Misexpression of wingless-related MMTV integration site 5A in mouse mammary gland inhibits the milk ejection response and regulates connexin43 phosphorylation

Abstract

Wingless-related MMTV integration site 5A (Wnt5a) is a noncanonical signaling WNT that is expressed in every stage of mouse mammary gland development except lactation. Using slow release pellets containing WNT5A as well as Wnt5a-null tissue, we previously showed that WNT5A acts to limit mammary development. Here, we generated transgenic mice that overexpress WNT5A in the mammary epithelium using the mouse mammary tumor virus promoter (M5a mice). Lactation was impaired in two high WNT5A-expressing lines. Lactation defects could not be explained by differences in apoptosis, lineage differentiation, milk synthesis, or secretion. Instead, misexpression of WNT5A led to a failure in oxytocin response and milk ejection. Noting the similarity between the M5a phenotype and that of mice with a mutation in connexin43 (Cx43; official gene symbol Gja1), we examined Cx43 phosphorylation and localization in M5a mice. In wild-type mice, Cx43 switched from a phosphorylated to a more hypophosphorylated form after parturition. In contrast, the phosphorylated form of Cx43 was maintained after parturition in M5a mice. Using a nontumorigenic breast cell line, MCF10A, we showed that, in addition to increasing the levels of phosphorylation of Cx43 on serine-368, ectopic expression of WNT5A reduced or blocked the amount of dye transferred between cells. In summary, we propose that WNT5A inhibits the response to oxytocin and prevents milk ejection through regulation of Cx43 function.

FIG. 1.

M5a mice overexpress WNT5A in the mammary gland. A) Human WNT5A cDNA was cloned into the MKbpAII vector containing the MMTV-LTR promoter, KCR intron, and polyA tail (pA). A 350 base pair segment is amplified for genotyping (primer sequences listed above diagram). A representative gel shows the specificity of the primers for the transgene in M5a mice versus their wild-type (WT) littermates. B) Semiquantitative RT-PCR confirms the expression of the human WNT5A transgene in the M5a3 line at 8 wk of age. Cycle numbers used for expression analysis are listed below the gel picture. β2-Microglobulin (b2m) was used to normalize the results. C) Transgenic WNT5A is detectable by Western blot analysis at 8 wk, 17.5 days into pregnancy (17.5 dpc), and 1 day postpartum (1 dpp). Beta-tubulin was used as a loading control.

FIG. 2.

M5a3 mice display only mild delays in mammary gland development. Whole mount staining of M5a3 glands was performed at several stages of development: 5 wk, 8 wk, 20 wk, and 17.5 dpc. At 5 wk of age, M5a3 epithelium extends normally, although the terminal end bud (TEB) size may be slightly reduced. M5a3 mammary glands at 8 wk are virtually indistinguishable from wild-type (WT) glands. By 20 wk, M5a3 glands may have slightly reduced branching. Lobulo-alveolar structures are apparent at 17.5 dpc, although they may be sparser than WT levels, perhaps due to mildly decreased branching. Overall, the development of M5a3 glands is only mildly impaired in hemizygous MMTV-WNT5A mice. Images captured using an Olympus SZX12 stereo microscope. Original magnification ×7 (left panels) and ×70 (right panels).

FIG. 3.

WNT5A overexpression leads to lactation deficits that correlate to differences in WNT5A expression. A) Pup survival after live birth decreases with M5a3 and M5a2 dams. Pups born to wild-type (WT) or M5a4 dams have 100% survival, while pups born to M5a3 dams have 0% survival. Pups born to M5a2 dams have 62% chance of survival. Both M5a3 and M5a2 lines are statistically different from WT (P < 0.001 by χ² analysis). B) WNT5A expression levels in each line correlate to pup survival. M5a3 has the highest level of expression by Western blot analysis, with M5a2 having a moderate level of expression and M5a4 having no detectable expression.

FIG. 4.

Luminal, alveolar, and myoepithelial cell differentiation is normal in M5a3 mammary glands. A) M5a3 histology by H&E staining resembles wild-type (WT) controls both during late pregnancy (17.5 dpc) and in early lactation (1 dpp). Ducts in M5a females are dilated with milk at 1 dpp, potentially indicating milk stasis (bottom right panel). B) Mammary glands in M5a3 females (bottom panel) contain dilated ducts that are visible grossly. Wild-type glands rarely demonstrate such extreme dilation (top panel). C) E-Cadherin staining indicates no difference in luminal cell morphology, differentiation, or localization in transgenic animals versus WT controls. D) Milk protein staining in M5a3 mammary glands mirrors WT staining during pregnancy. Milk appears in both WT and M5a3 alveolar structures at 1 dpp. E) Smooth muscle actin stains mammary myoepithelial cells. Staining is indistinguishable between WT and M5a3 mice at 17.5 dpc and 1 dpp. The similarity in staining patterns signifies normal differentiation and histology of myoepithelial cells in transgenic animals. F) Protein isolated from mammary epithelial cells reveals normal expression of myoepithelial cell markers: smooth muscle actin and p63. Additionally, M5a3 dams produce normal amounts of milk (beta-casein). G) TUNEL staining to identify apoptotic cells reveals no difference in cell death between WT and M5a3 epithelium during pregnancy at 17.5 dpc. However, increased TUNEL staining occurs at 1 dpp in M5a3 glands, possibly secondary to milk stasis. Images captured using an Olympus BX51 upright microscope (A, C–E, G) and an Olympus SZX12 stereo microscope (B). Original magnification ×7 (B), ×100 (A), and ×200 (C–E, G).

FIG. 5.

M5a3 mammary glands fail to respond to oxytocin and demonstrate altered Cx43 protein phosphorylation. A) Milk protein accumulates in the ducts of wild-type (WT) mammary glands in response to oxytocin treatment (+OXT, top). At the same time, milk is removed from the alveoli (+OXT, bottom), which can be seen as an increase in the empty spaces between alveoli as well as reduced opacity in the alveoli. However, M5a3 glands lack any visible response to oxytocin addition. PBS negative controls are also shown (+PBS). Images captured using an Olympus SZX12 stereo microscope. Original magnification ×7. B) Western blots demonstrate no differences in oxytocin receptor protein levels between WT and M5a3 mice. Two WT and two M5a3 samples are shown. Pan-cytokeratin was used as a loading control. C) Western blot for Cx43 phosphorylation shows two distinct bands: P0, the presumptive hypophosphorylated protein, and P1, the more phosphorylated Cx43 form. At 17.5 dpc, both WT and M5a3 glands have an abundance of P1. At 1 dpp, WT glands show both P0 and P1 forms about equally. M5a3 glands maintain predominantly the P1 form, which is similar to the pregnancy pattern. D) Western blot for the phospho368 form of Cx43 (Cx43P368) shows little to no expression in WT 1 dpp epithelium, whereas this form of Cx43 is readily visible in M5a3 epithelium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

FIG. 6.

Overexpression of WNT5A inhibits GJIC in MCF10A cells. A) Western blot showing increased levels of the Ser-638 phosphorylated form of Cx43 (Cx43P368) in WNT5A-expressing cells (W5a) compared to vector control cells (V). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B) A dye transfer experiment was performed to test GJIC in control (Vector) and WNT5A-expressing cells. Representative fields are shown. The left panel is a phase contrast image to show the confluency of the cells and the location of the scratch used to load the dyes. The middle image shows the red channel representing staining with rhodamine-dextran, which marks the cells that were initially loaded with dye. The right panel shows green staining of luciferase yellow. Green staining in cells separate from the red-stained cells indicates the level of dye transfer, which is a measure of GJIC. WNT5A-expressing cells demonstrate limited GJIC. Images captured using an Olympus CK40 inverted microscope. Original magnification ×50.