Project

Discipline

Wound healing; Cathelicidin; PTH1R; Parathyroid hormone-related peptide; Antimicrobial peptides; PTHrP

Lead
Lay summary
Our overall goal is to define the role of parathyroid hormone-related peptide (PTHrP) in skin innate immune defense. PTHrP is a peptide with homology to parathyroid hormone but produced in the skin and with largely unknown function. Our key preliminary findings are that PTHrP can strongly induce expression of the antimicrobial peptide cathelicidin in keratinocytes and that PTHrP/PTH-signaling can enhance protection against bacterial skin infection. Cathelicidins are small peptides with antimicrobial properties and important immunomodulatory functions. Recent scientific evidence has shown that cathelicidins are critically involved in several diseases: Relative absence of cathelicidin enhances the risk for certain types of skin infection, impaired wound healing and atopic dermatitis, whereas too much cathelicidin and abnormally processed forms thereof trigger inflammation in psoriasis and rosacea. Upstream factors that may control the expression of PTHrP or its receptor PTH1R in the skin are still unknown. Understanding these factors and the exact mechanisms involved would improve our understanding of the role of the PTHrP/PTH1R signaling pathway in controlling cathelicidin in skin biology and disease. This project aims at elucidating the molecular events that control PTHrP and PTH1R in the skin and at defining its role in wound healing.

Direct link to Lay Summary

Last update: 21.02.2013

Active participation

Number	Title	Start	Funding scheme

Our overall goal is to define the role of parathyroid hormone-related peptide (PTHrP) in skin innate immune defense. PTHrP is a peptide with homology to parathyroid hormone but produced in the skin and with largely unknown function. Our key preliminary findings for this proposal are that PTHrP can strongly induce expression of the antimicrobial peptide cathelicidin in keratinocytes and that PTHrP can enhance protection against bacterial skin infection. Cathelicidins are small peptides with antimicrobial properties and important immunomodulatory functions. Recent scientific evidence has shown that cathelicidins are critically involved in several diseases. Relative absence of cathelicidin enhances the risk for certain types of skin infection, impaired wound healing and atopic dermatitis, whereas too much cathelicidin and abnormally processed forms thereof trigger inflammation in psoriasis and rosacea. The factors that control cathelicidin expression are still largely unknown, though. Therefore, our recent finding that PTHrP regulates cathelicidin opens a promising field of research. In an ongoing research project, we could show that PTH1R is the critical receptor for PTHrP. However, upstream factors that may control the expression of PTHrP or its receptor PTH1R in the skin are still unknown. Understanding these factors and the exact mechanisms involved would improve our understanding of the role of the PTHrP/PTH1R signaling pathway in controlling cathelicidin in skin biology and disease. The main hypothesis driving this proposal is that molecular events that enhance expression of PTHrP or PTH1R in the skin will enhance cutaneous innate immune defense through induction of the antimicrobial peptide cathelicidin, and may therefore be important for cathelicidin-related cutaneous antimicrobial defense and wound healing. The following research plan aims at both elucidating the molecular events that control production of PTHrP (Aim 1) and PTH1R (Aim 2) in skin and defining its role in wound healing (Aim 3).Aim 1. To define the molecular regulation of keratinocyte PTHrP expression and its biological relevance in skin.In preliminary experiments we could show that PTHrP is highly expressed in the skin at sites of infection, and that human keratinocytes in culture overexpress PTHrP when stimulated with the TLR2-agonist LTA. We therefore propose our first sub-hypothesis that the response of keratinocytes to infectious agents in the skin comprises enhanced keratinocyte PTHrP expression. To this end, normal human epidermal keratinocytes (NHEK) will be exposed in culture to a panel of well defined pathogen-associated molecular patterns (PAMPs) and inflammatory cytokines to assess their ability to alter keratinocyte PTHrP expression at the mRNA (qPCR) and protein level (ELISA). Subsequently, specific inhibitors such as chloroquin for endosomal TLRs or siRNAs will be used in vitro to functionally dissect which pathogen response receptor (PRR) or cytokine(s) drives PTHrP expression. The functional relevance of PTHrP-induced antimicrobial defense induced by triggering the above identified signaling pathway(s) will then be tested in vitro in standardized lysate killing assays against living skin pathogens, and in vivo in PTHrP knockout mice. Simultaneously, PTHrP expression will be assessed by immunohistochemistry in human specimens of infectious and chronic-inflammatory skin diseases.Aim 2. To define the exact mechanisms that control PTH1R in skin.Further preliminary data indicates that 1,25-vitamin D3 (1,25-D3) can enhance PTH1R expression. Locally elevated 1,25-D3 levels in infected or wounded skin were published before (3, 9). Our second sub-hypothesis is that skin injury and infection via elevated 1,25-D3 induce PTH1R and thus amplify the response to PTHrP. To this end, we will stimulate NHEK in vitro with vitamin D3 and its metabolites, PAMPs and cytokines, and test their ability to alter PTH1R mRNA and protein expression. Human specimens of infectious and chronic-inflammatory skin disorders as well as normal skin will be immunostained for PTH1R expression. To further investigate the mechanism of 1,25-D3-induced PTH1R expression, we will perform a seek (BLAST) for vitamin D response elements (VDRE) in the human PTH1R gene promoter and test functional relevance of such VDREs by luciferase promoter assays. Aim 3. To define the role of PTHrP and its potential therapeutic use in wound healing.Cathelicidin is required for intact wound healing and our preliminary data show, that PTHrP can control cathelicidin expression. Our third sub-hypothesis is that inducing PTHrP/PTH1R in chronic wounds will increase cathelicidin and enhance wound healing. First experiments will assess PTHrP expression in human tissue specimens of chronic wounds by immunohistochemistry and in wound fluid by ELISA. To test function of PTHrP in vivo, we will then perform standardized sterile wounding experiments in WT mice treated with PTHrP or factors that induce PTHrP or PTH1R as identified in aims 1 and 2. This will show, if PTHrP ehnances wound healing in vivo. To test the requirement of PTH1R for PTHrP-enhanced wound healing in vivo, skin-specific conditional knock-out mice for PTH1R will be used. To confirm in vivo that PTHrP-enhanced wound healing acts by increasing cathelicidin, skin wounds will be co-stained for PTHrP and mouse cathelicidin CRAMP, and wound healing will be also tested in cathelicidin knock-out mice.