Studies of the role of the cathelicidin peptide LL-37 in epithelial biology

Abstract: The biology of epithelial immunity as well as of tissue repair is complex and highly regulated. A variety of molecules, cell types, and biological processes such as, cell differentiation, proliferation and migration, programmed cell death and antimicrobial mechanisms contribute to maintaining a balance between tissue damage and tissue repair. Different sources of evidence indicated that the cathelicidin hCAP18/LL-37 might, not only act as an antibacterial molecule, but as a key regulator of the above mentioned processes. We therefore tested this concept through in vivo and in vitro studies aimed at determining whether and how hCAP18/LL-37 could be such a multifunctional molecule. The goal of this thesis was to investigate the role of LL-37 in epithelial cell biology by addressing the following questions: How is the expression of the human cathelicidin gene (CAMP) controlled in primary keratinocytes? Does LL-37 contribute to tissue repair through either, regulation of cell proliferation, differentiation and/or programmed cell death? Which genes become expressed in the presence of LL-37 and what potential biological processes do they control? And does LL-37 contribute to defects in cell proliferation, differentiation and/or migration during pathological states such as cancer? We found that the active form of Vitamin D (VD) and its metabolites induced the expression of CAMP. As the biological effects of VD are mediated by the vitamin D receptor (VDR), which, once activated binds to response elements in the promoter region of target genes (VDRE), we tested the CAMP promoter for the presence of VDR binding sites. We identified one active VDRE binding site at about ~500 bp from the transcription start site. In vivo stimulation of human skin with the vitamin D analog calcipotriol resulted in high expression of hCAP18 at the mRNA and protein levels, compared to control skin samples from the same individuals (Paper I). Tissue homeostasis is maintained by, among others, the selective removal of cells through mechanisms such as apoptosis or programmed cell death. Because cell proliferation and differentiation are central to skin biology, we tested the role of LL-37 in the apoptosis of human keratinocytes. We found that LL-37 prevented camptothecin-induced apoptosis, which was associated with decreased caspase-3 activity and increased expression of PGE2 and IAP2, through a mechanism dependent on COX2 activity (Paper II). By using a microarray approach we found that LL-37 affected the gene expression profile of human keratinocytes with a signifficant effect on STC2. We investigated the mechanism of LL-37-induced STC2 upregulation and found evidence (paper III, manuscript) suggesting that unfolded protein response in keratinocytes might be triggered by expossure to LL-37. We measured the expression of the hCAP18/LL-37 in a panel of 104 breast cancer tumors and compared it to the levels found in control samples, as well as analyzed its relationship to clinical data. We also studied, in vitro, the effect of LL-37 on breast cancer cell migration and in anchorage independent colony formation. In addition, we explored the molecular mechanisms responsible for LL-37 effects on breast cancer cells. To evaluate the relevance of our findings in vivo, a xenograft model using severely compromised immunodeficient (SCID) mice was designed to test the effect of the transgenic expression of the hCAP18/LL-37 in tumor formation. The results from these experiments indicate that 1) hCAP18/LL-37 is functionally connected with ErbB2; 2) LL-37 alters breast cancer cell phenotype in vitro 3) LL-37 stimulates the migration of breast cancer cells and 4) LL-37 stimulates metastasis formation in SCDI mice in vivo (paper IV). Increasing evidence shows that besides its immune function, LL-37 has tissue-repair-like effects, promotes cell proliferation, migration and angiogenesis. Furthermore LL-37 has been implicated in the pathogenesis of inflammatory skin disease such as psoriasis. Based on our findings, we propose that LL-37 is a key regulator of epithelial homeostasis by influencing, tissue defense, tissue repair and maintenance through control of programmed cell death, in association with vitamin D and likely acting as an alarmin by activating processes such as the unfolded protein response. Thus, these effects become relevant for the study of pathological states such as chronic inflammation and cancer.