Abstract
Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b2 of the v-type H+ ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.
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Abbreviations
- LAMP:
-
lysosomal-associated membrane protein
- NF-κB:
-
nuclear factor kappaB
- ChIP:
-
chromatin immunoprecipitation
- CAPN:
-
calpain
- RNApol II:
-
RNA polymerase II
- PMCA2:
-
plasma membrane calcium ATPase
- COX IV:
-
cytochrome c oxidase IV
- VATB2:
-
vacuolar-type proton ATPase subunit B
- v-ATPase:
-
vacuolar H+-ATPase
- TGF-β1:
-
transforming growth factor beta 1
- TGN38:
-
trans-Golgi network protein 2
- RT-PCR:
-
reverse transcriptase-PCR
- RT-qPCR:
-
real-time quantitative PCR
- siRNA:
-
small interfering RNA
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Acknowledgements
This study was supported by grants from Ministerio de Ciencia e Innovación (BFU2010-18253 to JRV and PS09-02360 to ERG-T), Consellería de Educación (GVPROMETEO 2010-075) and Consellería de Sanidad (AP-085/11 to RZ). TA is the recipient of a pre-doctoral fellowship from Ministerio de Educación and IF-V is funded by Consellería de Educación (GVPROMETEO 2010-075). We thank Estefanía Fernández for technical advice on proteomic studies, Eva Serna for microarray experiments, Sonia Priego for acquiring confocal microscopy images, and Jaime Ferrer and Elisa Alonso-Yuste for tissue histology.
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Arnandis, T., Ferrer-Vicens, I., García-Trevijano, E. et al. Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization. Cell Death Differ 19, 1536–1548 (2012). https://doi.org/10.1038/cdd.2012.46
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DOI: https://doi.org/10.1038/cdd.2012.46
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