This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our lab has recently demonstrated an important role for ? adrenergic receptor (?AR) signaling in regulation of cardiomyocyte proliferation during the fetal and early postnatal period. We have also shown in developing animals that ?1AR expression is under control of a novel sequence element(s) including a homeodomain protein binding site, an E-box for binding of the bHLH factors myc and max and a consensus site for glucocorticoid receptor binding. Using nuclear run-on assays for measurement of transcription rate, we showed that there is a much higher ?1AR transcription rate during the proliferative period of cardiac growth than in older animals.1 This increase in ?1AR transcription rate is associated with a concomitant increase in transcription rate of the protooncogene c-myc. Transcription rate assays performed in rat fibroblast cell lines genetically engineered to over-express c-myc (myc+/+) show increased ?1AR expression, compared to the wild type cell line. Transient transfection of ?1AR promoter constructs in the myc+/+ cells demonstrated robust expression, which is abrogated by mutation of the myc/max binding site or by co-transfection with a c-myc antisense expression vector. While the bHLH transcription factor c-myc has classic DNA binding motifis, its direct demonstration as a transcription factor has proven more difficult. Our recent results suggest that the regulation of cardiac ?1AR transcription and expression of c-myc are tightly integrated. In this proposal we have sought to verify the physiological importance of this regulation using a well accepted experimental growth paradigm, in-vitro stretch of isolated cardiomyocytes. We also seek to identify the transcriptional mechanism(s) responsible for the developmentally regulated expression of the ?1AR. The original Specific Aims are listed below along with progress in each Aim.
Specific Aim 1 : Determine the effect of stretch on transcriptional activation of ?1AR and c-myc using promoter reporter gene constructs, Northern blot and transcription rate assays. a. Progress: Primary cardiomyocytes have been isolated from 2-3 day old rats as previously described.1,2 Cells were cultured on specially-designed collagen matrix for use with the flexor cell apparatus. After 24 hours overnight culturing and stabilization, the cells were stretched for time intervals ranging from 30 minutes to eight hours. Prior studies looking at the effect of stretch on c-myc activation indicated that peak expression of the protooncogene c-myc was seen after two hours of stretch. Total RNA was then isolated from the stretched or controlled cells and subjected to Northern blot analysis. The Northern blots are probed with random primary-labeled cDNAs for with c-myc or the ?1AR. The results are shown in Figure 1. In Panel A can be seen the effects of stretch for two hours on expression of the c-myc proto-oncogene. As can be seen, c-myc expression increased substantially by a two-hour phage. There was, however, no concomitant increase in c-myc expression at this early timepoint. te effects of stretch for 0, 2, 4 & 8 hours on ?1AR mRNA was then examined. There was a striking (5-10 fold) increase in ?1AR expression. The temporal sequence of increased c-myc expression followed by an increase in ?1AR expression is supportive of our preliminary data suggesting the ?1AR is downstream target of myc. This will be explored further in these cells using transient transfection assays to abrogate c-myc expression prior to stretching. We hypothesize that a downstream increase in transcription rate of the ?1AR will not be seen in cells in which the c-myc expression has been attenuated by co-transfection with a c-myc antisense expression vector.1 If the transfection efficiency is not sufficient to demonstrate such an effect, there are two alternative strategies we will explore. One of these is to attempt to abrogate c-myc expression using RNA interference. This, however, will be dependent on similar vagaries of transfection efficiency. Alternatively, we may construct an adenovirus expression vector containing the c-myc coding sequence in antisense orientation. We have had success with this strategy in transient transfection assays and luciferase reporters. The advantages of the adenovirus-mediated transfections will be to allow successful infection of a substantial proportion of the isolated cardiomyocytes and examination of the effect of abrogation of c-myc expression on ?1AR transcription rate.
Specific Aim 2 : Determine the role of c-myc as a DNA binding protein and transcriptional activator of the ?1AR gene by chromatin immunoprecipitation (ChIP) assays. a. Progress: As outlined in the original application, novel transcriptional elements regulate ?1 adrenergic receptor expression. A composite element we called glucocorticoid regulatory unit (GRU) is composed of a region for binding of homeodomain proteins, the E-box where myc and max bind and the glucocorticoid response element. We have recently had success identifying the putative homeodomain protein binding to the upstream region. Affinity chromatography was carried out with an oligonucleotide concatener of the homeodomain and the E-box binding region. Extract from isolated cells was first cleaned up by passage over a heparan column. The eluate was monitored using gel shift assays. The appropriate fractions were then applied to the DNA affinity column and eluted in stepwise fashion using a KCl concentrations varying from 0.1-1 mM. The column eluate was again monitored using the gel shift assays. A specific band was identified in the .3-.4 mm KCL fraction which was then pooled, concentrated using an Amicon filter and subjected to SDS page electrophoresis. A number of discrete bands were seen on the SDS page gel. In order to identify the specific band with DNA binding activity, the SDS page blot was transferred to a nylon membrane and probed with the radiolabelled oliqonucleotide sequence containing the homeodomain and the E-box region, a so-called Southwestern blot. The specific band identified by Southwestern blotting was eluted from the gel and subjected to analysis by mass spectrometry (MALDI-tof) at University of Massachusetts in Worcester. The identified protein is a previously recognized transcription factor known as the polypyrimidine tract binding protein splicing factor, PTB-psf ref 3. This transcription factor has been most extensively studied in its regulation of the insulin-like growth factor 1 (IGF1) promoter. It was originally isolated by Dr. Randall Urban (UT, Galveston). Dr. Urban has graciously provided a PTB-psf expression vector as well as antibodies to PTB-psf. We have co-expressed the GRU luciferase reporter construct with PTB-psf alone or in combination with c-myc. Studies were carried out in isolated three-day old primary cardiomyocytes. As can be seen, PTB-psf alone had only a modest effect on GRU-dependent luciferase activity. However, the combination of PTB-psf and c-myc resulted in a significant (5-6 fold) increase in the expression of the GRU luciferase construct. b. Future Studies: Studies are in progress using site-directed mutagenesis to abrogate the homeodomain binding region and to demonstrate that the effects of PTB-psf are indeed dependent on the intact transcriptional element. We are planning experiments using chromatin immunoprecipitation as outlined in the original proposal to identify the binding regions for myc and PTB-psf on the ?1AR and other promoters where they may cooperate. Likewise, we are carrying out single and double immunoprecipitation assays to see if we can demonstrate protein-protein interaction between PTB-psf and c-myc. Lastly, we believe that the level of ?1AR transcription at different stages of development dependent on the stoichiometry of the proteins PTB-psf, c-myc, max, mad and the glucocorticoid receptor. RNAse protection assays and Western immunoblotting are being conducted to determine the ontogeny of the levels of expression at the RNA and protein level of these important regulators of the ?1AR.
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