds in urine. 2. That thidglycolic acid (TDA) and TDA S-oxide are the major drug related compounds in urine. This present investigation has studied the metabolism of 750 mg of SCMC in 5 males at two different administration times (0O:OO h and 08:OO h) and analysed the 0-8and 8-16 h urine collections for drughirug related compounds. The administration of SCMC at 08:OO h and subsequent urine collection to 16:OO h resuIted in SCMC (1 1.4-30.5%of adrmnistered dose) and SCMC S-oxide (4.7-18.2%) being identified as the major drug related canrpounds m the urine. The 16:OO-0O:OOh (8-16 h) urine collection found that SCMC (5.3-15.1'70)and S-(carboxymethylthio)-L-cysteh~CMTC (2.1-5.1'70)were the major drug related compounds in the urine. When SCMC was administered at 0O:OO h.and the s u b s e q m t 0-8 h urine collection was analysed, SCMC (5.1-33.4%) and TDA (8.4-23.4%)were the major drug related compounds in the urine. However, the 08:OO-16:OOh (8-16 h) urine collection was
Androgens are important in the functions of the human prostate and the development and progression of prostatic diseases. Androgen effects are mediated through the androgen receptor (AR) which is a member of the nuclear hormone receptor superfamily of ligand dependent transcription factors. Our work has focused upon defining the mechanisms of transcriptional control exacted by the AR. Using a C-terminal region of the human AR in a yeast 2-hybrid screen, we have identified the histone acetyltransferase Tip60 as an AR interacting protein. Tip60, originally identified as a coactivator for the H N TAT protein enhances AR mediated transactivation in a ligand dependent manner in LNCaP and COS-1 cells. Additionally, Tip60 enhances transactivation through the estrogen and progesterone receptors in a ligand dependent manner. Our studies demonstrate that Tip60 co-immunoprecipitates with AR and SRC-1 in vitro and that Tip60 enhances transactivation to levels observed with the co-activators SRC-1, p300 and CBP. Further work, to be presented, indicates that Tip60 acts to recruit other co-activators to the promoter of AR target genes. The importance of such proteins in enhancing nuclear hormone receptor mediated transcriptional activation is widely accepted and this work suggests that Tip60 may play an equally important role.
A1 21
foundtocontainTDA(5.7-14.1%)~dTDAS-oxide (15.4-23.7%).Thus the production ofthe metabolites of SCMC appears to be under chronobiological control and thus may explain the di&rent metabolic profiles seen by different groups when studying the metabolism of SCMC in man.
