A mutation in the viral sensor 2'-5'-oligoadenylate synthetase 2 causes failure of lactation

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.This work was supported by grants from the Congress Directed Medical Research Program (BC995364 and DAMD17-01-1-0241), Cure Cancer Australia Foundation, NHMRC Australia (projects 1047149, Fellowships 1058356, 481310, 1043400), the Australian Research Council Discovery Project (DP110102288), Princeton University, NIH grant 1R01GM110161-01 (AK), Sidney Kimmel Foundation for Cancer Research (AK), Burroughs Wellcome Foundation (AK), Banque Nationale de Paris-Paribas Australia and New Zealand, Mostyn Family Foundation, Cue Clothing Co., Estee Lauder Australia, RT Hall Trust and Fellowships (ECF-13-08 and ECF-16-022) from the National Breast Cancer Foundatio

KIBRA interacts with discoidin domain receptor 1 to modulate collagen-induced signalling

AbstractMammary gland development is coupled to reproductive events by hormonal cues of ovarian and pituitary origin, which activate a genomic regulatory network. Identification of the components and regulatory links that comprise this network will provide the basis for defining the network's dynamic response during normal development and its perturbation during breast carcinogenesis. In this study KIBRA was identified as a transcript showing decreased expression associated with failed mammary gland development in Prlr knockout mammary epithelium. It is strongly up-regulated during pregnancy, falls during lactation and is again up-regulated during involution of the gland at weaning. A bioinformatic approach was undertaken to identify potential binding partners which interact with the WW domains of KIBRA. We show that KIBRA binds to a WW domain binding motif, PPxY, in the tyrosine kinase receptor DDR1, and dissociates upon treatment with the DDR1 ligands collagen type I or IV. In addition we show that KIBRA and DDR1 also interact with PKCz to form a trimeric complex. Finally, overexpression and knockdown studies demonstrate that KIBRA promotes the collagen-stimulated activation of the MAPK cascade. Thus KIBRA may play a role in how the reproductive state influences the mammary epithelial cell to respond to changing cell-context information, such as experienced during the tissue remodeling events of mammary gland development

A mutation in the viral sensor 2’-5’-oligoadenylate synthetase 2 causes failure of lactation

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation

Effects of knockdown of RNASEL, IRF7 and IRF3 on the effects of inducible expression of either mutant (mt) or wild type (wt) mouse <i>Oas2</i> in T47D cells.

<p><b>(A-G)</b> Provide the context of RNase L knockdown. <b>(A-C</b>) Demonstration of Doxycycline (DOX)-inducible expression of wt or mt <i>Oas2</i> in T47D cells, and effective knock-down of RNASEL (RNaL) in mt or wt expressing T47D cells by quantitative PCR (<b>B</b>) or western blot (<b>C</b>). <b>(D)</b> Effect of the induction of mt or wt OAS2 on RNase L activity <b>(E)</b> Effects of induction of mt and wt <i>Oas2</i> expression on apoptosis. <b>(F)</b> Effects of these treatments on interferon gamma protein production. <b>(G</b>) effects of these treatments on GM-CSF production. <b>(H</b>) Demonstration of effective knockdown of IRF7. <b>(I)</b> Effects of knockdown of IRF7 on mutant or wild type <i>Oas2</i>-driven apoptosis. <b>(J)</b> Demonstration of knockdown of IRF3. <b>(K)</b> Effects of knockdown of IRF3 on mutant or wild type <i>Oas2</i>-driven apoptosis.</p

Enzymatic properties of mutant OAS2.

<p><b>(A</b>) Details of the mutation in <i>Oas2</i> showing the ENU-induced SNP changing isoleucine to asparagine. <b>(B)</b> RNAseL activity measured as the abundance of RNase L-specific cleavage of tRNA-His-36 (upper panel) or rRNA (lower panel) at day 18 of pregnancy (d18pc) and two days post partum (2dpp). <b>(C</b>) Representative denaturing PAGE separating 2-5A species of different molecular weights synthesized in a cell free system by mutant (mt) or wild type (wt) mouse OAS2, in response to activation by different concentrations of the double-stranded RNA mimic polyI:C. <b>(D</b>) quantification of the data in panel C. <b>(E</b>) western blot demonstrating similar OAS2 protein input to the assay above.</p

The effects of inducible expression of mutant and wild type <i>Oas2</i> in T47D cells.

<p><b>(A</b>) pHUSH ProEx expression vector used to express either mutant (mt) or wild type (wt) mouse <i>Oas2</i> in T47D cells in response to doxycycline (DOX). <b>(B</b>) relative expression of mt and wt <i>Oas2</i>. <b>(C</b>) Western blot showing induction of mouse OAS2 (m) running just below endogenous human OAS2 protein, with both bands above a non-specific band (nsb). <b>(D</b>) Sensitivity of the cells lines to poly I:C (pl:C) with and without DOX induction of mt and wt <i>Oas2</i>. <b>(E</b>) Effect of mt and wt <i>Oas2</i> on adherent cell number after 72h. (<b>F</b>) Cell detachment (numbers of live cells in supernatant fraction) caused by mt <i>Oas2</i>. <b>(G</b>) Effects of mt or wt <i>Oas2</i> on replating of T47D cells in a 4 hour trypsin only replating assay after 48h of DOX. <b>(H</b>) Expression of β1 integrin (β1), E-cadherin (EC) and β-actin (βa) in response to induction of mt or wt <i>Oas2</i>. <b>(I</b>) apoptotic response to induction of mt or wt <i>Oas2</i>. Data represents the average of 7 independent experiments. <b>(J</b>) cell-cycle-phase distribution at the indicated times following induction of mt or wt <i>Oas2</i>. Data represents the average of 5 independent experiments. <i>*</i>p<0.01. ANOVA 4I and J. <b>(K)</b> <i>Oas2</i> expression in parental (p) normal mouse mammary HC11 cells or in cells constitutively expressing mt or wt <i>Oas2</i>. <b>(L)</b> Effect of wt or mt Oas2 on beta Casein in HC11s after 72 hours of prolactin (Prl) and Dexamethasone (Dex) stimulation. <b>(M)</b> Effect of mt or wt <i>Oas2</i> expression on cell death at 96 hours in HC11 cells after transient transfection. All data are representative of 3 independent experiments in response to 72h of DOX except otherwise specified. Paired t-tests 4B,E,F, G, L and M.</p

Discovery of a pedigree with dominant inheritance of failed lactation.

<p><b>(A)</b> Lactation performance of dams of the indicated genotypes (wild type; wt/wt mutant; mt/mt) assessed by pup weight-gain or survival (inset). Error bars show standard error of the mean for 4–5 litters per genotype of 7 pups each. wt/wt n = 35, wt/mt n = 28 and mt/mt n = 28 pups. <b>(B and C)</b> Whole mount histology of the 4^th inguinal mammary gland showing lobuloalveolar development at 2 days post partum (dpp) in wt/wt or mt/mt mice. <b>(D and E)</b> Corresponding haematoxylin-eosin histochemistry. <b>(F and G)</b> Corresponding immunohistochemistry for milk protein expression. <b>(H)</b> Corresponding Western blot for milk proteins. Molecular size is shown together with the established sizes of the indicated milk proteins [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007072#pgen.1007072.ref035" target="_blank">35</a>]. Lactoferrin (LF), serum albumin (SA), caseins α,κ,β,γ and ε, whey acidic protein (wap) and alpha lactalbumin (lac). <b>(I)</b> Quantification of <i>Wap</i> mRNA by qPCR at in wt/wt or mt/mt mice. <b>(J)</b> Quantification of β-casein (β-Cas) mRNA by qPCR. <b>(K)</b> Quantification of epithelial cell death by immunohistochemistry for cleaved caspase 3, results are the number of positively stained epithelial cells as a percentage as a percentage of total number of epithelial cells per field. <b>(L</b>) Quantification of epithelial cell proliferation by incorporated BrdU expressed as a percentage of total number of epithelial cells per field. <b>(M and N</b>) immunohistochemistry for phosphorylated (P) STAT1 at 2 days post partum (dpp) in wt/wt or mt/mt mice. <b>(O)</b> quantification of P-STAT1 in wt/wt or mt/mt mice by immunohistochemistry, results are the number of positively stained epithelial cells as a percentage of total epithelial area. <b>(P)</b> Quantification of P-STAT1 in wt/wt or mt/mt mammary transplants by immunohistochemistry, results are the number of positively stained epithelial cells as a percentage of total epithelial area. <b>(I-J and O)</b> wt/wt n = 4–5 mice, mt/mt n = 3–5 mice per time point <b>(P)</b> wt/wt n = 3–5 mice, mt/mt n = 2–5 per time point. Student’s t-test p values are given, error bars are standard error of the mean.</p

Effects of OAS2 mutation on global patterns of gene expression in the mammary gland.

<p>Whole mouse mammary glands from homozygous <i>Oas2</i> mutant (mt) or wild type (wt) animals were profiled using Affymetrix MTA arrays. Differential gene expression was ranked by the limma t-statistic and this was used as the input for gene set enrichment analysis to identify functional signatures. The enrichment-map plug in for Cytoscape was used to visualize the results. Each node represents a gene set and the expression of genes comprising the leading edge of some of these sets is shown as heat maps of the t-statistic. Labels indicate the function of the clustered gene sets. Gene expression in mt animals is compared with wt animals at 2dpp (node center color) or 18dpc (node edge color). Red indicates enrichment of expression the gene set and blue suppression of expression.</p

Taqman probes used for quantitative PCR indicating gene name, probe ID number and species specificity.

<p>Taqman probes used for quantitative PCR indicating gene name, probe ID number and species specificity.</p