nBMP2 is the name we gave the nuclear variant of BMP2. This variant was discovered in the Bridgewater Lab and first reported in 2010 . It is made by translation from a downstream alternative start codon, producing a protein that is missing the N-terminal signal that would have directed it to the endoplasmic reticulum to enter the secretory pathway. Instead, this alternate form contains a nuclear localization signal (Figure 1). We identified the nuclear localization signal and alternative start site using targeted mutagenesis of plasmids in cell culture assays, and we found that two other closely related BMP family members also have nuclear variants synthesized by similar mechanisms . This degree of evolutionary conservation suggests an important function.
To learn about the nuclear function of nBMP2 in the context of a whole animal, we generated a targeted mutant mouse line in which conventional BMP2 (the growth factor) is synthesized and secreted normally but nBMP2 is mutated so that it cannot enter the nucleus (Figure 2). The mice appear normal, but electrophysiology experiments on skeletal muscle revealed that their muscles relax more slowly than wild type mice after a stimulated contraction, suggesting aberrant intracellular calcium transport .
Because intracellular calcium transport also regulates the differentiation and activation of several different immune cell types, we examined immune function in the nBMP2 mutant mice by infecting them systemically with Staphylococcus aureus. The mutant mice had small spleens but were otherwise not much different than wild type upon initial infection, but a second infection a month later produced striking differences. Wild type mice were more resistant to the infection, having developed immunity after the initial infection as expected. The nBMP2 mutant mice, in contrast, were more vulnerable than before with higher bacterial loads in the bloodstream and more frequent mortality . Histological studies revealed that the spleens of mutant mice contained fewer hemosiderin-laden macrophages than wild type spleens, suggesting that macrophages lacking nBMP2 are less able to engulf red blood cells that are damaged by the hemolytic effects of S. aureus (Figure 3). We are now examining calcium transport within and the engulfment activity of mutant macrophages to test this hypothesis.
Like muscle contractions and immune cell activation, neuronal signaling is also dependent on intracellular calcium transport. We performed behavioral tests of learning and memory to determine whether nBMP2 mutant mice show impaired cognition. These tests include the Morris water maze, the novel object recognition test, and the T-maze. Results indicated that the nBMP2 mutant mice do, indeed, have impaired memory, consistent with compromised hippocampal function. Immunostaining of the hippocampus revealed that nBMP2 is detectable in the nuclei of hippocampal cells in wild type mice, but not in nBMP2 mutants (Figure 4). To determine whether the cognitive impairment is related to calcium transport, we performed electrophysiology experiments on the hippocampus to measure long-term potentiation (LTP), or “molecular memory.” LTP, which is dependent on intracellular calcium transport, was indeed compromised in brains from nBMP2 mutant mice. We also found that nBMP2 is detectable by immunofluorescence in the nuclei of hippocampal cells in wild type mice .
We are working now to uncover the mechanisms by which nBMP2 impacts all of these physiological processes by using flow cytometry and calcium imaging to study the impact of nBMP2 on intracellular calcium transport pathways.
1. 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, 11:20.
2. Bridgewater LC, Mayo JL, Evanson BG, Whitt ME, Dean SA, Yates JD, Holden DN, Schmidt AD, Fox CL, Dhunghel S et al: A Novel Bone Morphogenetic Protein 2 Mutant Mouse, nBmp2NLS(tm), Displays Impaired Intracellular Ca(2+) Handling in Skeletal Muscle. Biomed Res Int 2013, 2013:125492.
3. Olsen DS, Goar WA, Nichols BA, Bailey KT, Christensen SL, Merriam KR, Reynolds PR, Wilson E, Weber KS, Bridgewater LC: Targeted mutation of nuclear bone morphogenetic protein 2 (nBMP2) impairs secondary immune response in a mouse model. BioMed Research International 2015.
4. Cordner RD, Friend LN, Mayo JL, Badgley C, Wallmann A, Ventura JS, Chidsey BA, Rogers AC, Edwards JG, Bridgewater LC: The nuclear variant of bone morphogenic protein 2 (nBMP2) affects hippocampal function in a mouse model. In revision 2015.
Mutant mouse generation: Dr. Jeff Barrow, BYU Department of Physiology and Developmental Biology
Muscle electrophysiology: Dr. Chad Hancock, BYU Department of Nutrition, Dietetics, and Food Science
Brain electrophysiology: Dr. Jeff Edwards, BYU Department of Physiology and Developmental Biology
Immunology: Dr. Eric Wilson, BYU Department of Microbiology and Molecular Biology
Immunology, flow cytometry, and calcium imaging: Dr. Scott Weber, BYU Department of Microbiology and Molecular Biology
Histology: Dr. Paul Reynolds, BYU Department of Physiology and Developmental Biology