Uso de microvibración e inhibidores de la catepsina K en tratamientos de regeneración ósea dental / Use of micro-vibration and cathepsin K inhibitors in dental bone regeneration treatments

Introducción: el hueso está en remodelación constante para mantener la estructura del esqueleto, tener un ciclo de resorción por los osteoclastos y formación de hueso nuevo a cargo de los osteoblastos; el hueso también es susceptible a enfermedades sistémicas, traumas, edad y trastornos genéticos que afectarán el remodelado óseo, produciendo una pérdida masiva de masa ósea regulado por hormonas, citocinas, enzimas, etcétera. El objetivo es realizar una revisión sistemática de artículos que muestren cambio o alteración al utilizar tratamientos con microvibraciones y farmacológicos sobre la catepsina K en el hueso alveolar. Material y métodos: para realizar una comparación entre la efectividad del tratamiento a base de microvibraciones y con inhibidores de la catepsina K, se realizó una revisión sistemática en nueve bases de datos (Wiley Online Library, PubMed, Google Academic, Scopus, ScienceDirect, SciELO, Medline, EBSCO y Springer Link). La población de estudio fueron ratas y ratones. Resultados: en este estudio se incluyeron 20 artículos cuya investigación se realizó en estudios clínicos. En los resultados podemos observar cómo todos los tratamientos de alguna forma mejoran el proceso de remodelado óseo. Es difícil comparar cuál de los tratamientos dentro de cada grupo es mejor que otro, debido a que los resultados expresados son cualitativos. Conclusión: acorde a los resultados expresados se opta por realizar un tratamiento con microvibraciones debido a que el uso de inhibidores de la catepsina K aún no se encuentra completamente desarrollado y no se comprenden sus consecuencias debido a su manera sistémica de actuar (AU)

Introduction: the bone is in constant remodeling to maintain the skeletal structure, having a cycle of resorption by osteoclasts and formation of new bone by osteoblasts, the bone is also susceptible to systemic diseases, trauma, age and genetic disorders that affect bone remodeling, producing a massive loss of bone mass regulated by hormones, cytokines, enzymes, etcetera. The objective is to perform a systematic review of articles that show a change or alteration when using micro-vibration and pharmacological treatments on cathepsin K in the alveolar bone. Material and methods: in order to make a comparison between the effectiveness of micro-vibration and cathepsin K inhibitor treatments, a systemic review was carried out in nine databases (Wiley Online Library, PubMed, Google Academic, Scopus, ScienceDirect, SciELO, Medline, EBSCO and Springer Link). The study population was rats and mice. Results: this study included 20 articles whose research was carried out in clinical studies. In the results we can see how all the treatments in some way improve the bone remodeling process, it is difficult to compare which treatment within each group is better than the other, because the results expressed are qualitative. Conclusion: according to the results expressed, it is decided that it is better to perform a treatment with micro vibrations because the use of cathepsin K inhibitors are not yet fully developed and their consequences are not understood due to their systemic way of acting (AU)

[Effects of Cathepsin K on spatial learning and memory in rats].

OBJECTIVE: To investigate the effects of Cathepsin K(CatK) on spatial learning and memory in rat hippocampus and its mechanisms. METHODS: Twenty male SD rats were randomly divided into Control group and CatK inhibitor group(CatKⅡ group), which were microinjected with Cathepsin K specific inhibitor(0.5 µg/µl) and artificial cerebrospinal fluid in hippocampal DG area respectively with 5 days. The cultured hippocampal neuron cells were divided into control group (CON group), negative control group(NC group), siRNA interference group(siCatK group). Three re-wells were set for each group, and samples were collected 18～20 h after siRNA transfection. Morris water maze was used to evaluate spatial learning and memory function of rats. Meanwhile, dynamic changes of glutamate(Glu) content in extracellular fluid of DG region during learning and memory were observed by microdialysis and high performance liquid chromatography in conscious rats. Western blot was used to detect CatK-mediated Notch1 activation and other signal molecules. RESULTS: Animal experiments showed that compared with the control group, the spatial learning and memory ability were decreased significantly in CatKII group, and the hippocampus protein expressions of c-Notch1, p-Akt, p-CREB and BDNF were also decreased significantly(P＜0.05); the levels of Glu in DG area of control group and CatK II group were increased significantly with Morris water maze training days, but the increase of CatK II group was significantly weaker than that of control group(P＜ 0.05). The results of cell experiment showed that the expressions of CatK, c-Notch1, p-CREB and BDNF in siCatK group were significantly lower than other groups (P＜0.05). CONCLUSION: CatK can affect the spatial learning and memory function of rats by activating Notch1 and its memory related signal protein in hippocampus.

Function of Cathepsin K in the Central Nervous System of Male Mice is Independent of Its Role in the Thyroid Gland.

Cathepsin K deficiency in male mice (Ctsk-/-) results in decreased numbers of hippocampal astrocytes and altered neuronal patterning as well as learning and memory deficits. Additionally, cathepsin K carries essential roles in the thyroid gland where it contributes to the liberation of thyroid hormones (TH). Because TH are essential for brain development, in particular for the cerebellum, we investigated whether cathepsin K's function in the thyroid is directly linked to the brain phenotype of Ctsk-/- mice. Serum levels of thyroid stimulating hormone, brain concentrations of free TH, and deiodinase 2 (Dio2) activity in brain parenchyma as well as cerebellar development were comparable in Ctsk-/- and WT animals, suggesting regular thyroid states and TH metabolism. Despite unaltered transcript levels, protein expression of two TH transporters was enhanced in specific brain regions in Ctsk-/- mice, suggesting altered TH supply to these regions. Thyrotropin releasing hormone (Trh) mRNA levels were enhanced threefold in the hippocampus of Ctsk-/- mice. In the striatum of Ctsk-/- mice the mRNA for Dio2 and hairless were approximately 1.3-fold enhanced, while mRNA levels for monocarboxylate transporter 8 and Trh were reduced to 60% and 40%, respectively, pointing to altered striatal physiology. We conclude that the role of cathepsin K in the thyroid gland is not directly associated with its function in the central nervous system (CNS) of mice. Future studies will show whether the brain region-specific alterations in Trh mRNA may eventually result in altered neuroprotection that could explain the neurobehavioral defects of Ctsk-/- mice.

Cathepsin K-deficient osteocytes prevent lactation-induced bone loss and parathyroid hormone suppression.

Lactation induces bone loss to provide sufficient calcium in the milk, a process that involves osteoclastic bone resorption but also osteocytes and perilacunar resorption. The exact mechanisms by which osteocytes contribute to bone loss remain elusive. Osteocytes express genes required in osteoclasts for bone resorption, including cathepsin K (Ctsk), and lactation elevates their expression. We show that Ctsk deletion in osteocytes prevented the increase in osteocyte lacunar area seen during lactation, as well as the effects of lactation to increase osteoclast numbers and decrease trabecular bone volume, cortical thickness and mechanical properties. In addition, Ctsk deletion in osteocytes increased bone Parathyroid Hormone related Peptide (PTHrP), prevented the decrease in serum Parathyroid Hormone (PTH) induced by lactation, but amplified the increase in serum 1,25(OH)2D. The net result of these changes is to maintain serum and milk calcium levels in the normal range, ensuring normal offspring skeletal development. Our studies confirm the fundamental role of osteocytic perilacunar remodeling in physiological states of lactation and provides genetic evidence that osteocyte-derived Ctsk contributes not only to osteocyte perilacunar remodeling, but also to the regulation of PTH, PTHrP, 1,25-Dyhydroxyvitamin D (1,25(OH)2D), osteoclastogenesis and bone loss in response to the high calcium demand associated with lactation.

Cathepsin K Deficiency Ameliorates Systemic Lupus Erythematosus-like Manifestations in Faslpr Mice.

Cysteinyl cathepsin K (CatK) is expressed in osteoclasts to mediate bone resorption, but is also inducible under inflammatory conditions. Faslpr mice on a C57BL/6 background develop spontaneous systemic lupus erythematosus-like manifestations. Although normal mouse kidneys expressed negligible CatK, those from Faslpr mice showed elevated CatK expression in the glomeruli and tubulointerstitial space. Faslpr mice also showed elevated serum CatK levels. CatK deficiency in Faslpr mice reduced all tested kidney pathologies, including glomerulus and tubulointerstitial scores, glomerulus complement C3 and IgG deposition, chemokine expression and macrophage infiltration, and serum autoantibodies. CatK contributed to Faslpr mouse autoimmunity and pathology in part by its activity in TLR-7 proteolytic processing and consequent regulatory T (Treg) cell biology. Elevated TLR7 expression and proteolytic processing in Faslpr mouse kidneys and Tregs showed significantly reduced levels in CatK-deficient mice, leading to increased spleen and kidney Treg content. Purified CD4+CD25highFoxp3+ Tregs from CatK-deficient mice doubled their immunosuppressive activity against T effector cells, compared with those from CatK-sufficient mice. In Faslpr mice, repopulation of purified Tregs from CatK-sufficient mice reduced spleen sizes, autoantibody titers, and glomerulus C3 and IgG deposition, and increased splenic and kidney Treg contents. Tregs from CatK-deficient mice had significantly more potency than CatK-sufficient Tregs in reducing spleen sizes, serum autoantibody titers, and glomerulus C3 deposition, and in increasing splenic and kidney Treg content. This study established a possible role of CatK in TLR7 proteolytic activation, Treg immunosuppressive activity, and lupus autoimmunity and pathology.

Marcadores de remodelamiento óseo en pacientes con enfermedad periodontal / Bone remodeling biomarkers in patients with periodontal disease

La Fosfatasa Alcalina Ósea (FAO) es una isoforma de la Fosfatasa Alcalina (FAL). La medición de su actividad en saliva es una medida indirecta del proceso de formación ósea, más sensible y específica que la FAL. La catepsina K es la principal colagenasa del proceso de resorción ósea, es capaz de degradar al colágeno tipo I en varios sitios dando lugar a pequeños péptidos N- y C- terminales. El telopéptido C-terminal (CTx) es el marcador más sensible y específico en el aumento de la resorción ósea, ya que el colágeno tipo I constituye más del 90 por ciento de la matriz orgánica del hueso...

The silencing of cathepsin K used in gene therapy for periodontal disease reveals the role of cathepsin K in chronic infection and inflammation.

BACKGROUND AND OBJECTIVE: Periodontitis is a severe chronic inflammatory disease and one of the most prevalent non-communicable chronic diseases that affects the majority of the world's adult population. While great efforts have been devoted toward understanding the pathogenesis of periodontitis, there remains a pressing need for developing potent therapeutic strategies for targeting this dreadful disease. In this study, we utilized adeno-associated virus (AAV) expressing cathepsin K (Ctsk) small hairpin (sh)RNA (AAV-sh-Ctsk) to silence Ctsk in vivo and subsequently evaluated its impact in periodontitis as a potential therapeutic strategy for this disease. MATERIAL AND METHODS: We used a known mouse model of periodontitis, in which wild-type BALB/cJ mice were infected with Porphyromonas gingivalis W50 in the maxillary and mandibular periodontium to induce the disease. AAV-sh-Ctsk was then administrated locally into the periodontal tissues in vivo, followed by analyses to assess progression of the disease. RESULTS: AAV-mediated Ctsk silencing drastically protected mice (> 80%) from P. gingivalis-induced bone resorption by osteoclasts. In addition, AAV-sh-Ctsk administration drastically reduced inflammation by impacting the expression of many inflammatory cytokines as well as T-cell and dendritic cell numbers in periodontal lesions. CONCLUSION: AAV-mediated Ctsk silencing can simultaneously target both the inflammation and bone resorption associated with periodontitis through its inhibitory effect on immune cells and osteoclast function. Thereby, AAV-sh-Ctsk administration can efficiently protect against periodontal tissue damage and alveolar bone loss, establishing this AAV-mediated local silencing of Ctsk as an important therapeutic strategy for effectively treating periodontal disease.

Calcium-containing crystals enhance receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor-mediated osteoclastogenesis via extracellular-signal-regulated kinase and p38 pathways.

OBJECTIVE: Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, ß-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. METHODS: Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phosphorylation levels of signal transduction molecules. RESULTS: Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, ß-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. CONCLUSION: We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals.

Molecular and cellular basis of bone resorption.

Osteoclast research has an exciting history and a challenging future. More than 3 decades ago, it became evident that bone-resorbing osteoclasts are of hematopoietic origin and are ultimately linked to the "basic multicellular unit," where they team up with the other cell types, including bone-forming osteoblasts. Since 2 decades, we have learned about the signaling pathways controlling genes relevant for osteoclastogenesis and bone resorption. It took another decade until the hypothesized "osteoclast differentiation" factor was discovered and was translated into an approved pharmacologic strategy. Here, the focus is on another molecular target, cathepsin K, a cysteine protease being released by the osteoclast into the resorption compartment. Genetic deletion and pharmacological blocking of cathepsin K reduces bone resorption but with ongoing bone formation. This observation not only holds great promise to become a new pharmacologic strategy, but it also provides new insights into the coordinated work of cells in the "basic multicellular unit" and thus, bridges the history and future of osteoclast research. This article is a short primer on osteoclast biology for readers of the special issue on odanacatib, a cathepsin K inhibitor.

[Biological therapy for osteoporosis].

Osteoporosis is a disorder of bone formation and resorption balance. Advances in our knowledge of the molecular mechanisms of bone formation and resorption led to promising therapeutic targets for osteoporosis. In the novel biological drugs, denosumab, a monoclonal antibody against receptor activator of nuclear factor-κB ligand (RANKL) has been clinically applied by positive effect on bone mineral density, negative effect on bone resorption, preventive effect on fragility fractures and safety. Odanacatib, a cathepsin K inhibitor is drawing attention as an antiresorptive drug which has lower bone resorption potency than bisphosphoneate. On the other hand, BHQ-880, an anti-Dickkopf-1 (Dkk-1) antibody and romosozumab (AMG-785) , an anti-sclerostin antibody which activate Wnt/ß-catenin signaling pathway are drawing attention as bone formation accelerators with no bone resorption acceleration. Clinical studies of these drugs are now ongoing and their clinical applications are expected.

Bone turnover in hyperparathyroidism.

A narrow serum calcium level which is essential for many metabolic processes is regulated by the calcium-sensing receptor which regulates parathyroid hormone (PTH) release. Primary hyperparathyroidism is supposed to be the third most common endocrine disorder. Besides nephrolithiasis and an increased incidence of cardiovascular symptoms it is associated with bone loss and an increased risk of fracture. Several different classical bone turnover markers have been shown to be increased. However, there are many uncertainties in pathophysiology of PHPT. Hardly any conclusive data exist on the RANK (receptor activator of nuclear factor-kB)/RANKL (receptor activator of nuclear factor-kB ligand)/OPG (osteoprotegerin) system, cathepsin K, sclerostin, FGF-23 (Fibroblast growth factor-23), Klotho, and DKK 1 (Dickkopf 1) in patients suffering from PHPT.

Macrophage cathepsin K promotes prostate tumor progression in bone.

Bone marrow macrophages (BMMs) share common progenitors with osteoclasts and are critical components of bone-tumor microenvironment; however, their function in prostate tumor growth in the skeleton has not been explored. BMMs are the major source of inflammatory factors and proteases, including cysteine protease cathepsin K (CTSK). In this study, utilizing mice deficient in CTSK, we demonstrate the critical involvement of this potent collagenase in tumor progression in bone. We present the evidence that tumor growth and progression in the bone are impaired in the absence of CTSK. Most importantly, we show for the first time that BMM-supplied CTSK may be involved in CCL2- and COX-2-driven pathways that contribute to tumor progression in bone. Together, our data unravel novel roles for CTSK in macrophage-regulated processes, and provide evidence for close interplay between inflammatory, osteolytic and tumor cell-driven events in the bone-tumor microenvironment.

Cathepsin K knockout mitigates high-fat diet-induced cardiac hypertrophy and contractile dysfunction.

The cysteine protease cathepsin K has been implicated in pathogenesis of cardiovascular disease. We hypothesized that ablation of cathepsin K protects against obesity-associated cardiac dysfunction. Wild-type mice fed a high-fat diet exhibited elevated heart weight, enlarged cardiomyocytes, increased left ventricular wall thickness, and decreased fractional shortening. All these changes were reconciled in cathepsin K knockout mice. Cathepsin K knockout partly reversed the impaired cardiomyocyte contractility and dysregulated calcium handling associated with high-fat diet. Additionally, cathepsin K knockout alleviated whole-body glucose intolerance and improved insulin-stimulated Akt phosphorylation in high-fat diet-fed mice. High-fat feeding increased the expression of cardiac hypertrophic proteins and apoptotic markers, which were inhibited by cathepsin K knockout. Furthermore, high-fat feeding resulted in cathepsin K release from lysosomes into the cytoplasm. In H9c2 myoblasts, silencing of cathepsin K inhibited palmitic acid-induced release of cytochrome c from mitochondria and expression of proapoptotic signaling molecules. Collectively, our data indicate that cathepsin K contributes to the development of obesity-associated cardiac hypertrophy and may represent a potential target for the treatment to obesity-associated cardiac anomalies.

Potential role of odanacatib in the treatment of osteoporosis.

Cathepsin K is a key enzyme involved in the degradation of organic bone matrix by osteoclasts. Inhibition of bone resorption observed in human and animal models deficient for cathepsin K has identified this enzyme as a suitable target for intervention by small molecules with the potential to be used as therapeutic agents in the treatment of osteoporosis. Odanacatib (ODN) is a nonbasic selective cathepsin K inhibitor with good pharmacokinetic parameters such as minimal in vitro metabolism, long half-life, and oral bioavailability. In preclinical studies, ovariectomized monkeys and rabbits treated with ODN showed substantial inhibition of bone resorption markers along with increases in bone mineral density (BMD). Significant differences were observed in the effects of ODN treatment compared with those of other antiresorptive agents such as bisphosphonates and denosumab. ODN displayed compartment-specific effects on trabecular versus cortical bone formation, with treatment resulting in marked increases in periosteal bone formation and cortical thickness in ovariectomized monkeys whereas trabecular bone formation was reduced. Furthermore, osteoclasts remained viable. Phase I and II studies conducted in postmenopausal women showed ODN to be safe and well tolerated. After 5 years, women who received ODN 50 mg weekly continuously from year 1 (n = 13), showed BMD increases from baseline of 11.9% at the lumbar spine, 9.8% at the femoral neck, 10.9% at the hip trochanter, and 8.5% at the total hip. Additionally, these subjects maintained a low level of the urine bone resorption marker N-terminal telopeptide/creatinine (-67.4% from baseline) through 5 years of treatment, while levels of serum bone-specific alkaline phosphatase remained only slightly reduced relative to baseline (-15.3%). In women who were switched from ODN to placebo after 2 years, bone turnover markers were transiently increased and BMD gains reversed after 12 months off medication. Adverse experiences in the ODN-treated group were not significantly different from the placebo group. In conclusion, available data suggests that cathepsin K inhibition could be a promising intervention with which to treat osteoporosis. Ongoing studies are expected to provide information on the long-term efficacy in fracture reduction and safety of prolonged treatment with ODN.

Inhibition of cathepsin K for treatment of osteoporosis.

Cathepsin K is the protease that is primarily responsible for the degradation of bone matrix by osteoclasts. Inhibitors of cathepsin K are in development for treatment of osteoporosis. Currently available antiresorptive drugs interfere with osteoclast function. They inhibit both bone resorption and formation, due to the coupling between these processes. Cathepsin K inhibitors, conversely, target the resorption process itself and may not interfere with osteoclast stimulation of bone formation. In fact, when cathepsin K is absent or inhibited in mice, rabbits, or monkeys, bone formation is maintained or increased. In humans, inhibition of cathepsin K is associated with sustained reductions in bone resorption markers but with smaller and transient reductions in bone formation markers. The usefulness of cathepsin K inhibitors in osteoporosis is now being examined in phase 2 and phase 3 clinical trials of postmenopausal osteoporotic women.

Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes.

By secreting proteases such as cathepsins and matrix metalloproteinases (MMPs), macrophage foam cells may be a major cause of ruptured atherosclerotic plaques. The aims of the present study were to investigate in vitro role of human macrophage foam cells in degrading type I collagen, a major component of extracellular matrix (ECM) in plaques, and to establish whether the pro-inflammatory molecules, tumor necrosis factor (TNF)-alpha, and receptor activator of nuclear factor-κB ligand (RANK-L) increase this degradation. CD14+ monocytes isolated from peripheral blood were differentiated into macrophage foam cells and cultured on a type I collagen matrix in the presence of TNF-alpha and RANK-L. Matrix degradation was measured by the cathepsin K-generated C-terminal cross-linked telopeptide of type I collagen (CTX-I) and the MMP-generated carboxyterminal telopeptide of type I collagen (ICTP) in supernatants showing that macrophage foam cells secrete MMPs and cathepsin K, resulting in release of ICTP and CTX-I. Stimulation with TNF-alpha increased CTX-I and ICTP dose dependently, with ICTP levels increasing by 59% and CTX-I levels increasing by 43%. RANK-L enhanced the release of CTX-I and ICTP by 56% and 72%, respectively. This is, to our knowledge, the first data describing a simple in vitro system in which macrophage foam cells degradation of matrix proteins can be monitored. This degradation can be enhanced by cytokines since TNF-alpha and RANK-L significantly increased the matrix degradation. This in vitro system in part is a model system for the macrophage-mediated proteolytic degradation of the ECM, which is found in many diseases with an inflammatory component.

New targets for intervention in the treatment of postmenopausal osteoporosis.

Postmenopausal osteoporosis is a disease of high bone remodeling, with an imbalance of bone resorption over bone formation, resulting in decreased bone mineral density and disruption of bone microarchitecture. With our improved understanding of the molecular and cellular regulators and mediators of bone remodeling, new targets for therapeutic intervention have been identified. Receptor activator of nuclear factor κB ligand (RANKL) is the principal regulator of osteoclast differentiation, activity, and survival; denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and is approved for the treatment of women with postmenopausal osteoporosis at high risk of fractures. Cathepsin K is a protease produced by activated osteoclasts that degrades the protein matrix of bone. An inhibitor of cathepsin K, odanacatib, is in phase III clinical trials for the treatment of postmenopausal osteoporosis; it decreases bone resorption while seeming to suppress bone formation less than other antiresorptive agents. Sclerostin is a cytokine produced by osteocytes that inhibits osteoblastic bone formation; investigational monoclonal antibodies to sclerostin, such as AMG 785, have osteoanabolic properties with the potential to improve clinical outcomes in patients with osteoporosis. These and other novel interventions that target newly recognized regulators of bone remodeling are promising agents for the treatment of osteoporosis.

Cathepsin K deficiency aggravates lung injury in hyperoxia-exposed newborn mice.

Cathepsin K (CatK) is a potent collagenase and elastase and may be involved in the development of neonatal bronchopulmonary dysplasia. The authors evaluated the effects of CatK deletion on neonatal lung development and response to prolonged hyperoxic challenge. CatK deficiency resulted in thinner alveolar walls than wild-type littermates on postnatal day (PN) 7. However, no morphological difference could be detected between CatK-deficient and control groups on PN 14. Exposure to 90% oxygen for 7 days after birth caused intensive CatK expression in the bronchial epithelium and alveolar macrophages of wild-type mice. Hyperoxia caused fatal respiratory distress in both groups of mice. However, whereas ∼20% of wild-type mice survived for 2 weeks in hyperoxia, all CatK-deficient mice died within the first 9 postnatal days. Hyperoxia-exposed lungs of CatK-deficient mice contained high number of macrophages and multinucleated giant cells and had increased content of reduced glutathione, indicating intensified pulmonary oxidative stress. These results suggest that CatK is involved in pulmonary development and it may be an important host-defence protease in the oxygen-stressed newborn lung.

Regulation of chemerin chemoattractant and antibacterial activity by human cysteine cathepsins.

Chemerin, a ligand for the G-protein coupled receptor chemokine-like receptor 1, requires C-terminal proteolytic processing to unleash its chemoattractant activity. Proteolytically processed chemerin selectively attracts specific subsets of immunoregulatory APCs, including chemokine-like receptor 1-positive immature plasmacytoid dendritic cells (pDC). Chemerin is predicted to belong to the structural cathelicidin/cystatin family of proteins composed of antibacterial polypeptide cathelicidins and inhibitors of cysteine proteinases (cystatins). We therefore hypothesized that chemerin may interact directly with cysteine proteases, and that it might also function as an antibacterial agent. In this article, we show that chemerin does not inhibit human cysteine proteases, but rather is a new substrate for cathepsin (cat) K and L. cat K- and L-cleaved chemerin triggered robust migration of human blood-derived pDC ex vivo. Furthermore, cat K- and L-truncated chemerin also displayed antibacterial activity against Enterobacteriaceae. Cathepsins may therefore contribute to host defense by activating chemerin to directly inhibit bacterial growth and to recruit pDC to sites of infection.

Effect of the release from mechanical stress on osteoclastogenesis in RAW264.7 cells.

The effects of mechanical stress release on osteoclastogenesis may be as important as those of mechanical stress application. However, the direct effects of mechanical stress on the behavior of osteoclasts has not been thoroughly investigated and there is limited information on the results of the release from mechanical stress. In this study, the effects of mechanical stress application and its release on osteoclast differentiation were examined. The number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts derived from RAW264.7 cells were measured and the expression of osteoclast differentiation genes, which was altered in response to the release from mechanical stress according to the Flexercell tension system was evaluated by real-time PCR. Osteoclast differentiation and fusion were suppressed by mechanical stress application and were rapidly induced after mechanical stress release. The mRNA expression of the osteoclast specific genes, TRAP, matrix metalloproteinase-9 (MMP-9), cathepsin-K (cath-k), calcitonin receptor (CTR), ATPase H+ transporting vacuolar proton pump member I (ATP6i), chloride channel-7 (ClC7) and dendritic cell-specific transmembrane protein (DC-STAMP) was decreased with mechanical stress application, and increased up to 48 h after the release from it. These alterations in gene mRNA expression were associated with the number of osteoclasts and large osteoclasts. Inducible nitric oxide synthetase (iNOS) mRNA was increased with mechanical stress and decreased after its release. Nitric oxide (NO) production was increased with mechanical stress. Nuclear factor of activated T cells cytoplasmic (NFATc) family mRNAs were not altered with mechanical stress, but were up-regulated up to 48 h after the release from it. These findings indicate that the suppression of osteoclast differentiation and fusion induced by mechanical stress is the result of NO increase via iNOS, and that the promotion of osteoclast differentiation and fusion after the release from mechanical stress is related to the NFATc family genes, whose expression remained constant during mechanical stress but was up-regulated after the release from mechanical stress.