The Antimicrobial Peptide LL-37 Alters Human Osteoblast Ca²⁺ Handling and Induces Ca²⁺-Independent Apoptosis

Abstract

The human antimicrobial peptide cathelicidin LL-37 has, besides its antimicrobial properties, also been shown to regulate apoptosis in a cell type-specific manner. Mechanisms involved in LL-37-regulated apoptotic signaling are not identified. Here, we show that LL-37 reduces the human osteoblast-like MG63 cell number and cell viability in the micromolar concentration range with an IC₅₀ value of about 5 µ<smlcap>M</smlcap>. Treatment with 4 µ<smlcap>M</smlcap> LL-37 increased the number of annexin V-positive cells and stimulated activation of caspase 3 showing that LL-37 promotes apoptosis. Treatment with 4 µ<smlcap>M</smlcap> LL-37 caused an acute and sustained rise in intracellular Ca²⁺ concentration assessed by laser-scanning confocal microscopy of Fluo-4-AM-loaded MG63 cells. LL-37 increased Ca²⁺ also in the presence of the respective L- and T-type voltage-sensitive Ca²⁺ channel blockers nifedipine and NiCl₂. LL-37 had no effect on Ca²⁺ in cells incubated with Ca²⁺-free solution. LL-37 (4 and 8 µ<smlcap>M</smlcap>) reduced the MG63 cell number both in the presence and absence of Ca²⁺ in the medium. In conclusion, LL-37 reduces the osteoblast cell number by promoting apoptosis, and furthermore, LL-37 stimulates Ca²⁺ inflow via a mechanism independent of voltage-sensitive Ca²⁺ channels. Interestingly, LL-37-induced lowering of the cell number seems to be mediated via a mechanism independent of Ca²⁺.

Vitamin D-induced up-regulation of human keratinocyte cathelicidin anti-microbial peptide expression involves retinoid X receptor α

“…Indeed, we show here that both CAMP gene activity and the hCAP-18 protein level are enhanced in human keratinocytes in response to stimulation with 1,25D3. LL-37 is suggested to exert its anti-microbial activity through multiple mechanisms including the permeabilization of the bacterial cell wall, the neutralization of bacterial lipopolysaccharide, and the stimulation of chemokine production, but, additionally, LL-37 might seriously reduce host cell viability (Burton and Steel 2009;Säll et al 2013;Svensson et al 2016a). In patients suffering from chronic periodontitis, ulcerative colitis and rosacea micromolar concentrations of LL-37 are observed locally, and in lesions from patients suffering from psoriasis, the LL-37 concentration can exceed the several hundred micromolar level (Türkoğlu et al 2009;Ong et al 2002;Schauber et al 2006;Yamasaki et al 2007).…”

Probing the disparate effects of arginine and lysine residues on antimicrobial peptide/bilayer association

“…LL-37 is also able to bind to and neutralize lipopolysaccharide (LPS) [24, 25, 18], which helps protect the body against one source of septic shock. Despite these unique properties that make LL-37 an appealing template for future drug design, LL-37 is significantly hemolytic and cytotoxic to human cells [26, 27, 18, 28]. KR-12 is a truncated portion of LL-37 corresponding to residues 18–29 (sequence KRIVQRIKDFLR), and is the smallest section of the peptide that is as active as LL-37 against Gram-negative [29, 28, 13] and certain Gram-positive bacteria [28].…”

LL-37-induced host cell cytotoxicity depends on cellular expression of the globular C1q receptor (p33)

“…Under pathological conditions, high levels of LL-37 have been found to reduce cell viability and promote apoptosis in osteoblasts, vascular smooth muscle cells, periodontal ligament cells, neutrophils, airway epithelial cells and T cells [10][11][12][13][14][15]. Notably, very high concentrations, exceeding several 100 µM of LL-37, have been detected in lesions from patients suffering from the autoimmune diseases psoriasis, rosacea and ulcerative colitis, suggesting an important role of LL-37 in these disease processes [16][17][18].…”