Msi1 expression leads to progenitor cell expansion. (A) Stable expression of Msi1 in mouse mammary epithelial cell line COMMA-1D (Msi1) versus control cells (Vect). (Top) Increased Msi1 mRNA expression determined by RT-PCR. (Bottom) Increased Msi1 protein expression determined by Western blotting. (B and C) Dual fluorescence-activated cell sorting of cells stained for stem and progenitor cell markers. The percentage of cells expressing the markers is shown in each quadrant. COMMA/Msi1 cells (Msi1) exhibit a higher percentage of CD24^hi Sca-1⁺ cells (B) and CD24^hi CD29⁺ cells (C) than control (Vect) cells.

Msi1 increases expression of multipotential progenitor cells. (A) Fluorescence-activated cell sorting histogram of CK6⁺ cells. CK6 expression is increased in COMMA/Msi1 (Msi1) versus control (Vect) cells. (B) Immunofluorescence for CK19⁺ cells. CK19 expression is increased in COMMA/Msi1 cells (Msi1) versus control cells (Vect). (Right) Quantitation of the percentage of CK19⁺ cells (means ± standard errors [SE]) from five microscopic fields. (C, top) Immunofluorescence for CK14⁺ CK18⁺ cells. COMMA/Msi1 cells express an increased percentage of CK14⁺ CK18⁺ cells. CK14⁺ cells are stained green, and CK18⁺ cells are stained red; the merge shows that COMMA/Msi1 cells (Msi1) express a greater percentage of CK14⁺ CK18⁺ cells (yellow) than control cells (Vect). (Bottom) Quantitation of CK14⁺, CK18⁺, and CK14⁺ CK18⁺ cells. Each value is the mean ± SE of the percentage of positive cells from five microscopic fields.

Msi1 promotes S-phase proliferation and growth factor secretion. (A) Cell proliferation is increased in COMMA/Msi1 (Msi1) cells versus control (Vect) cells. Cells were grown in 96-well plates for 24 to 92 h, and cell density was determined by sulforhodamine B staining (means ± standard errors [SE]; n = 5; *, P < 0.05; **, P < 0.001). OD 560, optical density at 560 nm. (B) Conditioned media (CM) from COMMA/Msi1 cells (Msi1) increases colony formation in control (Vect) cells. Cells were seeded at 3,000 cells/100-mm dish and grown in a mixture containing 50% fresh medium and 50% CM from either COMMA/Vect or COMMA/Msi1 cells. (Left) Colony formation in the conditioned medium from control cells (+Vect/CM) was greater in COMMA/Msi1 (Msi1) cells than in control (Vect) cells; however, both COMMA/Msi1 and control cells exhibited increased colony formation when grown in conditioned medium from COMMA/Msi cells (+Msi1/CM). (Right) Colonies of >50 cells were counted in each dish (means ± SE; n = 3). (C, left) Increased PLF1 and reduced DKK3 mRNA expression in COMMA/Msi1 cells versus control cells, as determined by gene microarray and qRT-PCR analyses (means ± SE; n = 3). (Right) Increased PLF1 and reduced DKK3 protein levels in the conditioned medium from COMMA/Msi1 cells (Msi1) versus control cells (Vect), as determined by Western blotting. Sample loading was measured by Ponceau S staining.

Msi1 inhibits p21^Cip1 and GSK3β and activates ERK. (A) Msi increases ERK2 and cyclin D1 and D2 levels and reduces p21^Cip1 and pGSK3β but does not affect PDK1/Akt activation. Western blots of cell lysates from COMMA/Msi1 cells (Msi1) and control cells (Vect) are shown. (B) MEK inhibitor U0126 preferentially reduces cell growth of COMMA/Msi1 cells. COMMA/Msi1 cells (Msi1) and control cells (Vect) were treated for 48 h with 2 and 10 μM U0126, and pERK1/2 levels (top) and cell growth (bottom) were determined. (C) pERK is reduced in cells grown in PLF1-depleted conditioned medium from COMMA/Msi1 cells. Conditioned medium was concentrated and incubated with a PLF1 monoclonal antibody, and antigen-antibody complexes were removed with protein G-Sepharose. PLF1 and pERK1/2 were reduced in cells grown in PLF1-depleted conditioned medium (PLF1 Ab) versus medium treated with IgG (IgG control). pERK1/2 was quantitated with Scion Image software and normalized to β-actin. (D) Pertussis toxin inhibits ERK phosphorylation in COMMA/Msi1 cells. Cells were treated for 4 h with 0, 0.3, 1, and 3 ng/ml pertussis toxin, and pERK was measured by Western blotting. pERK1/2 was quantitated with Scion Imaging Software and normalized to β-actin. (E) PLF1 shRNA reduces pERK levels. COMMA/Msi1 cells were transfected with a PLF1 shRNA or control inactive shRNA and selected with G418 for 2 weeks. pERK was reduced only in cells expressing the PLF1 shRNA.

Msi1 activates the Wnt and Notch pathways. (A, top) Msi1 activates the Notch pathway. CBF1-luciferase reporter gene activity is increased sevenfold in COMMA/Msi1 cells (Msi1) versus control cells (Vect). (Middle) qRT-PCR analysis indicates increased expression of Notch1, Notch ligands Delta1 (DL1) and Jagged1 (Jag1), and the Notch target Hes1 gene (Hes1). Data are means ± standard errors (SE) (n = 3). (Bottom) CBF1 reporter gene activity in COMMA/Msi1 cells is preferentially inhibited by U0126. (B, top) Msi1 increases nuclear β-catenin localization. β-Catenin immunofluorescence indicates greater nuclear localization in COMMA/Msi1 cells (Msi1) than in control cells (Vect). Magnification, ×200. (Bottom) Quantitation of nuclear β-catenin based on the number of fluorescent nuclei in three microscopic fields (means ± SE; n = 3). (C) Msi1 activates the Wnt pathway. (Top) β-Catenin/TCF reporter gene activity is increased fivefold in COMMA/Msi1 cells (Msi1) versus control cells (Vect) (means ± SE; n = 3). (Bottom) β-Catenin/TCF-dependent reporter gene activity in COMMA/Msi1 cells is preferentially inhibited by U0126. (D) Reduction of DKK3 mRNA in control cells increases β-catenin/TCF-dependent and CBF1-luciferase reporter gene activities. (Top) qRT-PCR analysis of DKK3 mRNA in control cells treated with a control (C) or DKK3 (shDKK3) shRNA. (Middle) CBF1-luciferase activity is increased following DKK3 shRNA treatment. (Bottom) β-Catenin/TCF-dependent reporter gene activity is increased following DKK3 shRNA treatment. Statistical significance is indicated by P values of <0.05. (E) Reduction of PLF1 mRNA in COMMA/Msi1 cells reduces β-catenin/TCF-dependent and CBF1-luciferase reporter gene activities. (Top) qRT-PCR analysis of PLF1 mRNA in COMMA/Msi1 cells treated with a control (C) or PLF1 (shPLF1) shRNA. (Middle) CBF1-luciferase activity is reduced following PLF1 shRNA treatment. (Bottom) β-Catenin/TCF-dependent reporter gene activity is reduced following PLF1 shRNA treatment. Statistical significance is indicated by P values of <0.05.

Musashi1 signaling in mammary progenitor cell proliferation. Msi1 blocks translation of two known targets in mammalian cells, Numb and p21^Cip1. The present study indicates that Msi1 inhibits DKK3 protein secretion and mRNA levels, but it is unknown if this occurs by interference with nuclear processing and/or translation. Inhibition of Numb expression increases intracellular Notch and Notch-dependent transcription of genes such as the Delta1, Jagged1, Notch1, Hes1, and cyclin D1 genes. Inhibition of p21^Cip1 relieves its inhibitory effect on cyclin D-dependent protein kinases to increase G₁/S transit through the cell cycle. Loss of DKK3 expression activates the Wnt pathway, resulting in increased nuclear localization of β-catenin and transcription of β-catenin/TCF-dependent target genes such as the cyclin D1 and D2 and PLF1 genes, and possibly the Msi1 gene itself. PLF1 activates ERK, presumably through the IGF2R, which in turn inhibits GSK3β to further increase activation of the Notch and Wnt pathways through an autoregulatory loop. The net result of these processes is the stimulation of mammary progenitor cell proliferation. Dashed lines indicate inhibition; solid lines indicate stimulation.