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An apoptosis-associated mammary protein deficiency leads to enhanced production of IgM antibodies against multiple damage-associated molecules.

ABSTRACT: Milk fat globule epidermal growth factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and enhances the engulfment of apoptotic cells by macrophages. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in the MFG-E8-deficient (MFG-E8(-/-)) mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus. We found that the MFG-E8 deficiency was accompanied by the increased production of immunoglobulins. Further Western blot and ELISA analyses validated the increase in the IgM levels in the MFG-E8(-/-) mice. It was also revealed that the sera from the MFG-E8(-/-) mice cross-reacted with oxidation-specific epitopes generated upon incubation of serum albumin with the peroxidized lipids. Among the modified proteins with several unsaturated aldehydes of chain lengths varying from three to nine carbons, the MFG-E8(-/-) mice sera exclusively cross-reacted with the protein-bound 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ?6 polyunsaturated fatty acids. In addition, the IgM monoclonal antibodies (mAbs) that selectively cross-reacted with the ONE-modified proteins were generated from the MFG-E8(-/-) mice. A subset of the ONE-specific IgM mAbs significantly recognized the late apoptotic and necrotic cells and enhanced the phagocytosis by macrophages. These data demonstrate that the impairment of the phagocytic clearance of apoptotic cells through MFG-E8 can lead to the generation of natural antibodies, which may play a critical role in removing multiple damage-associated molecules, including oxidation-specific epitopes and late apoptotic/necrotic cells.

SUBMITTER: Chikazawa M

PROVIDER: S-EPMC3709889 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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An apoptosis-associated mammary protein deficiency leads to enhanced production of IgM antibodies against multiple damage-associated molecules.

Chikazawa Miho M Otaki Natsuki N Shibata Takahiro T Yasueda Takehiko T Matsuda Tsukasa T Uchida Koji K

PloS one 20130712 7

Milk fat globule epidermal growth factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and enhances the engulfment of apoptotic cells by macrophages. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in the MFG-E8-deficient (MFG-E8(-/-)) mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus. We found that the MFG-E8 deficiency was accompanied by the increased production of immunoglob ...[more]

PMID: 23874637

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Project description:HIF-1a activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HET HIF-1a fracture calluses are larger, stronger and stiffer than WT HIF-1a calluses due to decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. Introduction: Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia following fracture activates hypoxia-inducible factor 1 alpha (HIF-1a) leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1a is a key regulator of bone regeneration Materials and Methods: Fixed femoral fractures were generated in mice with partial HIF-1a deficiency (HET HIF-1a) and wild type littermates (WT HIF-1a). Fracture calluses and intact contralateral femurs from post fracture day (PFD) 21 and 28 (N=5-10) were subjected to MicroCT evaluation and 4-point bending in order to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10 and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis. Results: Surprisingly, fracture calluses from HET HIF-1a mice exhibit greater mineralization and are larger, stronger and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, demonstrating higher expression of pro-apoptotic PP2A and lower expression of anti-apoptotic BCL2 in WT HIF-1a calluses. Subsequent TUNEL staining demonstrates that WT HIF-1a calluses contain larger numbers of TUNEL positive chondrocytes and osteoblasts than HET HIF-1a calluses. Conclusions: We conclude that partial HIF-1a deficiency results in decreased chondrocytic and osteoblastic apoptosis; thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration. Keywords: Bone, Fracture, Apoptosis, Hypoxia, Microarray

Project description:UNLABELLED:HIF-1alpha activates genes under hypoxia and was hypothesized to regulate bone regeneration. Surprisingly, HIF-1alpha+/- fracture calluses are larger, stronger, and stiffer than HIF-1alpha+/+ calluses because of decreased apoptosis. These data identify apoptosis inhibition as a means to enhance bone regeneration. INTRODUCTION:Bone regeneration subsequent to fracture involves the synergistic activation of multiple signaling pathways. Localized hypoxia after fracture activates hypoxia-inducible factor 1alpha (HIF-1alpha), leading to increased expression of HIF-1 target genes. We therefore hypothesized that HIF-1alpha is a key regulator of bone regeneration. MATERIALS AND METHODS:Fixed femoral fractures were generated in mice with partial HIF-1alpha deficiency (HIF-1alpha+/-) and wildtype littermates (HIF-1alpha+/+). Fracture calluses and intact contralateral femurs from postfracture days (PFDs) 21 and 28 (N=5-10) were subjected to microCT evaluation and four-point bending to assess morphometric and mechanical properties. Molecular analyses were carried out on PFD 7, 10, and 14 samples (N=3) to determine differential gene expression at both mRNA and protein levels. Finally, TUNEL staining was performed on PFD 14 samples (N=2) to elucidate differential apoptosis. RESULTS:Surprisingly, fracture calluses from HIF-1alpha+/- mice exhibited greater mineralization and were larger, stronger, and stiffer. Microarray analyses focused on hypoxia-induced genes revealed differential expression (between genotypes) of several genes associated with the apoptotic pathway. Real-time PCR confirmed these results, showing higher expression of proapoptotic protein phosphatase 2a (PP2A) and lower expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL2) in HIF-1alpha+/+ calluses. Subsequent TUNEL staining showed that HIF-1alpha+/+ calluses contained larger numbers of TUNEL+ chondrocytes and osteoblasts than HIF-1alpha+/- calluses. CONCLUSIONS:We conclude that partial HIF-1alpha deficiency results in decreased chondrocytic and osteoblastic apoptosis, thereby allowing the development of larger, stiffer calluses and enhancing bone regeneration. Furthermore, apoptosis inhibition may be a promising target for developing new treatments to accelerate bone regeneration.

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An apoptosis-associated mammary protein deficiency leads to enhanced production of IgM antibodies against multiple damage-associated molecules.

Publications

An apoptosis-associated mammary protein deficiency leads to enhanced production of IgM antibodies against multiple damage-associated molecules.

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