Myosin II isoform switching mediates invasiveness after TGF-β-induced epithelial-mesenchymal transition

Abstract

Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

Fig. 1.

MHC isoforms are differentially expressed in normal mouse mammary gland and breast epithelial cell lines. (A) Normal mouse mammary tissue was fixed, sectioned, and immunostained for MHC IIA (Top, red), MHC IIB (Middle, green), or MHC IIC (Bottom, red). The sections were coimmunostained with smooth muscle actin to mark the basal layer (Top and Bottom, green) or keratin-8 to mark the luminal layer (Middle, red). (Scale bar, 100 μm.) (B) Whole-cell lysates of the indicated cell lines were subjected to Western blot analysis with the indicated antibodies.

Fig. 2.

MHC regulation during TGF-β and E1-shRNA treatment of NMuMG cells. (A) NMuMG, (B) E1-shRNA, and (C) E1⁺⁺ cells were untreated or treated with TGF-β for the indicated time. Whole-cell lysates were collected, normalized, and probed via Western blot with the indicated antibodies. Vimentin represents a positive control for EMT, and actin and GAPDH are shown as loading controls. (D) NMuMG cells were treated with TGF-β for the indicated time, after which RNA was collected and quantitative PCR was performed for the indicated myosin isoform. Data are the mean ± SEM from four independent experiments. *P < 0.05, Student’s t test, relative to the untreated. (E) RNA was collected from NMuMG, E1-shRNA, and E1⁺⁺ cells and quantitative PCR was performed for the indicated myosin isoform. For each cell line, relative isoform transcript level is expressed as a percentage of the total transcript abundance of all three isoforms. Data are the mean ± SEM. n = 3.

Fig. 3.

MHC IIB shRNA inhibits transmigration. (A and B) 2D migration and transwell assays were performed on uninfected, scramble shRNA cells or MHC IIB shRNA cells. Data were normalized to the mean of scramble shRNA cells. Data points are the mean ± SEM. For B, n ≥ 28 fields of view from three independent experiments. For C, n ≥ 11 fields of view from three independent experiments. *P < 0.001 relative to scramble shRNA cells. (C) Scramble shRNA and IIB shRNA cells were tested for their ability to invade a collagen gel in the presence (black bars) or absence (gray bars) of BAC macrophages. MDA-MB-231 cells were used as a positive control. For D, n ≥ 12 regions analyzed from five experiments performed on three independent dates. *P < 0.001 relative to invasion in the absence of BACs. Data points are the mean ± SEM.

Fig. 4.

MHC IIB localizes to the rear and perinuclear regions of migrating and transmigrating E1-shRNA cells. (A) E1-shRNA cells migrating in 2D were fixed and immunostained for MHC IIA (Top, green) and MHC IIB (Middle, red). The white arrow in the lower image indicates the direction of migration. (B) E1-shRNA cells actively moving through a transwell pore were fixed and immunostained for MHC IIA (green) and MHC IIB (red) and counterstained with DAPI (blue). Confocal z-stacks were collected. Images are orthogonal maximum intensity projections. Bottom: Merge 2 is a merge of DAPI, MHC IIA, and MHC IIB. Merge 1 excludes DAPI. The dotted line in merge 2 represents the position of the transwell membrane. (C) Same dataset used in Fig. 4B was separated into images above the membrane (Upper) and below the membrane (Lower). Each separated dataset was then averaged into a single image. (Scale bars, 20 μm.)