Hedgehog Signaling-related Proteins Regulate Innate Immune Response In The Epidermal Cells

The Hedgehog(Hh)signaling pathway is one of the five major signaling pathways in developmental biology,and its well-known function is the regulation of cell development.Therefore,it plays an important role in all kinds of biological processes,such as tissue development and tumor formation.However,the cell-autonomous immune regulation function of Hh signaling pathway in the epithelial cells still remain unclear.The Caenorhabditis elegans(C.elegans)epidermis can trigger a highly conserved and specific immune response against invading pathogens or physical damage,so it can be used as a model to study the mechanisms underlying innate immune response.C.elegans epidermis was utilized as the model in this work to study the Hh components-induced innate immunity,and to reveal the underlying molecular mechanisms.The strategies we used and the results we obtained were as follows:1)The Hh-related genes were inactivated by RNAi,and the expression of antimicrobial peptide(AMP)was examined.We knockdowned Hh-related genes and checked the immune response using the antimicrobial peptide Pnlp-29::GFP reporter.The results showed that most Hh components did not cause the upregulation of nlp-29 expression.Inactivation of ptc-3.qua-1,ptr-4,ptr-23 induced AMP production.2)The subcellular localization of Hh-related proteins inducing innate immune activation was observed by fluorescent labeling.PTC-3 and QUA-1 were expressed in the CeHDs region and the cytoplasm.3)The effects of Hh-related proteins on the C.elegans hemidesmosome(CeHDs)were detected by RNAi knockdown.Most Hh-related genes had no significant effect on the CeHDs structure.Loss of PTC-3 and PTR-4 disrupted the CeHDs structure.4)We examined most players known to be required for epidermal AMP induction upon external insults to find out through which signaling pathway the Hh-related proteins drive AMP production.The functional loss of SEK-1 did not block nlp-29 upregulation caused by ptc-3 inactivation,but with the sole exception of STA-2/STAT5B,suggesting that PTC-3-induced immune response requires a STAT protein,but not p38MAPK signaling pathway.5)We disrupted CeHDs structure by mup-4 knockdown,and observe the distribution of Hh-related protein.MUP-4 inactivation disrupted PTC-3 pattern,indicating that PTC-3 localization is subjected to CeHDs stability.6)The expression level of STA-2 was determined by fluorescent labeling and real-time quantitative PCR after Hh-related genes inactivation,and the results showed that RNAi against ptc-3 upregulated STA-2 expression.7)We employed an epidermal-injury approach by micro-needle puncture to introduce a single epidermal wound,and observed the spatial localization of Hh ligand and receptor after injury.Epidermal injury destoryed PTC-3 distribution,but had no obvious effect on QUA-1,indicating that PTC-3 likely contributes to physical damage.Epidermal damage resulted in the separation of PTC-3 and QUA-1,followed by activating innate immune response.8)We disrupted the Hh receptor in adult human epidermal keratinocytes(HEKa).We next quantified the expression levels of several key innate immune effectors produced by skin keratinocytes following disruption of Hh receptor.Knockdown of PTCH1,the mammalian homologue for C.elegans PTC-3,induced AMP production in primary human epidermal keratinocytes,suggesting that PTCH1 inhibits the innate immunity in epidermal cells.On the contrary,knockdown of Smoothened(SMO),which is normally suppressed by PTCH1,downregulated the expression of AMP.We hypothesized that the function of Hh signaling pathway to monitor the innate immunity in epidermal cells is likely to be evolutionarily conserved.In summary,we found that PTC-3-regulated immune response was mediated by STA-2 in the C.elegans epidermal cells.PTC-3 is likely involved in defense strategies for epidermal damage.When the epidermal cells were damaged or the CeHDs were disrupted,PTC-3 became separated from QUA-1 and activated the downstream pathway,and finally induced AMP expression through the STAT family protein STA-2.The Hh receptor PTCH1 in mammalian cells also participated in the regulation of innate immune response,so its function of immune surveillance may be conserved in evolution.Our results revealed that the Hh pathway possesses the physiological function of immune surveillance in addition to regulating cell development.Therefore,our research will provide a new understanding of the function of Hh signaling pathway in epidermal cells,and offer new evidences for the underlying mechanisms of pathological processes involved in Hh signaling pathway,such as epidermal cell inflammation and tumorigenesis.