Abstract
We previously identified a nuclear variant of bone morphogenetic protein 2 (BMP2), named nBMP2, that is translated from an alternative start codon. Decreased nuclear localization of nBMP2 in the nBmp2NLStm mouse model leads to muscular, neurological, and immune phenotypes—all of which are consistent with aberrant intracellular calcium (Ca2+) response. Ca2+ response in these mice, however, has yet to be measured directly. Because a prior study suggested impairment of macrophage function in nBmp2NLStm mutant mice, bone marrow derived (BMD) macrophages and splenic macrophages were isolated from wild type and nBmp2NLStm mutant mice. Immunocytochemistry revealed that nuclei of both BMD and splenic macrophages from wild type mice contain nBMP2, while the protein is decreased in nuclei of nBmp2NLStm mutant macrophages. Live-cell Ca2+ imaging and engulfment assays revealed that Ca2+ response and phagocytosis in response to bacterial supernatant are similar in BMD macrophages isolated from naïve (uninfected) nBmp2NLStm mutant mice and wild type mice, but are deficient in splenic macrophages isolated from mutant mice after secondary systemic infection with Staphylococcus aureus, suggesting progressive impairment as macrophages respond to infection. This direct evidence of impaired Ca2+ handling in nBMP2 mutant macrophages supports the hypothesis that nBMP2 plays a role in Ca2+ response.
Highlights
Our group has reported the existence of a nuclear variant of the growth factor bone morphogenetic protein 2 (BMP2), designated nBMP21
To determine whether nBMP2 is expressed in macrophages, bone marrow derived (BMD) macrophages and splenic macrophages were isolated from naïve wild type and nBmp2NLStm mutant mice and differentiated in vitro, and immunocytochemistry was performed using an anti-BMP2 antibody that binds to both BMP2 and nBMP2
ImageJ software quantification of immunofluorescence images showed that the density of nuclear BMP2 staining was significantly more intense in wild type compared to mutant macrophages in both BMD macrophages (p = 0.0005) and splenic macrophages (p < 0.0001) (Fig. 2)
Summary
Our group has reported the existence of a nuclear variant of the growth factor bone morphogenetic protein 2 (BMP2), designated nBMP21. The observation of fewer hemosiderin-laden macrophages in the spleens of mutant mice after a secondary infection suggested to us that macrophage phagocytic activity might be impaired in the absence of nBMP2, potentially providing us with an accessible cell type in which to directly test our hypothesis that intracellular Ca2+ response is disrupted in the absence of nBMP2. Live-cell Ca2+ imaging as well as bead engulfment assays were performed to measure intracellular Ca2+ response and phagocytic activity These analyses revealed deficient Ca2+ response and phagocytosis in splenic macrophages isolated from mutant mice after secondary systemic infection with S. aureus, but not in BMD macrophages from naïve mice, suggesting that as nBmp2NLStm mutant cells respond to infection over time, Ca2+ response is progressively impaired
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