Recently, using the yeast two-hybrid screening method, a number of coactivator proteins have been identified for the steroid receptor superfamily. Some of them are specific coactivator pro- teins for a particular receptor as in the case of PPAR-γ, which has a cold-inducible coactivator protein PGC-1 (PPAR gamma coactivator protein-1) (Puigserver et al., 1998). Most coactivator proteins have been shown to interact with a number of steroid receptors. Although PPARα is typical in this respect, so far no coactivator proteins have been found to be specific for PPARα. It is possible that some of these coactivators may be important in peroxisome proliferation me- diated by PPARα. Determination of the expression of PPARα and any such coactivator proteins
Time (hr:min:sec) U V ( ar b it a ry unit s)
Figure 3.53 FPLC of PPARα-LBDwt. His-tag purified proteins were dialysed overnight in TRIS-HCl solution to re- move contaminating small molecular weight solutes. Protein samples were then run on a weak cation exchanger (Econo-S, Bio- rad) FPLC column with isocratic flow using buffer A (0.1M Tris-HCl, pH 8.0) and buffer B (1M NaCl in 0.1M Tris-HCl, pH 8.0). Samples were eluted from the column using a flow rate of 1ml/min and a gradient of 0-50% buffer B over 10 minutes (from 2 minutes to 12 minutes).
in the liver acini may provide a clue as to which, if any, of the coactivators are important in the functioning of PPARα-mediated peroxisomal events that show zonal distribution. Also the ef- fect of a peroxisome proliferator (MCP) on the local expression of both PPARα and “general” coactivator proteins for steroid receptors has been examined by in situ hybridisation.
Section 3.9.1 Region-specific distribution of mPPARα mRNA
Expression and spatial distribution of mPPARα in the liver acini has been investigated using in
situ hybridisation. To determine the expression of each gene, sense and anti-sense oligo-nucle-
otides (45 nucleotides in length) were designed from the 5’-end of the gene and were synthe- sized using automated sequencer (Biomedical Synthesis and Analysis unit, QMC, Nottingham). Using a FASTA search on Genebank, the probe for each gene was checked to ensure that the designed probe shows only 100% homology to the particular mouse gene of interest.
Cryostat sections of frozen liver were treated with 35S ATP labelled sense and anti-sense probes and hybridised at 42 0C overnight. Hybridized probes were detected by coating the tissue sec- tions (for appropriate length of time) with emulsion and then developed as detailed in Methods. Hybridization to α-35S-labelled probes was visulized by bright-field microscopy, revealing pos- itive signals as black silver grains. Expression of glutamine synthase was used as a reliable pos- itive control which is highly expressed in the liver and shows distinct centrilobular distribution pattern (Moorman et al., 1994). Figure 3.54 shows the distribution of glutamine synthase ex- pression in the liver which is illustrated at high and low power magnification as observed under bright-field microscopy. As expected, sense control was unable to detect any mRNA messages while anti-sense probe specifically detected the expression of GS-mRNA in the centrilobular region (Figure 3.54 A,C, labelled C) of the liver acini. Expression of mPPARα mRNA was de- tected in the liver, and due to its low abundance, the radioactive signal was observed under high magnification only (Figure 3.55). The expression of mPPARα mRNA was localised in the nu-
clei but showed no specific pattern of distribution, i.e. mouse PPARα has panlobular distribu- tion. Dosing of mouse with MCP caused no observable change in the expression of liver PPARα when compared to untreated liver (not shown).
Figure 3.54 Distribution of glutamine synthase mRNA in mouse liver. Specific centrilobular distribution of glutamine synthase mRNA in the mouse liver were determined using in situ hybridisation. Radiolabelled anti-sense (A, C, E) and sense (B, D, F) probes were prepared as detailed in methods and hybridised to frozen liver sections of untreated mice. Hy- bridised probes in the tissue sections were localised by overlaying the sections with emulsion and then developed after 6 weeks of exposure in the dark at 4 0C. Hybridisation to a-35S-labelled probe was visualised by bright-field microscopy, revealing pos- itive signals as black silver grain (shown by the use of an arrow). For illustration purposes, results are presented in the pict ure format as viewed under light microscope using magnification: x200 (A, B): x800 (C, D): x2000 (E, F).
A B
C D
E F
anti-sense probe sense probe
C C P P C P C C
Section 3.9.2 Expression of co-activator proteins in the liver
The expression and distribution, at the mRNA level, of a number of coactivator proteins which have been shown to interact and enhance or modulate transcriptional activity of PPARα in vitro has been investigated in the mouse liver. These coactivators include PBP (peroxisome prolifer- ator binding protein), SRC-1 (steroid receptor coactivator-1), CBP/p300 (CREB binding pro- tein), PGC-1 (PPAR gamma binding protein-1) and RIP-140 (receptor interacting protein). Anti-sense probes for each gene were used for the detection of their mRNAs using in situ hy- bridisation while sense probe was used as a control. As shown in Figure 3.56, using anti-sense probes, low level expression of PBP, SRC-1 and CBP/300 was detectable in the liver but their expression was not confined to a particular pattern in the liver acini. As expected no signal was obtained using sense controls (not shown). Dosing of mouse with MCP caused no observable change in the expression of liver PBP, SRC-1 and CBP/300 mRNA when compared to untreated liver (not shown). The expression of the coactivator PGC-1 and RIP-140 mRNAs were not de- tectable in either untreated and MCP treated mouse liver.
Figure 3.55 In situ hybridisation with anti-sense (A) and sense (B) PPARα. Radiolabelled anti-sense probes were prepared as detailed in methods and hybridised to frozen liver sections of untreated mice. Hybridised probes in the tissue sections were localised by overlaying the sections with emulsion and then developed after 6 weeks of exposure in the dark at 4 0C. Hybridisation to α-35S-labelled probe was visualised by bright-field microscopy, revealing positive signals as black silver grain (shown by the use of an arrow). For illustration purposes, results are presented in the picture format as viewed under light microscope (x2000 magnification).
Figure 3.56 Localisation of coactivator mRNA expression in mouse liver. In situ hybridisation for the localisa-
tion of mRNAs for coactivator protein (A) PBP, (B) CPB/300, (C) SRC-1 and (D) PGC-1. Radiolabelled anti-sense probes were prepared as detailed in methods and hybridised to frozen liver sections of untreated mice. Hybridised probes in the tissue sec- tions were localised by coating the sections with emulsion and then developed after 6 weeks of exposure in the dark at 4 0C. Hybridisation to α-35S-labelled probe was visualised by bright-field microscopy, revealing positive signals as black silver grains (shown by the use of an arrow). For illustration purposes, results are presented in the picture format as viewed under light microscope (x2000 magnification).
A B