Indian hedgehog and β-catenin signaling: Role in the sebaceous lineage of normal and neoplastic mammalian epidermis

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   Fig. 1.

   Expression of proteins in the PTCH-SHH signaling pathway in K14ΔNLef1 transgenic mouse sebaceous tumors. Immunostaining was performed with antibodies to the proteins indicated. (a) Ptch1 C terminus. (b) Ptch1 N terminus. (c) SHH. (d) IHH. (e) DHH. (f) pan-HH. (g) GLI1 (h) GLI2. (Scale bar: 200 μm.)

   
   
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   Fig. 2.

   Expression of proteins in the SHH-PTCH signaling pathway in human sebaceous tumors. Immunostaining was performed with antibodies to the proteins indicated. (a) PTCH1 C terminus. (b) PTCH1 N terminus. (c) SHH. (d) IHH. (e) DHH. (f) pan-HH. (g) GLI1. (h) GLI2. (Scale bar: 200 μm.)

   
   
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   Fig. 3.

   Expression of IHH, Gli1, and Gli2 proteins in normal and K14ΔNLef1 transgenic mouse skin and in human pilomatricoma. (a) Immunostaining with IHH antibody in normal mouse skin. Note strong reactivity in the sebaceous glands (arrowheads). (b) In human pilomatricoma (a hair follicle-derived tumor) immunostaining for IHH was not detected. (c-f) Immunostaining for GLI1 (green, c and d) and GLI2 (green, e and f) proteins in whole mounts of tail epidermis of WT (c and e) and K14ΔNLef1 transgenic (d and f) mice. Note strong expression of GLI1 in the nuclei of cells in the normal sebaceous glands (arrowheads in c) and newly differentiating sebocytes along the deformed transgenic hair follicles (arrowheads in d). Note strong and predominantly cytoplasmic staining of GLI2 in the permanent portion of the follicle below the sebaceous glands in WT epidermis (e, brackets) and weaker, more uniform staining in transgenic epidermis (f, bracket indicates equivalent region to those shown in e). Note weak nuclear staining for GLI2 in normal sebaceous glands (e, arrowheads). Whole mounts were double-labeled for β1 integrins (red) to reveal the basal IFE, periphery of sebaceous glands and outer root sheath of the hair follicles. [Scale bars: 100 μm (a and b) and 50 μm (c-f).]

   
   
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   Fig. 4.

   Expression of IHH, PTCH1, GLI1, and GLI2 in undifferentiated and differentiated human SZ95 sebocytes in vitro. (a, c, and e) Undifferentiated cells. (b, d, and f) Postconfluent, differentiated cells. (g-i) Partially differentiated cultures. (a and b) Nile red staining of lipids. (b) Note dramatic increase in the number of lipid droplets in differentiated SZ95 sebocytes (arrows). (c and d) Immunostaining for IHH protein (green) and E-cadherin (to reveal cell-cell borders; red). (d) IHH protein levels are up-regulated in differentiated sebocytes (arrows). (e and f) Immunostaining for PTCH1 protein (green) with 4′,6-diamidino-2-phenylindole nuclear counterstain (blue). PTCH1 staining is up-regulated in the cytoplasm of differentiated sebocytes (arrows in f). Immunostaining for GLI1 (g and h) and GLI2 (i) (green) with 4′,6-diamidino-2-phenylindole nuclear counterstain (blue), showing nuclear and cytoplasmic GLI localization in areas where the cultures are still undifferentiated (g and i) and exclusively cytoplasmic localization in suprabasal, terminally differentiating sebocytes (h and i Inset, arrows). [Scale bar: 20 μm(a-i).] (j) Western blots of protein lysates prepared from undifferentiated and differentiated SZ95 sebocytes (Left and Center) and retrovirally infected differentiated cells (Right) probed with antibodies against PTCH1 C terminus (160 kDa), pan-Hh (processed form of ≈25 kDa) and, as a loading control, actin (42 kDa). ev, empty vector control; ΔNLef1, retroviral vector.

   
   
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   Fig. 5.

   Manipulating Hh and β-catenin signaling regulates growth and differentiation of human sebocytes in vitro.(a-c) Inhibition of Hh signaling in human sebocytes decreases proliferation and stimulates differentiation. (a) Growth curve of SZ95 cells treated with Hh inhibitor cyclopamine or solvent methanol. (b) Treatment of undifferentiated SZ95 cells with methanol did not affect differentiation as evaluated by Nile red staining, whereas treatment with cyclopamine (c) resulted in accumulation of Nile red-positive lipid droplets (arrows). (d-j) Retroviral transduction of SZ95 cells with pBabepuro (d and g), pBabeΔNLef1 (e and h), and ΔNβ-catenin/T2 (f and i). Transduced gene products were detected by immunostaining with anti-Myc tag (e) and antihemagglutinin tag (d and f). Cells were labeled with antibodies specific for E-cadherin (to reveal cell-cell borders; red, g-i) and IHH (green, g-i). ΔNLef1 increased production of IHH protein (arrows in h). [Scale bars: 20 μm (b-i).] (j) Growth curve of cells transduced with pBabepuro (ev), pBabeΔNLef1 (dnLef1), or ΔNβ-catenin (T2). (k) Model of interactions between β-catenin and Hh signaling in epidermis. SHH promotes proliferation of progenitor cells of the hair lineage, whereas IHH stimulates proliferation of sebocyte precursors. The IHH signal is proposed to be produced by differentiated sebocytes and act on the sebocyte progenitors in a paracrine fashion.