Regulation of Cathelicidin Antimicrobial Peptide Expression by an Endoplasmic Reticulum (ER) Stress Signaling, Vitamin D Receptor-independent Pathway

Theodora Mauro,Peter M. Elias,Walter M. Holleran,Yoshikazu Uchida,Kyungho Park,Yuko Oda,Donald Mackenzie

doi:10.1074/jbc.m111.250431

Abstract

Vitamin D receptor (VDR)-dependent mechanisms regulate human cathelicidin antimicrobial peptide (CAMP)/LL-37 in various cell types, but CAMP expression also increases after external perturbations (such as infection, injuries, UV irradiation, and permeability barrier disruption) in parallel with induction of endoplasmic reticulum (ER) stress. We demonstrate that CAMP mRNA and protein expression increase in epithelial cells (human primary keratinocytes, HaCaT keratinocytes, and HeLa cells), but not in myeloid (U937 and HL-60) cells, following ER stress generated by two mechanistically different, pharmacological stressors, thapsigargin or tunicamycin. The mechanism for increased CAMP following exposure to ER stress involves NF-κB activation leading to CCAAT/enhancer-binding protein α (C/EBPα) activation via MAP kinase-mediated phosphorylation. Furthermore, both increased CAMP secretion and its proteolytic processing to LL-37 are required for antimicrobial activities occur following ER stress. In addition, topical thapsigargin also increases production of the murine homologue of CAMP in mouse epidermis. Finally and paradoxically, ER stress instead suppresses the 1,25(OH)(2) vitamin D(3)-induced activation of VDR, but blockade of VDR activity does not alter ER stress-induced CAMP up-regulation. Hence, ER stress increases CAMP expression via NF-κB-C/EBPα activation, independent of VDR, illuminating a novel VDR-independent role for ER stress in stimulating innate immunity.

Highlights

Mammalian epithelial tissues, such as the respiratory system, gastrointestinal tract, and genitourinary tract, as well as mucosal epithelia and skin, are positioned at the interface with the environment, where they deploy multiple protective strategies that protect underlying tissues from exogenous microbial pathogens [1,2,3]
Because several stressors up-regulate cathelicidin antimicrobial protein (CAMP) expression and because endoplasmic reticulum (ER) stress/activation ubiquitously occurs in mammalian cells, we hypothesized here that ER stress-mediated signals could stimulate CAMP expression in epithelial cells exposed to external perturbations, thereby restoring or enhancing antimicrobial defense barrier(s)
We assessed whether Tg-mediated ER stress stimulates CAMP mRNA expression. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a significant increase in CAMP mRNA expression in both primary human KC and HaCaT KC following treatment with Tg (100 nM) (Fig. 1B)

Summary

ER Stress Induces CAMP Expression

Stimuli lead to either accumulation of unfolded protein or overaccumulation of folded protein [16], conditions that together are referred to as ER stress [15]. Excessive ER stress can overwhelm these mechanisms, triggering apoptosis [16], two key intracellular signal pathways, termed the unfolded protein response and the ER-overload response, restore normal ER functions [17]. Because several stressors up-regulate CAMP expression and because ER stress/activation ubiquitously occurs in mammalian cells, we hypothesized here that ER stress-mediated signals could stimulate CAMP expression in epithelial cells exposed to external perturbations, thereby restoring or enhancing antimicrobial defense barrier(s). Our results demonstrate that ER stress stimulates synthesis of CAMP/LL-37 via a novel NF-␬BC/EBP␣ pathway, independent of the VDR, in epithelial cells such as KC and HeLa cells, but not in myeloid cells. The insights from these studies could lead to the development of novel mechanism-based therapies for diseases with CAMP/ LL-37 deficiency, as well as for improving antimicrobial defense

EXPERIMENTAL PROCEDURES

RESULTS

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DISCUSSION