The Hedgehog (Hh) signaling network is critical for patterning and organogenesis in mammals, and has been implicated in a variety of cancers. Smoothened (Smo), the gene encoding the principal signal transducer, is overexpressed frequently in breast cancer, and constitutive activation in MMTV-SmoM2 transgenic mice caused alterations in mammary gland morphology, increased proliferation, and changes in stem/progenitor cell number. Both in transgenic mice and in clinical specimens, proliferative cells did not usually express detectable Smo, suggesting the hypothesis that Smo functioned in a non-cell autonomous manner to stimulate proliferation. Here, we employed a genetically tagged mouse model carrying a Cre-recombinase-dependent conditional allele of constitutively active Smo (SmoM2) to test this hypothesis. MMTV-Cre- or adenoviral-Cre-mediated SmoM2 expression in the luminal epithelium, but not in the myoepithelium, was required for the hyper-proliferative phenotypes. High levels of proliferation were observed in cells adjacent or in close-proximity to Smo expressing cells demonstrating that SmoM2 expressing cells were stimulating proliferation via a paracrine or juxtacrine mechanism. In contrast, Smo expression altered luminal cell differentiation in a cell-autonomous manner. SmoM2 expressing cells, purified by fluorescence activated cell sorting (FACS) via the genetic fluorescent tag, expressed high levels of Ptch2, Gli1, Gli2, Jag2 and Dll-1, and lower levels of Notch4 and Hes6, in comparison to wildtype cells. These studies provide insight into the mechanism of Smo activation in the mammary gland and its possible roles in breast tumorigenesis. In addition, these results also have potential implications for the interpretation of proliferative phenotypes commonly observed in other organs as a consequence of hedgehog signaling activation.
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