Essential role of Orai1 store-operated calcium channels in lactation

Abstract

The nourishment of neonates by nursing is the defining characteristic of mammals. However, despite considerable research into the neural control of lactation, an understanding of the signaling mechanisms underlying the production and expulsion of milk by mammary epithelial cells during lactation remains largely unknown. Here we demonstrate that a store-operated Ca(2+) channel subunit, Orai1, is required for both optimal Ca(2+) transport into milk and for milk ejection. Using a novel, 3D imaging strategy, we visualized live oxytocin-induced alveolar unit contractions in the mammary gland, and we demonstrated that in this model milk is ejected by way of pulsatile contractions of these alveolar units. In mammary glands of Orai1 knockout mice, these contractions are infrequent and poorly coordinated. We reveal that oxytocin also induces a large transient release of stored Ca(2+) in mammary myoepithelial cells followed by slow, irregular Ca(2+) oscillations. These oscillations, and not the initial Ca(2+) transient, are mediated exclusively by Orai1 and are absolutely required for milk ejection and pup survival, an observation that redefines the signaling processes responsible for milk ejection. These findings clearly demonstrate that Ca(2+) is not just a substrate for nutritional enrichment in mammals but is also a master regulator of the spatiotemporal signaling events underpinning mammary alveolar unit contraction. Orai1-dependent Ca(2+) oscillations may represent a conserved language in myoepithelial cells of other secretory epithelia, such as sweat glands, potentially shedding light on other Orai1 channelopathies, including anhidrosis (an inability to sweat).

Keywords: calcium channels; calcium signaling; lactation; mammary gland; store-operated calcium entry.

Fig. 1.

Orai1 KO mice have reduced milk Ca²⁺. (A) TG-induced Ca²⁺ entry in mammary luminal cells isolated from Orai1^+/+ (n = 82 cells) and Orai1^−/− (n = 86 cells) mice. Cells were bathed in nominally Ca²⁺-free HBSS supplemented with 1,2-bis(o-aminophenoxy)ethane-N,N,N′, N′-tetraacetic acid (BAPTA, 500 μM) for 2 min and treated with TG (2 μM) to deplete ER Ca²⁺ stores before Ca²⁺ readdition (2 mM). Peak ratio responses to (B) TG (150–270 s) and (C) Ca²⁺ readdition (1,000–1,180 s). Total Ca²⁺ and protein concentrations in milk collected from (D and E) lactating (day 3) Orai1^−/− mice versus control genotypes and (F and G) mice with conditional deletion of Orai1 in the mammary epithelia (Orai1^fl/−;MMTV-Cre) versus control genotypes (n = 3–4 mice). Data represent mean ± SEM; *P < 0.05, Student’s t test (B and C) or one-way ANOVA with Bonferroni posttests (D–G).

Fig. 2.

Pups nursed by Orai1 KO mice fail to thrive. (A) Average weight of pups born to and nursed by Orai1^−/− or control mothers (n = 3 litters), and (B) representative images of PND 4 pups from each litter. (Scale bar, 10 mm.) (C) Average weight of pups from Orai1^+/+ and Orai1^−/− litters fostered on PND 1 to lactating CD-1 foster mothers (n = 2 litters). (D) Milk output in Orai1^+/+ and Orai1^−/− dams (n = 5 mice). (E) Average weight of pups nursed by control mice and mice with conditional deletion of Orai1 in the mammary epithelium (Orai1^fl/−;MMTV-Cre, n = 3–4 litters). Data represent mean ± SEM; *P < 0.05, two-way ANOVA with Bonferroni posttests (A and E) or Student’s t test (D).

Fig. 3.

Orai1 KO mice demonstrate normal mammary gland development and secretory activation but impaired milk let down. (A) Mammary whole mounts from lactating Orai1^−/− mice versus control genotypes (days 1 and 4 of lactation) and cKO mice (Orai1^fl/−;MMTV-Cre) versus control genotypes (day 4 lactation, n = 3). (Scale bar, 400 µm.) (B) H&E staining of mammary glands from Orai1^−/− or control mice on gestation day 18 (i–iii), lactation day 1 (iv–vi), and lactation day 4 (vii–ix) (n = 2–3). [Scale bars, 300 µm (60 µm, higher magnification image).] Lu, alveolar lumen. Arrows, secretory epithelium; arrowheads, cytoplasmic lipid droplets. (C) Actin staining of myoepithelial structures with phalloidin and (D) Oxtr mRNA levels in lactating mammary glands (n = 3). Data represent mean ± SEM; P > 0.05, Student’s t test.

Fig. 4.

Reduced oxytocin-mediated Ca²⁺ oscillations and alveolar unit contractility in Orai1^−/− mice. (A) TG-induced Ca²⁺ entry in mammary myoepithelial cells isolated from Orai1^+/+ (n = 159 cells) and Orai1^−/− (n = 128 cells) mice. Cells were bathed in nominally Ca²⁺-free HBSS supplemented with BAPTA (500 μM) for 2 min and treated with TG (2 μM) to deplete ER Ca²⁺ stores before readdition of Ca²⁺ (2 mM). Peak ratio responses to (B) TG (150–270 s) and (C) Ca²⁺ readdition (1,000–1,180 s). Single-cell ratio responses to oxytocin (50 nM) in (D) Orai1^+/+ and (E) Orai1^−/− myoepithelial cells loaded with fura-5F; data binning periods are shown in Inset (n = 57 cells). (F) Average number of oxytocin-induced Ca²⁺ oscillations per data bin in Orai1^+/+ myoepithelial cells with 2 mM Ca²⁺ (filled circle), Orai1^−/− myoepithelial cells with 2 mM Ca²⁺ (filled triangle), and Orai1^+/+ myoepithelial cells in the absence of extracellular Ca²⁺ (open circle) (n = 3 coverslips). Analyses of alveolar unit contractions in live tissue, showing (G) percentage of alveoli responding to oxytocin (50 nM) and (H) frequency, (I) amplitude, and (J) latency of alveolar unit contractions (n = 3 mice). Data represent mean ± SEM; *P < 0.05, Student’s t test (B, C, and G–J) or two-way ANOVA with Bonferroni posttests (F).