Molecular mechanism of Indian Hedgehog signalling in human sebocytes

The hedgehog signalling pathway plays a crucial role during regulation of hair cycle and homeostasis of mammalian skin. Furthermore, misregulation of this pathway is attributed to a range of skin diseases and cancers. In previous studies Indian hedgehog was identified to be the only Hedgehog ligand expressed in sebaceous glands. Moreover, modulation of hedgehog pathway activity was shown to affect size and number of sebaceous glands in the skin. Thereby, local inactivation of the canonical Wnt pathway seems to be prerequisite for correct sebaceous gland development. However, the underlying molecular mechanism has not been clarified, yet. Therefore, we hypothesise that Indian hedgehog signalling regulates proliferation and/or differentiation in human sebocytes. Thereby, activation of Hedgehog pathway and inactivation of canonical Wnt signalling might be cross-linked. To test this, we applied the human sebocyte cell line SZ95 as in vitro model by comparing three distinct sebocyte populations depending on their differentiation state: “undifferentiated”, “differentiating” and “terminally differentiated” after induction by arachidonic acid treatment. Terminally differentiated sebocytes displayed increased expression of IHH without endogenous pathway activation. In contrast, undifferentiated sebocyte displayed increased proliferation rate upon pathway activation by GLI1 and GLI2 expression. Additionally, our findings point to a role of the lipid metabolism in regulating these processes as shown by modulation of gene expression by addition of arachidonic acid. Importantly, overexpression of GLI1, GLI2 and GLI3 also revealed that each GLI transcription factors preferentially activates a distinct set of established and potentially new Hedgehog target genes in human sebocytes. Additionally, we identified a new mechanism of mutual regulation between Hedgehog and Wnt pathways on protein level in human sebocytes. More precisely, overexpression of GLI3 and GLI2 transcription factors resulted in accumulation of non-phosphorylated, active β-CATENIN, although Wnt pathway activity was not increased. Conversely, augmented levels of β-CATENIN in combination with GLI1 activator dramatically increased Gli reporter activity. In summary, we propose the following model where undifferentiated sebocytes are the target cells of Hedgehog signals originating from mature sebocytes and GLI repressors might block endogenous Wnt pathway by interference with β-CATENIN