Dr. Laura Bridgewater, Department of Microbiology & Molecular Biology

The goal of this project was to follow up on preliminary data suggesting that nBmp2 directly activated transcription. A few months of follow-up experiments, however, showed that the original transcription data was not reproducible. At that point we turned our attention to two different questions about nBmp2 function: 1) Does nBMP-2 affect the cell cycle? 2) Is nBMP-2 involved in the immune response? These questions have each yielded interesting and important results that are leading toward publications and grant proposals.

Achievement of academic objectives

The question of whether nBMP-2 affects the cell cycle was triggered by some early results showing that cells transfected with an nBmp2 expression vector behaved abnormally in tissue culture. To address this question we overexpressed nBmp2 in two different cell types, human kidney cells and human colon cancer cells. We then analyzed cells by flow cytometry to determine what percentage of each population was in G1 phase (resting) vs. in S and G2 phases (preparing for cell division). We found that nBmp2 overexpression increases the percentage of cells in S phase of the cell cycle—an indication that it may act as an oncogene in tumor cells. Furthermore, we found that cancer cells were even more sensitive to this effect than were the kidney cells. This work has opened the way for a new study that directly focuses on the role of nBmp2 in carcinogenesis in mice, using a mouse with a targeted mutation of nBmp2. Wild type and nBmp2 mutant mice are being treated with a carcinogen over a period of several weeks to induce colon cancer. We predict that because nBmp2 overexpression increases cell division, mice that are lacking nBmp2 will conversely be more resistant to cancer induction. The cell cycle analysis project will be presented at the annual meeting of the American Society of Biochemistry and Cell Biology (ASBMB) in Boston next April.

We considered the question of immune function in nBmp2 mutant mice because immune response depends on intracellular calcium transport, and we previously showed that nBmp2 mutant mice have impaired calcium transport in muscle cells and neurons. Wild type and mutant mice were injected intravenously with a dose of bacteria, and blood and organ samples were collected on the third day. Organs were weighed and cultured to detect bacteria in the organ. No differences were detectable between wild type and mutant on the third day, but the spleen weights were significantly different. Mutant spleens failed to enlarge in response to the infection. This suggests that a particular immune cell type found in the spleen is unable to rally and mount a strong response against infection. This work has led to two posters that will be presented at the ASBMB in Boston next April, and we’re now moving forward with experiments to identify the unresponsive cell type, to see if longer-term immune responses are compromised, and to compare the levels of various cytokines between wild type and mutants.

Evaluation of the mentoring environment

My lab operated well over the past year as a successful mentoring environment. We have two master’s students, three senior undergraduates who have been in my lab for as much as two years, seven newer undergraduates who have worked closely with the senior undergraduates to learn techniques, lab safety, and experimental design and interpretation, and one high school intern who works with a graduate student. We hold lab meeting on Friday afternoons, and each student presents his or her results from the past week’s experiments. If a student has had technical problems, we work together on trouble-shooting. Besides providing each student with regular feedback on their work, this meeting also has been invaluable in broadening each student’s understanding of science in general and of the other projects going on in the lab. It has been gratifying to see the students grow and develop, increasing in independence as they become more comfortable performing techniques and begin looking at the bigger picture of the work we’re doing. I also hold individual meeting with each graduate student each week, where we discuss not only their projects but also the undergraduate projects they’re helping to mentor. I work with all the undergraduate students individually as needed, discussing their research and their future plans. I also spend 1-2 hours each day in the lab, checking on progress and helping students through roadblocks they have encountered.

Students involved and academic deliverables produced

Students involved

Publications in revision, which have been supported in part by this MEG grant

• Cordner, R.D., Mayo, J.L., Badgley, C., Wallmann, A., Ventura, J.S., Chidsey, B.A., Barrow, J.R., Edwards, J.G. and Bridgewater, L.C. A novel mouse bearing a targeted mutation of nuclear bone morphogenetic protein 2 (nBmp2) develops impaired memory. In revision
• Bridgewater, L.C., Mayo, J.L., Evanson, B.G., Schmidt, A.D., Fox, C.L., Dhungel, S., Steed, K.S., Adam, M.M., Nichols, C.A., Loganathan, S.K., Barrow, J.R. and Hancock, C.R. A novel bone morphogenetic protein 2 mutant mouse, nBmp2NLStm, displays impaired intracellular Ca2+ handling in skeletal muscle. In revision.

Posters presented

• Rogers, A.C., Nichols, B.A., Loos, T.J., and Bridgewater, L.C. “Nuclear Bmp4 interacts with the SCF E3 ubiquitin ligase complex and inhibits the cell cycle.” American Society for Biochemistry and Molecular Biology Annual Meeting, San Diego, CA (2012).
• Ricks, M.L., Falk, D.J., Farrell, J.T., Rees, M.B., Carr, J.M., Williams, D.T., Kooyman, D.L., Bridgewater, L.C., and Seegmiller, R.E. “Premature osteoarthritis in the temporomandibular joint of heterozygous disproportionate micromelia mice.” American Society for Biochemistry and Molecular Biology Annual Meeting, San Diego, CA (2012).

Description of Results/Findings

See description under “Achievement of Academic Objectives” above.

Summary of spending

The budget for this project was spent primarily on supplies and animal care costs, as follows:

• Animal care costs, WIDB mouse facility ($10,000)
• PCR enzymes and primers for genotyping the mice ($2,000)
• Cell cycle analysis stains and flow cytometer charges ($1,000)
• Tissue culture media, serum, flasks, and transfection reagents ($5,000)
• Microbiological plates and culture media ($2,000)