The Antimicrobial Peptide, LL-37, Inhibits in vitro Osteoclastogenesis

Uncoupled bone resorption leads to net alveolar bone loss in periodontitis. The deficiency of LL-37, the only human antimicrobial peptide in the cathelicidin family, in patients with aggressive periodontitis suggests that LL-37 may play a pivotal role in the inhibition of alveolar bone destruction in periodontitis. We aimed to investigate a novel function of LL-37 in osteoimmunity by blocking osteoclastogenesis in vitro. Human osteoclast progenitor cells were isolated from a buffy coat of blood samples. The cells were cultured in the presence of various concentrations of LL-37 during an in vitro induction of osteoclastogenesis. Non-toxic doses of LL-37 could block multinuclear formation of the progenitor cells and significantly diminish the number of tartrate-resistant acid-phosphatase-positive cells and the formation of resorption pits (p < 0.05), whereas these concentrations induced cellular proliferation, as demonstrated by increased expression of proliferating cell nuclear antigen. Expression of several osteoclast genes was down-regulated by LL-37 treatment. It was demonstrated that nuclear translocation of nuclear- factor-activated T-cells 2 (NFAT2) was blocked by LL-37 treatment, consistent with a significant reduction in the calcineurin activity (p < 0.005). Collectively, our findings demonstrate that LL-37 inhibits the in vitro osteoclastogenesis by inhibiting the calcineurin activity, thus preventing nuclear translocation of NFAT2. Abbreviations: CALCR, calcitonin receptor; ClC-7, chloride-proton exchanger; CTSK, cathepsin K; DAPI, 4′,6-diamidino-2-phenylindole; EGTA, ethylene glycol tetraacetic acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M-CSF/CSF1, macrophage-colony- stimulating factor; MMP-9, matrix metalloproteinase-9; MTT, [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]; NFAT2, nuclear factor of activated T-cells 2; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PCR, polymerase chain reaction; RANK, receptor activator of nuclear factor kappa-B; RANKL, receptor activator of nuclear factor kappa-B ligand; RT-PCR, reverse-transcription polymerase chain- reaction; TBS, Tris-buffered saline; TCIRG1, T-cell, immune regulator 1, ATPase, H+ transporting, lysosomal V0 subunit A3; TRAcP, tartrate-resistant acid phosphatase

Regulation of pH During Amelogenesis

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation

Involvement of the P2X(7) purinergic receptor and c-Jun N-terminal and extracellular signal-regulated kinases in cyclooxygenase-2 and prostaglandin E-2 induction by LL-37

Periodontal disease is caused by microorganisms and host-derived inflammation involving increased cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production. We previously demonstrated that human β-defensin-3 induces COX-2 and PGE2 in human gingival fibroblasts (HGFs). We, therefore, aimed to examine the inducible effects of LL-37, the only cathelicidin expressed in humans, on COX-2 expression and PGE2 synthesis in HGFs and to elucidate the relevant signaling pathways. The COX-2 expression was upregulated by LL-37 in dose- and time-dependent manners. Accordingly, the synthesis of PGE2 in cell-free culture supernatants was raised by LL-37 (p < 0.01) and blocked by NS-398, a specific COX-2 inhibitor (p < 0.01). P2X inhibitors and a neutralizing antibody against P2X7 purinergic receptor significantly abrogated COX-2 induction and PGE2 production by LL-37 (p < 0.01). LL-37 upregulated COX-2 expression and PGE2 synthesis via activation of extracellular signal-regulated kinase (ERK) and p46 c-Jun N-terminal kinase (JNK), while interleukin-1β did so via nuclear factor-ĸB and all three mitogen-activated protein kinases. In summary, LL-37 can control arachidonic acid metabolism by induction of COX-2 expression and PGE2 synthesis via the P2X7 receptor, ERK, and p46 JNK. The pro-inflammatory effects of LL-37 may be essential for initiating oral mucosal inflammation in periodontal disease

ClC-7 expression levels critically regulate bone turnover, but not gastric acid secretion

Mutations in the 2Cl(-)/1H(+)-exchanger ClC-7 impair osteoclast function and cause different types of osteoclast-rich osteopetrosis. However, it is unknown to what extent ClC-7 function has to be reduced to become rate-limiting for bone resorption. In osteoclasts from osteopetrosis patients expression of the mutated ClC-7 protein did not correlate with disease severity and resorption impairment. Therefore, a series of transgenic mice expressing ClC-7 in osteoclasts at different levels was generated. Crossing of these mice with Clcn7(-/-) mutants rescued the osteopetrotic phenotype to variable degrees. One resulting double transgenic line mimicked human autosomal dominant osteopetrosis. The trabecular bone of these mice showed a reduction of osteoblast numbers, osteoid, and osteoblast marker gene expression indicative of reduced osteoblast function. In osteoclasts from these mutants ClC-7 expression levels were 20 to 30% of wildtype levels. These reduced levels not only impaired resorptive activity, but also increased numbers, size and nucleus numbers of osteoclasts differentiated in vitro. Although ClC-7 was expressed in the stomach and PTH levels were high in Clcn7(-/-) mutants loss of ClC-7 did not entail a relevant elevation of gastric pH. In conclusion, we show that in our model a reduction of ClC-7 function by approximately 70% is sufficient to increase bone mass, but does not necessarily enhance bone formation. ClC-7 does not appear to be crucially involved in gastric acid secretion, which explains the absence of an osteopetrorickets phenotype in CLCN7-related osteopetrosis

Dev Biol

Bone development is dependent on the functionality of three essential cell types: chondrocytes, osteoclasts and osteoblasts. If any of these cell types is dysfunctional, a developmental bone phenotype can result. The bone disease osteopetrosis is caused by osteoclast dysfunction or impaired osteoclastogenesis, leading to increased bone mass. In ClC-7 deficient mice, which display severe osteopetrosis, the osteoclast malfunction is due to abrogated acidification of the resorption lacuna. This study sought to investigate the consequences of osteoclast malfunction on bone development, bone structure and bone modeling/remodeling in ClC-7 deficient mice. Bones from wildtype, heterozygous and ClC-7 deficient mice were examined by bone histomorphometry and immunohistochemistry. ClC-7 deficient mice were found to have a severe developmental bone phenotype, characterized by dramatically increased bone mass, a high content of cartilage remnants, impaired longitudinal and radial growth, as well as lack of compact cortical bone development. Indices of bone formation were reduced in ClC-7 deficient mice; however, calcein labeling indicated that mineralization occurred on most trabecular bone surfaces. Osteoid deposition had great regional variance, but an osteopetrorickets phenotype, as observed in oc/oc mice, was not apparent in the ClC-7 deficient mice. A striking finding was the presence of very large abnormal osteoclasts, which filled the bone marrow space within the ClC-7 deficient bones. The development of these giant osteoclasts could be due to altered cell fate of the ClC-7 deficient osteoclasts, caused by increased cellular fusion and/or prolonged osteoclast survival. In summary, malfunctional ClC-7 deficient osteoclasts led to a severe developmental bone phenotype including abnormally large and non-functional osteoclasts. Bone formation paremeters were reduced; however, bone formation and mineralization were found to be heterogenous and continuing

Science

During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl-) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead mediates Cl-/proton (H+) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl- conductor. Despite maintaining lysosomal conductance and normal lysosomal pH, these Clcn7(unc/unc) mice showed lysosomal storage disease like mice lacking ClC-7. However, their osteopetrosis was milder, and they lacked a coat color phenotype. Thus, only some roles of ClC-7 Cl-/H+ exchange can be taken over by a Cl- conductance. This conductance was even deleterious in Clcn7(+/unc) mice. Clcn7(-/-) and Clcn7(unc/unc) mice accumulated less Cl- in lysosomes than did wild-type mice. Thus, lowered lysosomal chloride may underlie their common phenotypes

CLCN7 and TCIRG1 mutations differentially affect bone matrix mineralization in osteopetrotic individuals

Osteopetrosis is an inherited disorder of impaired bone resorption with the most commonly affected genes being CLCN7 and TCIRG1, encoding the Cl(-) /H(+) exchanger CLC-7 and the a3 subunit of the vacuolar H(+)-ATPase, respectively. We, and others have previously shown that the disease is frequently accompanied by osteomalacia, and that this additional pathology is also found in Tcirg1-deficient oc/oc mice. The remaining question was, whether osteoid enrichment is specifically associated with TCIRG1 inactivation, or whether CLCN7 mutations would also cause skeletal mineralization defects. Here we describe a complete osteologic assessment of one family carrying a novel mutation in CLCN7 (D145G), which impairs the activation and relaxation kinetics of the CLC-7 ion transporter. The two siblings carrying the mutation in the homozygous state displayed high bone mass, increased serum levels of bone formation markers, but no impairment of calcium homeostasis when compared to the other family members. Most importantly however, undecalcified processing of an iliac crest biopsy from one of the affected children clearly demonstrated a pathological increase of trabecular bone mass, but no signs of osteomalacia. Given the potential relevance of these findings we additionally performed undecalcified histology of iliac crest biopsies from seven additional cases with osteopetrosis caused by a mutation in TNFRSF11A (n = 1), CLCN7 (n = 3) or TCIRG1 (n = 3). Here we observed that all cases with TCIRG1-dependent osteopetrosis displayed severe osteoid accumulation and decreased calcium content within the mineralized matrix. In contrast, there was no detectable bone mineralization defect in the cases with TNFRSF11A- or CLCN7-dependent osteopetrosis. Taken together, our analysis demonstrates that CLCN7 and TCIRG1 mutations differentially affect bone matrix mineralization, and that there is a need to modify the current classification of osteopetrosis

Lack of cathelicidin processing in Papillon-Lefèvre syndrome patients reveals essential role of LL-37 in periodontal homeostasis.

BACKGROUND Loss-of-function point mutations in the cathepsin C gene are the underlying genetic event in patients with Papillon-Lefèvre syndrome (PLS). PLS neutrophils lack serine protease activity essential for cathelicidin LL-37 generation from hCAP18 precursor. AIM We hypothesized that a local deficiency of LL-37 in the infected periodontium is mainly responsible for one of the clinical hallmark of PLS: severe periodontitis already in early childhood. METHODS To confirm this effect, we compared the level of neutrophil-derived enzymes and antimicrobial peptides in gingival crevicular fluid (GCF) and saliva from PLS, aggressive and chronic periodontitis patients. RESULTS Although neutrophil numbers in GCF were present at the same level in all periodontitis groups, LL-37 was totally absent in GCF from PLS patients despite the large amounts of its precursor, hCAP18. The absence of LL-37 in PLS patients coincided with the deficiency of both cathepsin C and protease 3 activities. The presence of other neutrophilic anti-microbial peptides in GCF from PLS patients, such as alpha-defensins, were comparable to that found in chronic periodontitis. In PLS microbial analysis revealed a high prevalence of Aggregatibacter actinomycetemcomitans infection. Most strains were susceptible to killing by LL-37. CONCLUSIONS Collectively, these findings imply that the lack of protease 3 activation by dysfunctional cathepsin C in PLS patients leads to the deficit of antimicrobial and immunomodulatory functions of LL-37 in the gingiva, allowing for infection with A. actinomycetemcomitans and the development of severe periodontal disease