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

Abstract

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.

Fig 1. Discovery of a pedigree with dominant inheritance of failed lactation.

(A) 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 and C) 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. (D and E) Corresponding haematoxylin-eosin histochemistry. (F and G) Corresponding immunohistochemistry for milk protein expression. (H) Corresponding Western blot for milk proteins. Molecular size is shown together with the established sizes of the indicated milk proteins [35]. Lactoferrin (LF), serum albumin (SA), caseins α,κ,β,γ and ε, whey acidic protein (wap) and alpha lactalbumin (lac). (I) Quantification of Wap mRNA by qPCR at in wt/wt or mt/mt mice. (J) Quantification of β-casein (β-Cas) mRNA by qPCR. (K) 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. (L) Quantification of epithelial cell proliferation by incorporated BrdU expressed as a percentage of total number of epithelial cells per field. (M and N) immunohistochemistry for phosphorylated (P) STAT1 at 2 days post partum (dpp) in wt/wt or mt/mt mice. (O) 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. (P) 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. (I-J and O) wt/wt n = 4–5 mice, mt/mt n = 3–5 mice per time point (P) 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.

Fig 2. Effects of OAS2 mutation on global patterns of gene expression in the mammary gland.

Whole mouse mammary glands from homozygous Oas2 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.

Fig 3. Enzymatic properties of mutant OAS2.

(A) Details of the mutation in Oas2 showing the ENU-induced SNP changing isoleucine to asparagine. (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). (C) 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. (D) quantification of the data in panel C. (E) western blot demonstrating similar OAS2 protein input to the assay above.

Fig 4. The effects of inducible expression of mutant and wild type Oas2 in T47D cells.

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

Fig 5. Effects of knockdown of RNASEL, IRF7 and IRF3 on the effects of inducible expression of either mutant (mt) or wild type (wt) mouse Oas2 in T47D cells.

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