Broc McCune and Dr. Laura Bridgewater, Microbiology and Molecular Biology
Main Text
Bone morphogenetic protein 2 (Bmp2) is a well-characterized secreted transforming growth factor. Researchers in the lab of Dr. Laura Bridgewater, Brigham Young University, discovered a novel nuclear variant of Bmp2 (nBmp2)1. Preliminary data indicates that mice not expressing nBmp2 have difficulty regulating intracellular Ca2+ (unpublished). Seeking to find a molecular explanation for this observation, I performed a yeast two-hybrid (Y2H) screen to identify putative binding-partners of nBmp2. My results indicate nBmp2 binds Phospholipid scramblase 1 (PLSCR1). Zhou et al2 have shown that PLSCR1 directly binds a 15 DNA base pair sequence (hereafter called P1) ~100 base pairs upstream of the Inositol 1,4,5-triphosphate receptor 1 (Itpr1) gene. This sequence was found in the promoter (DNA that regulates the expression of a gene) for Itpr1. The interaction between Plscr1 and P1 increases the expression of Itpr1 protein in mice. Itpr1 is an additional important regulator of intracellular Ca2+ levels. I hypothesized that one significant way nBmp2 affects Ca2+ regulation is by controlling the transcription of Itpr1 through manipulating the interaction between Plscr1 and P1. I sought to test this possibility by constructing a transcriptional activation reporter assay.
A transcriptional activation reporter assay measures the relative levels of a reporter protein that is under the control of the promoter being studied. Over-expressing different proteins in cells containing plasmid DNA with the reporter downstream of the promoter-of-interest allows the researcher to see the action of those proteins on the promoter by looking at reporter levels in the cells (or indirectly by looking at the reporter’s enzymatic activity). My experimental design required five plasmids, two of which required me constructing them by introducing foreign DNA into plasmids. Plscr1 and nBmp2 were expressed using the mammalian expression vector pcDNA3. Plscr1/pcDNA3 was engineered in Dr. Peter Sims’ lab (University of Rochester) and he graciously allowed me to use his plasmid. I constructed nBmp2/pcDNA3. I prepared my reporter plasmid by inserting a 212-base pair sequence from the Itpr1 promoter upstream of luciferase in the luciferase reporter vector pGL3-Control. The pGL3-Control plasmid expresses luciferase at a medium level. This was chosen to allow me to learn whether binding of nBmp2 to Plscr1 and P1 enhances or represses transcription of the reporter.
Zhou, et al, used Mouse Embryonic Fibroblast (MEF) cells when they showed Plscr1 upregulates Itpr1 expression2. To this end I used MEF cell culture, in addition to Human Embryonic Kidney (HEK), and 10T1/2 cell cultures, two cell lines that are normally maintained in the Bridgewater lab. Dr. Jeff Barrow (BYU, Department of Physiology and Developmental Biology) generously donated MEF cells for my use. The appropriate DNA was incubated with transfection reagent then added to the cells. The cells were given time to express the proteins, whereupon they were harvested and the protein isolated. Luciferase and beta-galactosidase levels were quantified by assaying the level of their enzymatic activity. Luciferase levels were normalized by dividing by beta-galactosidase levels.
The preliminary data obtained reflects a need to optimize the experiment. The most significant challenge was to recapitulate the previous results showing that Plscr1 up-regulates Itpr1 expression. Using MEF, HEK, and 10T½ cells, and varying the transfection conditions, I was unable to obtain these results.
A number of strategies may be used in the future to optimize the experiment. First, pGL3-Control plasmid was used in order to allow for up or down regulation of the reporter gene. However, it is conceivable that the plasmid’s capacity to show up-regulated reporter expression was maxed out. Using the pGL3-Promoter plasmid purportedly would give more room on the upward bound of expression. Second, transfection efficiencies, or the measure of how well the cells took up the DNA, using 10T½ and MEF cells were very poor; HEK was fairly good. The very low levels of DNA taken into the cells could skew the reporter data so it does not reflect the true biological relationship. Because our lab has historically struggled with transfections, it may be necessary to obtain fresh cells or receive help from other labs.
Should the data be proven significant, it appears that while nBmp2 and Plscr1 may moderately activate Itpr1 expression, the presence of them both reduces Itpr1 expression to basal levels. A multitude of models could predict this outcome if it is a true phenomenon. First, while both nBmp2 and Plscr1 activate Itpr1 expression, likely under different conditions, it may be necessary to repress Itpr1 expression when both are activated. nBmp2 may degrade or interfere with repressive transcription factors, though its affinity to Plscr1 is greater. These possibilities will be investigated should the experiments be optimized and the data prove significant.
This work was not possible without the generosity of donors which funded my ORCA grant. A portion of this research was presented as a poster at the Experimental Biology meeting in Anaheim, CA, April 24-28.
References
- Felin JE, Mayo JL, Loos TJ, Jensen JD, Sperry DK, Gaufin SL, Meinhart CA, Moss JB, Bridgewater LC. Nuclear variants of bone morphogenetic proteins. BMC Cell Biol 2010 Mar 15;11:20.
- Zhou Q, Ben-Efraim I, Bigcas J, Junqueira D, Wiedmer T, Sims PJ. Phospholipid scramblase 1 binds to the promoter region of the inositol 1,4,5-triphosphate receptor type 1 gene to enhance its expression. J Biol Chem 2005 October 14, 2005;280(41):35062-8.