The human antimicrobial protein hCAP18/LL37 in wound healing and cell proliferation

Abstract

Epithelia constitute the principal barriers separating the host and the potentially injurious environment, and defence of this interface is therefore essential. Antimicrobial peptides are key effector molecules of the innate immune system. hCAP18, the sole cathelicidin antimicrobial protein in humans, is present in leukocytes and is expressed in skin and other epithelia, indicative of a central role in barrier protection. The C-terminal peptide LL-37, enzymatically released from the holoprotein hCAP18, has the capacity to kill a wide range of pathogens as well as to modulate host cell responses.

The aim of this thesis was to explore the role of LL-37 in barrier protection by investigating the expression of hCAP18/LL-37 in response to epithelial injury and, in addition, to investigate regulatory aspects of its expression.

In this context we studied physiological wound healing in human skin in vivo and demonstrated significant upregulation of hCAP18/LL-37 upon wounding. The upregulation of hCAP18/LL-37 in the reepithelializing front following wounding in vivo was reproduced in a wound model of human skin ex vivo, indicating that this induction does not require inflammation. In contrast to the acute wounds, hCAP18/LL-37 levels in chronic ulcers were low and the protein was lacking in the wound edge epithelium. Inhibition studies employing anti-LL-37 serum to the ex vivo wounds hindered reepithelialization in a concentration-dependent manner. These results imply that LJ-37 is required for reepithelialization and that the deficiency of LL-37 in chronic ulcers may contribute to their failure to heal.

Antimicrobial peptides have also been suggested to be involved in host defence against tumors. In this perspective we examined hCAP18/LL-37 expression in a number of breast carcinomas and discovered that hCAP18/LL-37 was distinctly expressed in tumor cells and not in the surrounding stroma. Not all tumor cells were positive for hCAP18/LL-37 and strongly positive cells were detected adjacent to cells devoid of hCAP18/LL-37, suggesting an intricate and tightly controlled expression of hCAP18/LL-37 in breast tumors.

To investigate whether hCAP18/LL-37 would provide growth privilege for the tumor cells, human epithelial cells were treated with synthetic biologically active LL-37 peptide which resulted in significant increase in cell proliferation. Furthermore, transgenic overexpression of hCAP18 in human epithelial cells resulted in a significantly higher proliferation compared with control cells. These results do not support that hCAP18/LL37 acts as an anti-tumor agent, but rather suggest a role for hCAP18/LL-37 in promoting tumor growth.

The molecular machinery commanding hCAP18/LL-37 gene expression is still unclear. With the purpose of investigating agents known to affect proliferation and differentiation of human skin, we treated human neonatal keratinocytes with vitamin D3 and biologically active analogues and observed marked upregulation of hCAP18 mRNA and protein syntheses. Pretreatment with calcium increased the hCAP18 expression and was synergistic with vitamin D3. Promoter studies revealed that vitamin D directly induced transcription of the hCAP18 gene by binding functional vitamin D-responsive elements in hCAP18 gene promoter. The effect was verified in vivo on intact human skin and in acute surgical wounds. These findings suggest that vitamin D is a key regulator of hCAP18/LL-37 in the skin.

In conclusion, our results reveal novel properties of hCAP18/LL-37 demonstrating its critical participation in reepithelialization of human skin, its absence in chronic ulcers, its potential role in tumor growth and the discovery that vitamin D directly regulates hCAP18 gene expression in both intact and injured human skin in vivo.