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The Key Interplay of Fatty Acid Oxidation and Apoptosis Resistance of Macrophages in Pulmonary Fibrosis
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A6361 - The Key Interplay of Fatty Acid Oxidation and Apoptosis Resistance of Macrophages in Pulmonary Fibrosis
Author Block: L. Gu1, J. L. Casey2, D. Davis1, A. B. Carter3; 1Medicine, University of Alabama at Birmingham, Birmingham, AL, United States, 2Medicine, Univeristy of Alabama at Birmingham, Birmingham, AL, United States, 3Birmingham VAMC, University of Alabama at Birmingham, Birmingham, AL, United States.
Rationale:
We previously showed that expression of the mitochondrial calcium uniporter (MCU) polarizes lung macrophages to a pro-fibrotic phenotype. Mitochondrial bioenergetics in macrophages are unique for their function; however, it is not known if metabolic reprogramming to fatty acid oxidation (FAO) in lung macrophages promotes fibrotic repair. Moreover, the link of FAO to apoptosis resistance and regulatory effects of MCU on these processes remains to be elucidated. In this study, we sought to determine the regulation of MCU on FAO and apoptosis resistance by targeting the interaction of carnitine palmitoyltransferase 1α (CPT-1α) and Bcl-2.
Methods:
Mice overexpressing dominant negative MCU (DN-MCU) in macrophages were generated to evaluate the modulation of MCU on FAO and apoptosis resistance. Plasmids expressing full length or various segmented CPT-1α or Bcl-2 were generated. Pulldown and immunoblot analysis was utilized to map the actual binding site(s) between CPT-1α to Bcl-2 and the effects on resistance to apoptosis in lung macrophages.
Results:
Bleomycin injury triggered activation of FAO by increasing CPT-1α and apoptosis resistance by increasing Bcl-2 in lung macrophages from WT mice. However, CPT-1α and Bcl-2 were decreased below normal levels in DN-MCU mice, which were protected from pulmonary fibrosis. To understand the interplay between FAO and apoptosis resistance, we transfected plasmids expressing CPT-1α of various lengths. V5-His pulldown and immunoblot analysis of Bcl-2 showed that only full length of CPT-1α bound with endogenous Bcl-2, indicating the importance of CPT-1α conformation integrity in retaining the interaction. Expression of full-length Bcl-2 bound with endogenous CPT-1α. Overexpressing full length CPT-1α in macrophages triggered increase in mitochondrial Bcl-2, and inhibition of mitochondrial permeability transition pore (mPTP) opening and cytochrome c release. Moreover, active caspase-3 activity was potently abrogated. These results were attenuated with siRNA to CPT-1α or Bcl-2 in lung macrophages.
Conclusions:
The data collectively demonstrate that Bcl-2 binds with CPT-1α. The binding is necessary to mediate apoptosis resistance of lung macrophages. This study suggests that a therapeutic analog to inhibit the interaction of CPT-1α and Bcl-2 may halt fibrotic repair.
Research Funding Source: This work was supported, in whole or in part, by National Institutes of Health Grants 2R01ES015981-10 and 1R56ES027464-01.
Author Block: L. Gu1, J. L. Casey2, D. Davis1, A. B. Carter3; 1Medicine, University of Alabama at Birmingham, Birmingham, AL, United States, 2Medicine, Univeristy of Alabama at Birmingham, Birmingham, AL, United States, 3Birmingham VAMC, University of Alabama at Birmingham, Birmingham, AL, United States.
Rationale:
We previously showed that expression of the mitochondrial calcium uniporter (MCU) polarizes lung macrophages to a pro-fibrotic phenotype. Mitochondrial bioenergetics in macrophages are unique for their function; however, it is not known if metabolic reprogramming to fatty acid oxidation (FAO) in lung macrophages promotes fibrotic repair. Moreover, the link of FAO to apoptosis resistance and regulatory effects of MCU on these processes remains to be elucidated. In this study, we sought to determine the regulation of MCU on FAO and apoptosis resistance by targeting the interaction of carnitine palmitoyltransferase 1α (CPT-1α) and Bcl-2.
Methods:
Mice overexpressing dominant negative MCU (DN-MCU) in macrophages were generated to evaluate the modulation of MCU on FAO and apoptosis resistance. Plasmids expressing full length or various segmented CPT-1α or Bcl-2 were generated. Pulldown and immunoblot analysis was utilized to map the actual binding site(s) between CPT-1α to Bcl-2 and the effects on resistance to apoptosis in lung macrophages.
Results:
Bleomycin injury triggered activation of FAO by increasing CPT-1α and apoptosis resistance by increasing Bcl-2 in lung macrophages from WT mice. However, CPT-1α and Bcl-2 were decreased below normal levels in DN-MCU mice, which were protected from pulmonary fibrosis. To understand the interplay between FAO and apoptosis resistance, we transfected plasmids expressing CPT-1α of various lengths. V5-His pulldown and immunoblot analysis of Bcl-2 showed that only full length of CPT-1α bound with endogenous Bcl-2, indicating the importance of CPT-1α conformation integrity in retaining the interaction. Expression of full-length Bcl-2 bound with endogenous CPT-1α. Overexpressing full length CPT-1α in macrophages triggered increase in mitochondrial Bcl-2, and inhibition of mitochondrial permeability transition pore (mPTP) opening and cytochrome c release. Moreover, active caspase-3 activity was potently abrogated. These results were attenuated with siRNA to CPT-1α or Bcl-2 in lung macrophages.
Conclusions:
The data collectively demonstrate that Bcl-2 binds with CPT-1α. The binding is necessary to mediate apoptosis resistance of lung macrophages. This study suggests that a therapeutic analog to inhibit the interaction of CPT-1α and Bcl-2 may halt fibrotic repair.
Research Funding Source: This work was supported, in whole or in part, by National Institutes of Health Grants 2R01ES015981-10 and 1R56ES027464-01.
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