Weaning induces NOS-2 expression through NF-κB modulation in the lactating mammary gland: importance of GSH

Abstract

At the end of lactation the mammary gland undergoes involution, a process characterized by apoptosis of secretory cells and tissue remodelling. To gain insight into this process, we analysed the gene expression profile by oligonucleotide microarrays during lactation and after forced weaning. Up-regulation of inflammatory mediators and acute-phase response genes during weaning was found. Expression of IκBα (inhibitory κBα), a protein known to modulate NF-κB (nuclear factor-κB) nuclear translocation, was significantly up-regulated. On the other hand, there was a time-dependent degradation of IκBα protein levels in response to weaning, suggesting a role for NF-κB. Furthermore, we have demonstrated, using chromatin immunoprecipitation assays, binding of NF-κB to the NOS-2 (inducible nitric oxide synthase) promoter at the early onset of events triggered during weaning. The three isoforms of NOS are constitutively present in the lactating mammary gland; however, while NOS-2 mRNA and protein levels and, consequently, NO production are increased during weaning, NOS-3 protein levels are diminished. Western blot analyses have demonstrated that protein nitration is increased in the mammary gland during weaning, but this is limited to a few specific tyrosine-nitrated proteins. Interestingly, inhibition of GSH synthesis at the peak of lactation partially mimics these findings, highlighting the role of NO production and GSH depletion during involution.

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).…”

“…Chromatin from mammary tissue was fixed and immunoprecipitated as described previously. 3,38 The following antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were used: p65 (sc-109), p300 (sc-585) and RNA pol II (sc-899). After DNA purification, Input, Immunoprecipitated and Unrelated Ab fractions were analyzed by PCR with specific primers for either Real-time quantitative PCR (RT-qPCR) analysis and reverse transcriptase (RT)-PCR.…”

“…To determine if NF-kB binding activity was involved in the current transcription of these genes, the same cross-linked chromatin samples were used for RNA polymerase II (RNApol II) ChIP. 3 As shown in Figure 1b, no signal was noted in lactation; however, after weaning, the RNApol II was bound to the coding region of both genes.…”

“…Over the last decade, genomic approaches have shed light into the key regulators of the involution phase, [1][2][3] with nuclear factor kappaB (NF-kB) playing a key role in both mammary gland development and involution. [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.…”

Retinoids induce MMP-9 expression through RARα during mammary gland remodeling

“…(34). The antibodies used in this case were p300 and RAR␣ (both from Santa Cruz Biotechnology, Santa Cruz, CA).…”