Nitric oxide triggers mammary gland involution after weaning: remodelling is delayed but not impaired in mice lacking inducible nitric oxide synthase

Abstract

During mammary gland involution, different signals are required for apoptosis and tissue remodelling. To explore the role of NO in the involution of mammary tissue after lactation, NOS2 (inducible nitric oxide synthase)-KO (knockout) mice were used. No apparent differences were observed between NOS2-KO and WT (wild-type) animals during pregnancy and lactation. However, upon cessation of lactation, a notable delay in involution was observed, compared with WT mice. NOS2-KO mice showed increased phosphorylation of STAT (signal transducer and activator of transcription) 5 during weaning, concomitant with increased beta-casein mRNA levels when compared with weaned WT glands, both hallmarks of the lactating period. In contrast, activation of STAT3, although maximal at 24 h after weaning, was significantly reduced in NOS2-KO mice. STAT3 and NF-kappaB (nuclear factor kappaB) signalling pathways are known to be crucial in the regulation of cell death and tissue remodelling during involution. Indeed, activation of both STAT3 and NF-kappaB was observed in WT mice during weaning, concomitant with an increased apoptotic rate. During the same period, less apoptosis, in terms of caspase 3 activity, was found in NOS2-KO mice and NF-kappaB activity was significantly reduced when compared with WT mice. Furthermore, the activation of the NF-kappaB signalling pathway is delayed in NOS2-KO mice when compared with WT mice. These results emphasize the role of NO in the fine regulation of the weaning process, since, in the absence of NOS2, the switching on of the cascades that trigger involution is hindered for a time, retarding apoptosis of the epithelial cells and extracellular matrix remodelling.

Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization

“…[3][4][5][6] NF-kB regulates the expression of a large number of genes involved in multiple biological processes, including apoptosis. We have shown that both Capn1 and 2 are regulated by NF-kB during mammary gland involution (Figure 1a).…”

“…Caspase-3 activity was measured in mammary tissue lysates using the 'Caspase-3/CPP32 Colorimetric Assay Kit' (#K106, BioVision, Mountain View, CA, USA) as previously described. 5 Statistics. Data are presented as means±S.E.M.…”

“…Total protein was extracted from snap-frozen mammary glands (0.1 g) by homogenization in a buffer containing detergents and protease inhibitors as described previously. 5 Equal amounts of protein (15 mg) were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Protran, Whatman, Dassel, Germany). After addition of the corresponding primary antibody, immunocomplexes were revealed by using a HRP-conjugated secondary antibody (Dako), as appropriate, and subsequent peroxidase-induced enhanced chemiluminescence reaction (ECL Detection Kit, GE Healthcare, Uppsala, Sweden).…”

“…[4][5][6] Interestingly, calpain (CAPN)-1 and -2 were found among the 268 NF-kB target genes identified by chromatin immunoprecipitation (ChIP)-onchip experiments in 48-h-weaned glands. 7 CAPNs are a family of calcium-dependent cysteine proteases.…”

“…NF-kB is a key transcription factor that becomes activated early during involution and remains activated thereafter. 5,6 To identify new NF-kB target genes, our group performed ChIP-on-chip experiments during weaning; among those genes calpain-1 and calpain-2 were identified. 7 ChIP showed binding of NF-kB (p65 subunit) and its coactivator p300 to capn1 and capn2 promoters after 48 h of weaning, while no NF-kB DNA binding was detectable at the peak of lactation (Figure 1a).…”