The cJUN NH2-terminal kinase (JNK) pathway contributes to mouse mammary gland remodeling during involution

Involution returns the lactating mammary gland to a quiescent state after weaning. The mechanism of involution involves collapse of the mammary epithelial cell compartment. To test whether the cJUN NH2-terminal kinase (JNK) signal transduction pathway contributes to involution, we established mice with JNK deficiency in the mammary epithelium. We found that JNK is required for efficient involution. JNK deficiency did not alter the STAT3/5 or SMAD2/3 signaling pathways that have been previously implicated in this process. Nevertheless, JNK promotes the expression of genes that drive involution, including matrix metalloproteases, cathepsins, and BH3-only proteins. These data demonstrate that JNK has a key role in mammary gland involution post lactation.

-/+ Rosa mT/mG reporter mice were stained with antibodies to GFP, keratin 5 (K5), or keratin 8 (K8), and then counter-stained with DAPI. The GFP antibody stains Cre + cells in the reporter mice. The images presented are representative of sections prepared from mammary glands of 7 mice. Scale bar = 50 µm.
(b,c) Cre-mediated recombination of Mapk8 LoxP (b) and Mapk9 LoxP (c) alleles was confirmed by PCR analysis of genomic DNA isolated from mammary epithelial cells isolated from mice after 10 days of lactation.

Sections of mammary glands from JNK
WT and JNK KO female mice on day 0, 3, 7, and 14 of involution were stained with H&E. The images are representative of sections taken from n=5 JNK WT mice and n=5 JNK KO mice for each condition. Scale bar = 50 µm.
Supplemental Figure S3. Epithelial cell populations in mammary glands after involution.
(a,b) Mammary gland sections were stained with antibodies to smooth muscle actin (SMA, a) or with antibodies to keratin 5 and 8 (K5 and K8, b), and counterstained with DAPI. Representative images are presented (n=4 JNK WT mice and n=4 JNK KO mice). Scale bars = 50 µm (a) and 30 µm (b).

Supplemental Figure S4. Caspase 3 activation in mammary glands after 7 days of involution.
Sections prepared from mammary glands of JNK WT mice and JNK KO mice on day 7 of involution were stained with an antibody to cleaved caspase 3 (c-C3 + ). Representative images are presented. c-C3 + cells were quantitated in 6 fields (40x) per section and presented as the % of total cells. Statistical significance was calculated using an unpaired, two-tailed t-test (mean ± SEM; n=5 JNK WT mice and n=5 JNK KO mice; p=0.59). Scale bars = 100 µm.
(a,b) Sections prepared from mammary glands of 5 JNK WT mice and 5 JNK KO mice on involution day 3 were stained with an antibody to p-STAT5 (a) or p-SMAD2/3 (B) and counterstained with hematoxylin. Representative images are presented. Scale bars = 100 µm. Figure S6. Summary of RNA sequencing analysis.

Supplemental
(a) RNA sequencing analysis of mammary glands isolated from JNK WT and JNK KO mice is summarized. Each biological group comprises 3 independent replicates.
(b,c) Biotypes were determined for both detected gene expression and genes that are differentially expressed (log 2 |Fold Change|>1, q<0.01) between day 0 and day 3 of involution in JNK WT mice (b) and JNK KO mice (c).

Supplemental Figure S7. Enrichment of AP1 binding sites with genes that exhibit JNK-dependent expression during involution.
Genes expressed in a JNK-dependent manner (green) or JNK-independent manner (yellow) after 3 days of involution ( Figure 5) were compared with genes identified by ChIP-Seq analysis (cJUN, JUND, or JUN plus JUND) to determine overlap between JNK-dependent gene expression and genes with AP1 binding sites. Pearson's Chi-squared test was used to determine statistical significance.
Supplemental Figure S8. Mammary gland expression of Mmp and Timp genes.
(a-c) The mRNA expression of Ctsb (a), Ctsl (b), and Serpina3g (c) was measured by RNA-seq analysis. The data are presented as fragments per kilobase of exon model per million mapped fragments (FPKM) (mean ± SEM; n=3 JNK WT mice and n=3 JNK KO mice). The Benjamini-Hochberg method was used to calculate q-values.