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Background and purpose Adiponectin (APN) is an adipokine secreted from adipocytes that binds to APN receptors AdipoR1 and AdipoR2 and exerts an anti-inflammatory response through mechanisms not fully understood. There is a need to develop small molecules that activate AdipoR1 and AdipoR2 and to be used to inhibit the inflammatory response in lipopolysaccharide (LPS)-induced endotoxemia and other inflammatory disorders. Experimental approach We designed 10 new structural analogues of an AdipoR agonist, AdipoRon (APR), and assessed their anti-inflammatory properties. Bone marrow-derived macrophages (BMMs) and peritoneal macrophages (PEMs) were isolated from mice. Levels of pro-inflammatory cytokines were measured by reverse transcription and real-time quantitative polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA) and microarray in LPS-induced endotoxemia mice and diet-induced obesity (DIO) mice in which systemic inflammation prevails. Western blotting, immunohistochemistry (IHC), siRNA interference and immunoprecipitation were used to detect signalling pathways. Key results A novel APN receptor agonist named adipo anti-inflammation agonist (AdipoAI) strongly suppresses inflammation in DIO and endotoxemia mice, as well as in cultured macrophages. We also found that AdipoAI attenuated the association of AdipoR1 and APPL1 via myeloid differentiation marker 88 (MyD88) signalling, thus inhibiting activation of nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK) and c-Maf pathways and limiting the production of pro-inflammatory cytokines in LPS-induced macrophages. Conclusion and implications AdipoAI is a promising alternative therapeutic approach to APN and APR to suppress inflammation in LPS-induced endotoxemia and other inflammatory disorders via distinct signalling pathways.

Objective In this study, we aimed to investigate the therapeutic potential of miR-335-5p lipidoid nanocomplexes coated on Titanium (Ti) SLActive surface by lyophilization. Design In our model, we coated miR-335-5p/Lipidoid nanoparticles on titanium implant, seeded GFP-labelled mouse bone marrow stromal cells (BMSCs) onto the functionalized Ti implant surface, and analyzed the transfection efficiency, cell adhesion, proliferation, and osteogenic activity of the bone-implant interface. Results The Ti SLActive surface displayed a suitable hydrophilicity ability and provided a large surface area for miRNA loading, enabling spatial retention of the miRNAs within the nanopores until cellular delivery. We demonstrated a high transfection efficiency of miR-335-5p lipidoid nanoparticles in BMSCs seeded onto the Ti SLActive surface, even after 14 days. Alkaline phosphatase (ALP) activity and cell vitality were significantly increased in BMSCs transfected with miR-335-5p at 7 and 14 days as opposed to cells transfected with negative controls. When miR-335-5p transfected BMSCs were induced to undergo osteogenic differentiation, we detected increased mRNA expression of osteogenic markers including Alkaline phosphatase (ALP), collagen I (COL1), osteocalcin (OCN) and bone sialoprotein (BSP) at 7 and 14 days as compared with negative controls. Conclusion MiR-335-5p lipidoid nanoparticles could be used as a new cost-effective methodology to increase the osteogenic capacity of biomedical Ti implants.

MicroRNAs (miRNAs) and the Wnt signaling pathway play critical roles in regulating bone development and homeostasis. Our previous study revealed high expression of miR-335-5p in osteoblasts and hypertrophic chondrocytes in mouse embryos and the ability of miR-335-5p to promote osteogenic differentiation by downregulating Wnt antagonist Dickkopf-1 (DKK1). The purpose of this study was to investigate the effects of miR-335-5p constitutive overexpression on bone formation and regeneration in vivo. To that end, we generated a transgenic mouse line specifically over-expressing miR-335-5p in osteoblasts lineage by the osterix promoter and characterized its bone phenotype. Bone histomorphometry and μCT analysis revealed higher bone mass and increased parameters of bone formation in transgenic mice than in wild-type littermates. Increased bone mass in transgenic mice bones also correlated with enhanced expression of osteogenic differentiation markers. Upon osteogenic induction, BMSCs isolated from transgenic mice displayed higher mRNA expression of osteogenic markers than wild-type mice BMSCs cultures. Protein expression of Runx2 and Osx was also upregulated in BMSC cultures of transgenic mice upon osteogenic induction, while that of DKK1 was down-regulated. Most importantly, BMSCs from transgenic mice were able to repair craniofacial bone defects as demonstrated by μCT analysis, H&E staining and OCN immunohistochemistry of newly formed bone in defects treated with BMSCs. Taken together, our results demonstrate constitutive overexpression of miR-335-5p driven by an osterix promoter in the osteoblast lineage induces osteogenic differentiation and bone formation in mice and support the potential application of miR-335-5p-modified BMSCs in craniofacial bone regeneration. This article is protected by copyright. All rights reserved

Background and objective Periodontitis is a prevalent oral disease responsible for tooth loss. MicroRNAs have been proven crucial in bone disorders over the past decades. Promotive effect on osteogenic activities by microRNA-335-5p (miR-335-5p) has been well demonstrated, but its role involved in the pathogenesis of periodontitis remains elusive. In this study, we established experimental periodontitis (EP) on transgenic mice overexpressing miR-335-5p (335-Tg) to investigate the novel effects of miR-335-5p on periodontal inflammation and bone loss. Methods Experimental periodontitis was established via ligation. The expression of inflammatory and osteoclastic genes was examined by quantitative real-time PCR (qPCR). Morphology of alveolar bone was analyzed by microcomputed tomography (μCT). Hematoxylin and eosin (H&E), tartrate-resistant acid phosphatase (TRAP), and Toll-like receptor 4 (TLR4) immunohistochemistry (IHC) staining were conducted for histological analysis. Results The expression of miR-335-5p decreased significantly in the periodontal tissues of EP. Compared to the WT-EP group, μCT analysis showed less bone loss in the 335-Tg-EP group accompanying with a decreased number of TRAP-positive osteoclasts. H&E and IHC staining exhibited attenuated inflammation and TLR4 expression in the 335-Tg-EP group. Furthermore, reduced expressions of IL-1β, IL-6, TNF-α, and TLR4 were also detected in the 335-Tg-EP group. Overexpression of miR-335-5p in vivo weakened the periodontal bone destruction and inflammation compared with the WT-EP group. Conclusions Our data exhibit novel roles of miR-335-5p in preventing bone loss and inflammation in experimental periodontitis.

Central adiponectin (APN) in either the globular (gAPN) or full-length (fAPN) forms decrease sympathetic tone and increase trabecular bone mass in mice through the hypothalamus. It is known that cannabinoid type-1 (CB1) receptors are expressed in the hypothalamic ventromedial nucleus (VMN) and participate in energy metabolism by controlling sympathetic activity. However, whether central APN could influence endocannabinoid signaling through CB1 receptor to regulate bone metabolism has not been characterized. Here we demonstrate that gAPN down-regulated CB1 expression in embryonic mouse hypothalamus N1 cells in vitro. gAPN intracerebroventricular (icv) infusions also decreased hypothalamic CB1 expression and bone formation parameters in APN-knockout (APN-KO) and wild-type (WT) mice. Most importantly, mice pretreated with icv infusions with the CB1 receptor agonist arachidonyl-2′-chloroethylamine (ACEA) or antagonist rimonabant attenuated or enhanced respectively central APN induction of bone formation. We then investigated whether epigenetic signaling mechanisms were involved in the downregulation of hypothalamic CB1 expression by gAPN. We found gAPN enhanced expression levels of various histone deacetylases (HDACs), especially HDAC5. Furthermore, chromatin immunoprecipitation assays revealed HDAC5 bound to the transcriptional start site TSS2 region of the CB1 promoter. In summary, our study identified a possible novel central APN-HDAC5-CB1 signaling mechanism that promotes peripheral bone formation through epigenetic regulation of hypothalamic CB1 expression.

Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. JMJD3, a JmjC family histone demethylase necessary for M2 polarization is also required for effective induction of multiple M1 genes by lipopolysaccharide (LPS). However, the effects of JMJD3 to inflammation in the context of obesity remains unknown. To address this deficiency, we firstly examined the expression of JMJD3 in macrophage isolated from bone marrow and adipose tissue of diet induced obesity (DIO) mice. The results indicated that JMJD3 was down-regulated in obesity. Adiponectin (APN), a factor secreted by adipose tissue which is down-regulated in obesity, functions to switch macrophage polarization from M1 to M2, thereby attenuating chronic inflammation. Intriguingly, our results indicated that APN contributed to JMJD3 up-regulation, reduced macrophage infiltration in obese adipose tissue, and abolished the up-regulation of JMJD3 in peritoneal macrophages isolated from DIO mice when challenged with Porphyromonas gingivalis LPS (pg.lps). To elucidate the interaction of APN and JMJD3 involved in macrophage transformation in the context of inflammation, we designed the loss and gain-function experiments of APN in vivo with APN(-/-) mice with experimental periodontitis and in vitro with macrophage isolated from APN(-/-) mice. For the first time, we found that APN can help to reduce periodontitis-related bone loss, modulate JMJD3 and IRF4 expression, and macrophage infiltration. Therefore, it can be inferred that APN may contribute to anti-inflammation macrophage polarization by regulating JMJD3 expression, which provides a basis for macrophage-centered epigenetic therapeutic strategies.

Specific microRNAs (miRs) and the Wnt signaling pathway play critical roles in regulating bone development and homeostasis . Our previous studies revealed the ability of miR-335-5p to promote osteogenic differentiation by downregulating Wnt antagonist Dickkopf-1 (DKK1). The purpose of this study was to use nano-materials to efficiently deliver miR-335-5p into osteogenic cells for tissue engineering applications. We synthesized and screened a library of 12 candidate nano-lipidoids，of which L8 was identified as the preferred biodegradable lipidoid for miRNA molecule delivery into cells. We then investigated whether a lipidoid-miRNA formulation of miR-335-5-p (LMF-335) could successfully deliver miR-335-5-p into cells to promote osteogenesis in vitro and calvarial bone healing in vivo . Transfection of C3H10T1/2 cells and bone marrow stromal cells (BMSCs) with LMF-335 led to decreased expression of DKK1 and increased expression of the key osteogenic genes. LMF-335 and LMF-335-transfected BMSCs were then used in combination with silk scaffolds to evaluate healing of critical-size calvarial bone defects in mice. The results revealed significant new bone formation in the defects in LMF-335 groups as compared with control groups. In conclusion, this first report supports the notion that lipidoid delivery of miRNA can be used to induce osteogenic differentiation of stem cells and bone regeneration .

Irisin is a polypeptide hormone derived from the proteolytic cleavage of fibronectin-type III domain-containing 5 (FNDC5) protein. Once released to circulation upon exercise or cold exposure, irisin stimulates browning of white adipose tissue (WAT) and uncoupling protein 1 (UCP1) expression, leading to an increase in total body energy expenditure by augmented UCP1-mediated thermogenesis. It is currently unknown whether irisin is secreted by bone upon exercise or whether it regulates bone metabolism in vivo. In this study, we found that 2 weeks of voluntary wheel-running exercise induced high levels of FNDC5 messenger RNA as well as FNDC5/irisin protein expression in murine bone tissues. Increased immunoreactivity due to exercise-induced FNDC5/irisin expression was detected in different regions of exercised femoral bones, including growth plate, trabecular bone, cortical bone, articular cartilage, and bone–tendon interface. Exercise also increased expression of osteogenic markers in bone and that of UCP1 in WAT, and led to bodyweight loss. Irisin intraperitoneal (IP) administration resulted in increased trabecular and cortical bone thickness and osteoblasts numbers, and concurrently induced UCP1 expression in subcutaneous WAT. Lentiviral FNDC5 IP administration increased cortical bone thickness. In vitro studies in bone cells revealed irisin increases osteoblastogenesis and mineralization, and inhibits receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis. Taken together, our findings show that voluntary exercise increases irisin production in bone, and that an increase in circulating irisin levels enhances osteogenesis in mice.

The purpose of this study was to determine the effects of bone sialoprotein (BSP) overexpression in bone metabolism in vivo by using a homozygous transgenic mouse line that constitutively overexpresses mouse BSP cDNA driven by the cytomegalovirus (CMV) promoter. CMV-BSP transgenic (TG) mice and wildtype mice were weighed, and their length, BMD, and trabecular bone volume were measured. Serum levels of RANKL, osteocalcin, osteoprotegerin (OPG), TRACP5b, and PTH were determined. Bone histomorphometry, von Kossa staining, RT-PCR analysis, Western blot, MTS assay, in vitro mineralization assay, and TRACP staining were also performed to delineate phenotypes of this transgenic mouse line. Compared with wildtype mice, adult TG mice exhibit mild dwarfism, lower values of BMD, and lower trabecular bone volume. TG mice serum contained increased calcium levels and decreased PTH levels, whereas the levels of phosphorus and magnesium were within normal limits. TG mice serum also exhibited lower levels of osteoblast differentiation markers and higher levels of markers, indicating osteoclastic activity and bone resorption. H&E staining, TRACP staining, and bone histomorphometry showed that adult TG bones were thinner and the number of giant osteoclasts in TG mice was higher, whereas there were no significant alterations in osteoblast numbers between TG mice and WT mice. Furthermore, the vertical length of the hypertrophic zone in TG mice was slightly enlarged. Moreover, ex vivo experiments indicated that overexpression of BSP decreased osteoblast population and increased osteoclastic activity. Partly because of its effects in enhancing osteoclastic activity and decreasing osteoblast population, BSP overexpression leads to an uncoupling of bone formation and resorption, which in turn results in osteopenia and mild dwarfism in mice. These findings are expected to help the development of therapies to metabolic bone diseases characterized by high serum level of BSP.

In this review, benefits and side-effects of current and emerging therapies to treat and prevent pathological bone loss are described. Bisphosphonates are the antiresorptive compounds most widely used in the treatment of bone-loss associated diseases. They are generally well-tolerated although have recently been associated with osteonecrosis of the jaw and other complications. Therapies modulating estrogen receptor activation are indicated in the prevention and treatment of either breast cancer or osteoporosis in postmenopausal women. Thus, hormone replacement therapy is effective in prevention of osteoporosis, but its long-term use can increase the risk of breast cancer, stroke and embolism. Tamoxifen benefits all stages of breast cancer, but its use may lead to uterine cancer and thromboembolism. Raloxifene is approved in prevention of breast cancer and treatment of postmenopausal osteoporosis, but its use can increase the risk of fatal stroke. Aromatase inhibitors are superior to tamoxifen at advanced stages of disease and as adjuvants, but their use increase fracture incidence. Fulvestrant is as effective as aromatase inhibitors in the treatment of advanced breast cancer and does not cause bone fractures. Another antiresorptive available for the treatment of postmenopausal osteoporosis, Paget's disease and hypercalcemia is calcitonin, which also exhibits analgesic effects. A promising antiresorptive agent currently in clinical trials is denosumab. Aditional therapies for osteoporosis that decrease fracture risk consist of PTH-like anabolic agents and the dual action bone agent strontium ranelate. Antiseptics and antibiotics are used extensively in periodontal disease intervention to target bacterial biofilm, although host-directed therapies are also being developed.

Dlx5 plays an important role in the embryonic development of mineralized tissues. We hypothesized that Dlx5 also functions in regulating post-natal bone formation in mice. To prove this hypothesis, we infected 5-day-old bone sialoprotein (BSP)/avian retroviral receptor gene (TVA) transgenic mice with replication-competent retroviral vectors expressing wild-type Dlx5 (RCAS- Dlx5WT) and mutated Dlx5 at arginine (R) 31 of its homeodomain (RCAS- Dlx5RH). Immunohistochemistry indicated that RCAS- Dlx5WT increased BSP and osteopontin (OPN) expression, whereas it decreased that of osteocalcin (OC). RCAS- Dlx5RH mediated opposite effects. Semi-quantitative RT-PCR confirmed these results. Ex vivo overexpression of RCAS- Dlx5WT in BSP/TVA calvarial cells promoted, whereas that of RCAS- Dlx5RH inhibited, mineralized nodule formation as compared with that in control cells. Our results suggest that Dlx5 promotes expression of early markers of osteogenic differentiation and increases mineralization post-natally.

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice, osteoblast differentiation is impaired, and bone formation is absent. We hypothesized that overexpression of Osx in bone marrow–derived mesenchymal stem cells (BMSCs) would enhance osteogenic differentiation during bone regeneration in vivo. Overexpression of Osx in mouse BMSCs was achieved using retroviral infection together with a green fluorescent protein (GFP) vector to monitor transduction efficiency and determine the source of regenerative cells in implantation studies. Bone regeneration in vivo was evaluated by implanting BMSCs overexpressing Osx into 4-mm calvarial bone defects in adult mice using type I collagen sponge as a carrier. New bone formation in the defects was quantified using radiological and histological procedures 5 weeks after implantation. The results showed that implantation of Osx-transduced BMSCs resulted in 85% healing of calvarial bone defects as detected using radiological analyses. Histological examination of the implants demonstrated that the Osx-transduced group exhibited amounts of newly formed bone that was five times as high as in a group transduced with the empty vector. Immunohistochemistry for GFP showed positive immunoreaction localized to areas of newly engineered bone in the Osx-transduced group. Immunohistochemistry with antibodies against the extracellular matrix protein bone sialoprotein resulted in strong staining in areas of new bone formation. In addition, the clonal BMSCs showed an osteogenic potential similar to that of primary cultures of BMSCs, suggesting the usefulness of this model in bone tissue engineering. These results indicate that ex vivo gene therapy of Osx is a useful therapeutic approach in regenerating adult bone tissue.

Emerging evidence suggests an important role for epigenetic mechanisms in modulating signals during macrophage polarization and inflammation. JMJD3, a JmjC family histone demethylase necessary for M2 polarization is also required for effective induction of multiple M1 genes by lipopolysaccharide (LPS). However, the effects of JMJD3 to inflammation in the context of obesity remains unknown. To address this deficiency, we firstly examined the expression of JMJD3 in macrophage isolated from bone marrow and adipose tissue of diet induced obesity (DIO) mice. The results indicated that JMJD3 was down-regulated in obesity. Adiponectin (APN), a factor secreted by adipose tissue which is down-regulated in obesity, functions to switch macrophage polarization from M1 to M2 (Jenke et al., 2014), thereby attenuating chronic inflammation. Intriguingly, our results indicated that APN contributed to JMJD3 up-regulation, reduced macrophage infiltration in obese adipose tissue, and abolished the up-regulation of JMJD3 in peritoneal macrophages isolated from DIO mice when challenged with Porphyromonas gingivalis LPS (pg.lps). To elucidate the interaction of APN and JMJD3 involved in macrophage transformation in the context of inflammation, we designed the loss and gain-function experiments of APN in vivo with APN−/− mice with experimental periodontitis and in vitro with macrophage isolated from APN −/− mice. For the first time, we found that APN can help to reduce periodontitis-related bone loss, modulate JMJD3 and IRF4 expression and macrophage infiltration. Therefore, it can be inferred that APN may contribute to anti-inflammation macrophage polarization by regulating JMJD3 expression, which provides a basis for macrophage-centered epigenetic therapeutic strategies.

Progressive nerve and brain cell damage associated with Alzheimer's disease, has been attributed to the build-up of aggregated proteins. Detection of amyloid aggregation, through the measuring thei...

Wentworth Institute of Technology is an undergraduate teaching institution that emphasizes experiential learning and career success by implementing Externally-collaborative, Project-based, Interdis...

Osteopontin (OPN) is a non-collagenous extracellular matrix (ECM) protein expressed and secreted by several human cancers. This study investigated the expression pattern of OPN during development of oral squamous-cell carcinoma by using 7,12-dimethylbenz[a]anthracene (DMBA)-induced squamous-cell carcinomas in buccal pouch of syrian golden hamsters. We first identified the hamster OPN cDNA sequence by screening of a hamster calvariae cDNA library with a rat OPN cDNA probe. The resulting 1,449 bp of hamster OPN cDNA led to a deduced protein sequence of 305 amino acids containing several putative binding sites to integrins, CD44 receptors, calcium ions and hydroxyapatite, as well as multiple sites for phosphorylation, glycosylation and sulphation. Hamster OPN cDNA was then used as a probe to analyze the expression of OPN mRNA by Northern blot and in situ hybridization analyses of normal and malignant tissues. OPN mRNA was detected in several non-mineralized tissues as well as in mineralized tissues, but was not present in normal hamster buccal epithelium. DMBA-treated hamster buccal pouches expressed OPN mRNA as early as 4 weeks and displayed the highest level of expression at 15 weeks. The specimens treated with DMBA for 15 weeks exhibited histological features of squamous-cell carcinoma, presented microcrystalline deposits and showed OPN expression associated with malignant epithelium and tumor-associated macrophages. To summarize, our results suggest that buccal-pouch carcinogenesis of Syrian golden hamster may constitute an excellent experimental model to study the mechanisms by which OPN is associated with oral cancer pathogenesis, and to validate OPN-based therapeutic approaches to ameliorate oral cancer progression and metastasis.

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase, bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of alpha1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.

Periodontal disease infection with oral biofilm microorganisms initiates host immune response and signs of periodontitis, including bone resorption. This review delineates some mechanisms underlying the host immune response in periodontal infection and alveolar bone resorption. Activated T lymphocytes have been historically implicated in experimental periodontal bone resorption. An experimental rat adoptive transfer/gingival challenge periodontal disease model has been demonstrated to require antigen-specific T lymphocytes and gingival instillation of antigen and LPS for bone resorption. Interference with costimulatory interactions between T cells and antigen-presenting cells abrogated bone resorption, further emphasizing the significance of immune response in periodontal disease. Receptor activator of nuclear factor kappaB ligand (RANKL), a critical osteoclast differentiation factor, is expressed on T lymphocytes in human periodontal disease as determined by immunohistochemical and confocal microscopic analyses. Interference with RANKL by systemic administration of osteoprotegerin (OPG), the decoy receptor for (and inhibitor of) RANKL, resulted in abrogation of periodontal bone resorption in the rat model. This finding indicated that T cell-mediated bone resorption is RANKL-dependent. In additional experiments, treatment of T cell-transferred rats with kaliotoxin (a scorpion venom potassium channel inhibitor) resulted in decreases in T-cell RANKL expression, diminished induction of RANKL-dependent osteoclastogenesis, and abrogation of bone resorption, implicating an important role of immune response/RANKL expression in osteoclastogenesis/bone resorption. In other experiments, adoptive transfer of antigen-specific, RANKL-expressing B cells, and infection with the antigen-bearing Actinobaccillus actinomycetemcomitans gave rise to periodontal bone resorption, indicating that B cells also have the capacity to mediate bone resorption, probably via RANKL expression. In humans, prominent T lymphocytes have been identified in periodontal disease, and diseased tissues showed elevated RANKL mRNA expression, as well as decreased OPG mRNA expression. Mononuclear cells from periodontal lesions involving T cells and B cells of patients induced osteoclastogenesis in vitro. In summary, a biofilm interface initiates immune cell infiltration, stimulating osteoclastogenesis/bone resorption in periodontal disease. This resorption can be ameliorated by inhibition of RANKL activity or by diminishing immune cell stimulation. These two procedures, if localized, have the potential to lead to the prevention or therapeutic management of periodontal disease and therefore require further study.

Host immune response has been demonstrated to be of considerable importance in oral infections, namely dental caries and periodontal diseases. The molecular pathogenesis of dental caries has been well recognized and evidence indicates that salivary antibodies can modify the course of infection caused by mutans streptococci. The use of specific antigens from mutans streptococci and passive antibody administration has been shown to result in protection from dental caries. Thus, dental caries can be considered a conventional bacterial infectious disease where the major causative organism is recognized. However, periodontal disease is a more specialized type of infectious disease, related to a constellation of oral biofilm microorganisms. These organisms initiate a host immune response and produce symptoms of periodontitis. Antibodies from gingival crevicular fluid have shown the potential to alter the nature of the pathogenic flora. Recent findings suggest that lymphocytes can be the key to bone resorption in the pathogenesis of periodontal disease. Activated lymphocytes that infiltrate the gingival/periodontal tissues upregulate receptor activator of NF-κB ligand (RANKL) and subsequently induce osteoclastogenesis. Interference with antigen-specific T and B cell activation should provide new approaches for amelioration of inflammatory bone resorption in periodontal diseases.

The receptor tyrosine kinase Axl has been shown to be activated by its ligand Gas6 and by oxidative stress in the form of hydrogen peroxide. However, the regulatory mechanisms controlling the levels of Axl upon Gas6 binding or oxidative stress have not been elucidated. This report demonstrates that Gas6-induced downregulation of Axl is blocked by inhibitors of endocytosis and lysosomal degradation, but not by inhibitors of proteosomal activity. Furthermore, it is shown that binding of Axl to Gas6 induces the phosphorylation and ubiquitination of Axl and the interaction of Axl with the ubiquitin ligase c-Cbl. Importantly, hydrogen peroxide induces Axl tyrosine phosphorylation but not its ubiquitination, determining the inhibition of Axl downregulation. These results suggest that as shown for other receptor tyrosine kinases, ubiquitination of Axl is needed to ensure its proper degradation in the lysosome, and that oxidative stress may inhibit Axl ubiquitination and downregulation.

Inflammatory lesions of periodontal disease contain all the cellular components, including abundant activated/memory T- and B-cells, necessary to control immunological interactive networks and to accelerate bone resorption by RANKL-dependent and -independent mechanisms. Blockade of RANKL function has been shown to ameliorate periodontal bone resorption and other osteopenic disorders without affecting inflammation. Development of therapies aimed at decreasing the expression of RANKL and pro-inflammatory cytokines by T-cells constitutes a promising strategy to ameliorate not only bone resorption, but also inflammation. Several reports have demonstrated that the potassium channels Kv1.3 and IKCa1, through the use of selective blockers, play important roles in T-cell-mediated events, including T-cell proliferation and the production of pro-inflammatory cytokines. More recently, a potassium channel-blocker for Kv1.3 has been shown to down-regulate bone resorption by decreasing the ratio of RANKL-to-OPG expression by memory-activated T-cells. In this article, we first summarize the mechanisms by which chronically activated/memory T-cells, in concert with B-cells and macrophages, trigger inflammatory bone resorption. Then, we describe the main structural and functional characteristics of potassium channels Kv1.3 and IKCa1 in some of the cells implicated in periodontal disease progression. Finally, this review elucidates some recent advances in the use of potassium channel-blockers of Kv1.3 and IKCa1 to ameliorate the clinical signs or side-effects of several immunological disorders and to decrease inflammatory bone resorption in periodontal disease. ABBREVIATIONS: AICD, activation-induced cell death; APC, antigen-presenting cells; B(K), large conductance; CRAC, calcium release-activated calcium channels; DC, dendritic cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-γ, interferon-γ; IP3, inositol (1,4,5)-triphosphate; (K)ir, inward rectifier; JNK, c-Jun N-terminal kinase; I(K), intermediate conductance; LPS, lipopolysaccharide; L, ligand; MCSF, macrophage colony-stimulating factor; MHC, major histocompatibility complex; NFAT, nuclear factor of activated T-cells; RANK, receptor activator of nuclear factor-κB; TCM, central memory T-cells; TEM, effector memory T-cells; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; OPG, osteoprotegerin; Omp29, 29-kDa outer membrane protein; PKC, protein kinase C; PLC, phospholipase C; RT-PCR, reverse-transcriptase polymerase chain-reaction; S(K), small conductance; TCR, T-cell receptor; and (K)v, voltage-gated.

Most proliferating cells follow a series of orderly transitions from one phase to another. These transitions are usually controlled by timed degradation of cell cycle regulators by the ubiquitin-proteasome pathway (UPP). There are no published reports regarding the timing of phases of the human lens cell cycle or regarding cell cycle-related changes in UPP components. Objectives of this study were to characterize the timing of the phases of the human lens epithelial cell cycle and to explore potential functions of critical components of the UPP in controlling lens cell cycle. Human lens epithelial cells were synchronized at G0/G1 phase by contact inhibition. Cell cycle progression upon subculturing was monitored by FACS analysis. It took approximately 40 hr for HLEC to complete one cell cycle, approximately 20 hr for G1 phase, approximately 8-10 hr for S phase and approximately 10 hr for the combination of G2 and M phases. Proteasome-dependent degradation of p21WAF and p27Kip, the dominant Cdk inhibitors, was associated with the G1/S phase transition in these cells. Proteasome inhibition experiments indicate that proteolysis is the predominant process which is responsible for the variations in these regulators during the cell cycle. Levels of specific ubiquitin conjugating enzymes, Ubc7 and Ubc10, increased 6 and 2-fold at the G2/M phase and S/G2/M phases, respectively. Levels of these E2s decreased precipitously upon completion of the M phase. In contrast, levels of ubiquitin activating enzyme (E1) and Ubc3 remained constant during the cell cycle. Cul1, a component of the SCF (an E3), remained relatively constant during cell cycle. The up-regulation of Ubc7 and Ubc10 during the G2/M and S/G2/M phases suggests that these enzymes may be involved in controlling the cell cycle progression at this phase. Taken together, the data indicate that expression of key components of the UPP in the human lens epithelial cells is regulated in a cell cycle-dependent manner. Some of the variations in levels of ubiquitin conjugating enzymes are suggestive of previously undescribed functions.

Axl is a receptor tyrosine kinase that is activated by Gas6, a growth factor that belongs to the vitamin K-dependent protein family. Although Gas6 binding to Axl has been shown to transmit mitogenic and/or antiapoptotic signals to a variety of cell types, the role of the Axl-Gas6 system in normal and pathological lens biology is not known. We demonstrate for the first time that Axl protein is expressed in normal rat and bovine lens and that its ligand, Gas6, is present in bovine aqueous humor. In addition, we have detected tyrosine-phosphorylated Axl in normal rat and bovine lens epithelial tissues. We further show that human recombinant Gas6 is able to act as a growth factor in cultured human lens epithelial cells by activating Axl and then the AKT signaling pathway. Gas6 mediates a survival and anti-apoptotic response in cultured human lens epithelial cells subjected to serum-starvation (48-72hr), or treated with transforming growth factor beta1 (5 ng ml(-1), 48hr) or tumor necrosis alpha (100 ng ml(-1), 48hr), as demonstrated by increased number of viable cells, and decreased DNA condensation or caspase-3 activity. In contrast, Gas6 is not able to block apoptosis induced by staurosporin (1microM, 5-24hr) in human lens epithelial cells. Taken together, these data suggest that the Gas6/Axl signaling plays an important role in the control of lens epithelial cell growth and survival and hence in the maintenance of lens homeostasis.

The effects of the potassium channel (Kv1.3) blocker kaliotoxin on T-cell-mediated periodontal bone resorption were examined in rats. Systemic administration of kaliotoxin abrogated the bone resorption in conjunction with decreased RANKL mRNA expression by T-cells in gingival tissue. This study suggests a plausible therapeutic approach for inflammatory bone resorption by targeting Kv1.3. Introduction: Kv1.3 is a critical potassium channel to counterbalance calcium influx at T-cell receptor activation. It is not known if Kv1.3 also regulates RANKL expression by antigen-activated T-cells, and consequently affects in vivo bone resorption mediated by activated T-cells. Materials and Methods:Actinobacillus actinomycetemcomitans 29-kDa outer membrane protein-specific Th1-clone cells were used to evaluate the expression of Kv1.3 (using reverse transcriptase-polymerase chain reaction [RT-PCR] and Western blot analyses) and the effects of the potassium channel blocker kaliotoxin (0–100 nM) on T-cell activation parameters ([3H]thymidine incorporation assays and ELISA) and expression of RANKL and osteoprotegerin (OPG; flow cytometry, Western blot, and RT-PCR analyses). A rat periodontal disease model based on the adoptive transfer of activated 29-kDa outer membrane protein-specific Th1 clone cells was used to analyze the effects of kaliotoxin in T-cell-mediated alveolar bone resorption and RANKL and OPG mRNA expression by gingival T-cells. Stimulated 29-kDa outer membrane protein-specific Th1 clone cells were transferred intravenously on day 0 to all animals used in the study (n = 7 animals per group). Ten micrograms of kaliotoxin were injected subcutaneously twice per day on days 0, 1, 2, and 3, after adoptive transfer of the T-cells. The control group of rats was injected with saline as placebo on the same days as injections for the kaliotoxin-treated group. The MOCP-5 osteoclast precursor cell line was used in co-culture studies with fixed 29-kDa outer membrane protein-specific Th1-clone cells to measure T-cell-derived RANKL-mediated effects on osteoclastogenesis and resorption pit formation assays in vitro. Statistical significance was evaluated by Student's t-test. Results: Kaliotoxin decreased T-cell activation parameters of 29-kDa outer membrane protein-specific Th1 clone cells in vitro and in vivo. Most importantly, kaliotoxin administration resulted in an 84% decrease of the bone resorption induced in the saline-treated control group. T-cells recovered from the gingival tissue of kaliotoxin-treated rats displayed lower ratios of RANKL and OPG mRNA expression than those recovered from the control group. The ratio of RANKL and osteoprotegerin protein expression and induction of RANKL-dependent osteoclastogenesis by the activated T-cells were also markedly decreased after kaliotoxin treatments in vitro. Conclusion: The use of kaliotoxin or other means to block Kv1.3 may constitute a potential intervention therapy to prevent alveolar bone loss in periodontal disease.

Adiponectin is an adipokine that sensitizes the body to insulin. Low levels of adiponectin have been reported in obesity, diabetes and periodontitis. In this study we established experimental periodontitis in male adiponectin knockout and diet-induced obesity mice, a model of obesity and type 2 diabetes, and aimed at evaluating the therapeutic potential of adiponectin. We found that systemic adiponectin infusion reduced alveolar bone loss, osteoclast activity and infiltration of inflammatory cells in both periodontitis mouse models. Furthermore, adiponectin treatment decreased the levels of pro-inflammatory cytokines in white adipose tissue of diet-induced obesity mice with experimental periodontitis. Our in vitro studies also revealed that forkhead box O1, a key transcriptional regulator of energy metabolism, played an important role in the direct signaling of adiponectin in osteoclasts. Thus, adiponectin increased forkhead box O1 mRNA expression and its nuclear protein level in osteoclast-precursor cells undergoing differentiation. Inhibition of c-Jun N-terminal kinase signaling decreased nuclear protein levels of forkhead box O1. Furthermore, over-expression of forkhead box O1 inhibited osteoclastogenesis and led to decreased nuclear levels of nuclear factor of activated T cells c1. Taken together, this study suggests that systemic adiponectin application may constitute a potential intervention therapy to ameliorate type 2 diabetes-associated periodontitis. It also proposes that adiponectin inhibition of osteoclastogenesis involves forkhead box O1.

Adiponectin (APN), the most abundant adipocyte-secreted adipokine, regulates energy homeostasis and exerts well-characterized insulin-sensitizing properties. The peripheral or central effects of APN regulating bone metabolism are beginning to be explored but are still not clearly understood. In the present study, we found that APN-knockout (APN-KO) mice fed a normal diet exhibited decreased trabecular structure and mineralization and increased bone marrow adiposity compared with wild-type (WT) mice. APN intracerebroventricular infusions decreased uncoupling protein 1 (UCP1) expression in brown adipose tissue, epinephrine and norepinephrine serum levels, and osteoclast numbers, whereas osteoblast osteogenic marker expression and trabecular bone mass increased in APN-KO and WT mice. In addition, centrally administered APN increased hypothalamic tryptophan hydroxylase 2 (TPH2), cocaine- and amphetamine-regulated transcript (CART), and 5-hydroxytryptamine (serotonin) receptor 2C (Htr2C) expressions but decreased hypothalamic cannabinoid receptor-1 expression. Treatment of immortalized mouse neurons with APN demonstrated that APN-mediated effects on TPH2, CART, and Htr2C expression levels were abolished by downregulating adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif (APPL)-1 expression. Pharmacological increase in sympathetic activity stimulated adipogenic differentiation of bone marrow stromal cells (BMSC) and reversed APN-induced expression of the lysine-specific demethylases involved in regulating their commitment to the osteoblastic lineage. In conclusion, we found that APN regulates bone metabolism via central and peripheral mechanisms to decrease sympathetic tone, inhibit osteoclastic differentiation, and promote osteoblastic commitment of BMSC.

Adiponectin (APN) is an adipocyte-secreted adipokine that exerts well-characterized antidiabetic properties. Patients with type 2 diabetes (T2D) are characterized by reduced APN levels in circulation and impaired stem cell and progenitor cell mobilization from the bone marrow for tissue repair and remodeling. In this study, we found that APN regulates the mobilization and recruitment of bone marrow-derived mesenchymal stem cells (BMSCs) to participate in tissue repair and regeneration. APN facilitated BMSCs migrating from the bone marrow into the circulation to regenerate bone by regulating stromal cell-derived factor (SDF)−1 in a mouse bone defect model. More importantly, we found that systemic APN infusion ameliorated diabetic mobilopathy of BMSCs, lowered glucose concentration, and promoted bone regeneration in diet-induced obesity mice. In vitro studies allowed us to identify Smad1/5/8 as a novel signaling mediator of APN receptor (AdipoR)−1 in BMSCs and osteoblasts. APN stimulation of MC3T3-E1 osteoblastic cells led to Smad1/5/8 phosphorylation and nuclear localization and increased SDF-1 mRNA expression. Although APN-mediated phosphorylation of Smad1/5/8 occurred independently from adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1, it correlated with the disassembly of protein kinase casein kinase 2 and AdipoR1 in immunoprecipitation experiments. Taken together, this study identified APN as a regulator of BMSCs migration in response to bone injury. Therefore, our findings suggest APN signaling could be a potential therapeutic target to improve bone regeneration and homeostasis, especially in obese and T2D patients. Stem Cells 2015;33:240–252

Melanocyte stimulating hormone (MSH) plays an important role in determining the cutaneous response to ultraviolet radiation and may also influence melanoma progression. We have previously shown that variants of the melanocortin receptor present on melanocytes, MC1R, are associated with sun sensitivity and red hair in a UK population and therefore now consider the gene as a candidate for melanoma susceptibility. We have compared the frequency of known MC1R variants in the second and seventh transmembrane domains in 43 melanoma cases and 44 controls. MC1R variants were more common in cases than controls (chi 2 = 6.75, 1 d.f.; P = 0.0094) with a relative risk to carriers of variant alleles compared with normal homozygotes of 3.91 (95% c.l.: 1.48-10.35), and a population risk attributable to carriers of 34.6% (95% c.i. 10.7-52.1%). The Asp84Glu variant was only present in melanoma cases and appears to be of particular significance. The contribution of variant MC1R alleles was largely independent of skin type. Variants of the MC1R gene are likely to be causally associated with the development of melanoma.

Melatonin was found to have a small inhibitory effect on tyrosinase activity and a slight stimulatory action on dopachrome tautomerase activity in B16 mouse melanoma cells. These effects were time and dose dependent, with the maximal response being observed after 24-48 h treatment and at concentrations of melatonin higher than the physiologic levels of the circulating hormone. Although these effects on the melanogenic activities were modest, incubation of melanocytes with melatonin prior to the addition of the melanotropin mediated a dramatic inhibition of alpha-melanocyte-stimulating-hormone-(alpha-MSH)-induced melanogenesis. This inhibitory effect was evident at melatonin concentrations as low as 10 nM. Inhibition was nearly total at 0.1 mM melatonin, even at high concentrations of alpha-MSH (1 microM). The inhibitory effect of melatonin on alpha-MSH stimulation of melanogenesis was investigated. Melatonin appeared to act at least at two stages. Pharmacological concentrations of melatonin diminished the number of alpha-MSH receptors to about 75% of the control values without an apparent effect on receptor affinity, as determined by receptor-binding studies using 125I-[N-Leu4-D-Phe7]alpha-MSH as a probe. Physiological concentrations of melatonin also appeared to interfere with the intracellular events coupling increased cAMP levels and induction of the c locus tyrosinase, since it strongly inhibited the theophylline-mediated stimulation of melanogenesis. The inhibition of tyrosinase stimulation was higher in the microsomal than in the melanosomal fractions of cells which were treated with melatonin, then exposed to either alpha-MSH (1 microM) or theophylline (1 mM), suggesting that one of the main effects of melatonin might be inhibition of the induction of tyrosinase de novo synthesis.

Mammalian tyrosinase exists in a variety of subcellular locations and maturation states that result from a complex post-translational processing with possible regulatory implications. So far, SDS-PAGE has proven to be the method of choice for the resolution of tyrosinase isoforms. However, the relatively poor sensitivity of the currently available specific activity stain based on incubation of the gels with L-dopa until the formation of melanin has severely limited the use of electrophoresis in regulation studies. Two alternative staining procedures are presented and discussed. The first one involves the fluorographic detection of radioactive melanin after incubation of the gels in the presence of L-[3-14C]-dopa. A similar method has already been used by others (Tsukamoto et al., 1992, Pigment Cell Res. [Suppl.] 2:84-89), but its performance has not yet been compared to the one of the dopa procedure. The sensitivity of this method can be varied by adjusting the isotopic dilution of the tracer and/or the time of exposure of the gel, but it is at least ten times higher than the one of the colorimetric stain. Moreover, the intensity of the bands is proportional to the initial tyrosinase activity over a wide range. Using this procedure, the activity present in the different subcellular fractions of melanocytes in culture can be easily detected. The second procedure involves the formation of a colored adduct between dopaquinone and MBTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Although tyrosinase has been considered for a long time the only enzyme involved in mammalian melanosynthesis, it has been shown that mouse melanoma melanosomes contain high levels of dopachrome tautomerase (DCT2), an enzyme catalyzing DC tautomerization to DHICA. At least in B16 mouse melanoma, DCT is present in higher catalytic amounts than tyrosinase. Moreover, it can be anticipated that tyrosinase and DCT should be very difficult to resolve by most conventional biochemical techniques because of the structural similarity between these enzymes, as predicted from the sequence of their corresponding cDNAs. It is shown that the presence of DCT can cause serious artifacts when tyrosinase activity is determined by most of the currently available methods, such as the Dopa oxidase and melanin formation assays. We describe a simple and convenient method for the preparation of tyrosinase devoid of DCT. The method takes advantage of the different thermal stability of both enzymes. Heating of crude melanosomal extracts at 60 degrees C for 1 hr results in a complete denaturation of DCT, while tyrosinase activity is recovered almost quantitatively. The resulting tyrosinase preparation is considerably purified and the electrophoretic, immunologic and kinetic characteristics of the enzyme appear unaltered. Because if its high yield and simplicity, the method can be used for the microscale partial purification of DCT-free tyrosinase from mammalian malignant melanocytes grown in culture.

Receptor activator of nuclear factor-kappaB (RANKL)-mediated osteoclastogenesis plays a pivotal role in inflammatory bone resorption. The aim of this study was to identify the cellular source of RANKL in the bone resorptive lesions of periodontal disease. The concentrations of soluble RANKL, but not its decoy receptor osteoprotegerin, measured in diseased tissue homogenates were significantly higher in diseased gingival tissues than in healthy tissues. Double-color confocal microscopic analyses demonstrated less than 20% of both B cells and T cells expressing RANKL in healthy gingival tissues. By contrast, in the abundant mononuclear cells composed of 45% T cells, 50% B cells, and 5% monocytes in diseased gingival tissues, more than 50 and 90% of T cells and B cells, respectively, expressed RANKL. RANKL production by nonlymphoid cells was not distinctly identified. Lymphocytes isolated from gingival tissues of patients induced differentiation of mature osteoclast cells in a RANKL-dependent manner in vitro. However, similarly isolated peripheral blood B and T cells did not induce osteoclast differentiation, unless they were activated in vitro to express RANKL; emphasizing the osteoclastogenic potential of activated RANKL-expressing lymphocytes in periodontal disease tissue. These results suggest that activated T and B cells can be the cellular source of RANKL for bone resorption in periodontal diseased gingival tissue.

There is now consensus that the accumulation of oxidatively modified proteins is cytotoxic and causally related to several age-related diseases, including the amyloid diseases and age-related cataracts. There is also general agreement that proteolytic pathways provide a quality control mechanism to limit accumulation of damaged proteins. Although many researchers assume that the ubiquitin pathway is involved in recognition and proteolytic removal of oxidatively modified proteins, which are produced upon cellular stress, there has been no direct evidence to support this hypothesis. In this work, we used a novel proteolysis-resistant ubiquitin variant to demonstrate that ubiquitin conjugates isolated from oxidatively stressed mammalian cells are enriched 3.3-15-fold for oxidatively modified proteins and that failure to execute ubiquitin-dependent proteolysis renders various cell types more susceptible to oxidative stress-related cytotoxicity. These results were corroborated using several inhibitors of the ubiquitin proteasome pathway, including PS-341, an anticancer drug in clinical use. Taken together the data indicate that the ubiquitin proteolytic pathway recognizes and removes oxidatively modified proteins, and that failure of this system, as occurs upon aging or stress, may be involved in and exacerbate cytotoxicity and age-related syndromes in which accumulation of ubiquitinated and oxidatively modified proteins has an etiologic role.

RANKL and BSP are upregulated in several bone resorptive disorders. However, the mechanisms by which these two factors might induce osteoclastogenesis and bone resorption synergistically under pathological conditions remain largely unknown. Introduction: RANKL and bone sialoprotein II (BSP) have been shown to be upregulated in the serum of individuals with abnormally high osteoclastogenic and bone resorptive activities. Here we provide experimental evidence that RANKL and BSP induce osteoclastogenesis and bone resorption synergistically but mediate opposite effects in osteoclast survival and apoptosis. Materials and Methods: RAW264.7 cells and mouse bone marrow-derived monocytes/macrophages were treated with human recombinant BSP in the presence and absence of RANKL. TRACP stainings, bone resorption assays, Western blotting, immunoprecipitation analyses, and semiquantitative RT-PCR were used to evaluate the effects of BSP in osteoclast differentiation and bone resorption. Survival, DNA condensation, and caspase activity assays were used to determine the putative effects of BSP in osteoclast survival and apoptosis. Results and Conclusions: RANKL induced osteoclast differentiation and bone resorption at a higher extent in the presence than in the absence of BSP in RAW264.7 cells and bone marrow-derived monocytes/macrophages. c-Src-dependent c-Cbl phosphorylation was 8-fold higher in RAW264.7 cells treated with BSP and RANKL than in those treated with RANKL alone. Furthermore, BSP and RANKL activated the master regulator of osteoclastogenesis nuclear factor of activated T cells (NFAT)-2 and increased the mRNA expression of other differentiation markers such as cathepsin K or TRACP. Inhibition of c-Src activity or chelating intracellular calcium inhibited the synergistic effects in bone resorption and the phosphorylation of the c-Src substrate c-Cbl. Inhibition of calcineurin or intracellular calcium elevation inhibited the synergistic effects in osteoclastogenesis and decreased NFAT-2 nuclear levels. On the other hand, BSP and RANKL mediated opposite effects in osteoclast survival and apoptosis. Thus, BSP increased survival and decreased apoptosis markers in differentiated RANKL-treated RAW267.5 cells and RANKL/macrophage-colony stimulating factor (M-CSF)-treated bone marrow-derived monocytes/macrophages. In addition, RAW267.5 cells treated with BSP and RANKL exhibited decreased activation of the proapoptotic Jun N-terminal kinase pathway and increased activation of anti-apoptotic AKT pathway than cells treated with RANKL or BSP alone. Taken together, our findings suggest that BSP contributes to RANKL-mediated bone resorption by inducing osteoclastogenesis and osteoclast survival and decreasing osteoclast apoptosis.

WW domain-containing proteins are found in all eukaryotes and play an important role in the regulation of a wide variety of cellular functions such as protein degradation, transcription, and RNA splicing. The cloning and characterization of a novel human WW domain-containing gene, hWW45, which encodes a protein of approximately 45 kDa consisting of 2 WW domains and a coiled-coil region is reported here. The murine homologue cDNA, mWW45, displays a different 3*-untranslated region and predicts a protein identity of 93% to hWW45. Northern blot and RT-PCR analysis demonstrated that both mWW45 and hWW45 transcripts are ubiquitously expressed in adult tissues. The mouse embryonic expression is first seen at 7 days post coitum in Northern blot analysis of whole embryos. Chromosomal localization by radiation hybrid mapping revealed that hWW45 is localized at chromosome 14, 10.31cR from the marker D14S269. © 2000 Academic Press

Abstract —We have previously demonstrated that the cell-specific expression of Kv1.5 promoter is regulated by a silencer (Kv1.5 repressor element; KRE) containing a dinucleotide-repetitive element, (GT) 19 (GA) 1 (CA) 15 (GA) 16 . Electromobility gel shift assays (EMSAs) of KRE with GH3 nuclear extracts detected a unique DNA-protein complex, which was not detectable in Chinese hamster ovary or COS-7 cells. We further delineated KRE and determined that a 52-bp fragment that contained a (GT) 10 (GA) 1 (CA) 10 dinucleotide-repetitive element was sufficient for silencer activity. EMSAs using nuclear extracts isolated from the heart and from GH3 cells demonstrated that the 52-bp element formed specific and identical gel shift effects. These complexes were not detectable in EMSA experiments with liver nuclear extracts. Magnetic DNA affinity purification and UV cross-linking experiments identified a 27-kDa KRE binding factor (KBF) in GH3 cell nuclear extracts. Purified KBF reacted specifically with double-stranded KRE, abolishing the formation of multimeric KRE-DNA complexes. Thus, the interaction between KRE and KBF may play an important role in regulating the GH3- and cardiac-specific expression of Kv1.5.

Reactive oxygen species (ROS) are potentially cytotoxic and several mechanisms have evolved to protect against their damaging effects. In melanocytes, tyrosinase may have such a role by utilising the superoxide anion (O2-) in the production of melanin. In the present study, we have examined the cytotoxic effects of O2- and hydrogen peroxide (H2O2) in human melanocytes both before and following the activation of tyrosinase. Xanthine oxidase (XO, 5-150 mU.ml-1) and glucose oxidase (GO, 0.1-20 mU.ml-1) were used to generate the O2- and H2O2 respectively, and the cytotoxic effects assessed by measuring cell survival using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay. 3 h later, dose-related decreases in melanocyte survival were seen. Similar experiments with keratinocytes and fibroblasts showed that these cells were more resistant to the cytotoxic effects of O2- than were the melanocytes. The effect of increasing tyrosinase activity was examined by growing the melanocytes in the presence of an analogue of melanocyte-stimulating hormone (MSH) Nle4DPhe7 alpha-MSH (10(-8) M), for 48 h. This increased tyrosinase activity, melanin content, the ability to trap O2- and the resistance of the melanocytes to the cytotoxic effects of this ROS, but failed to alter their susceptibility to the damaging effects of H2O2.Nle4DPhe7 alpha-MSH had no effect on the resistance of keratinocytes and fibroblasts to either O2- or H2O2. After 3 h, XO, as opposed to GO, also increased the melanin content of human melanocytes; this effect was not accompanied by an increase in tyrosinase activity. The present results suggest that tyrosinase may utilise O2- to produce melanin and that this process may protect melanocytes from the potentially damaging effects of this ROS.

Melanin pigmentation protects the skin from the damaging effects of ultraviolet radiation (UVR). There are two types of melanin, the red phaeomelanin and the black eumelanin, both of which are present in human skin. Eumelanin is photoprotective whereas phaeomelanin, because of its potential to generate free radicals in response to UVR, may contribute to UV-induced skin damage. Individuals with red hair have a predominance of phaeomelain in hair and skin and/or a reduced ability to produce eumelanin, which may explain why they fail to tan and are at risk from UVR. In mammals the relative proportions of phaeomelanin and eumelanin are regulated by melanocyte stimulating hormone (MSH), which acts via its receptor (MC1R), on melanocytes, to increase the synthesis of eumelanin and the product of the agouti locus which antagonises this action. In mice, mutations at either the MC1R gene or agouti affect the pattern of melanogenesis resulting in changes in coat colour. We now report the presence of MC1R gene sequence variants in humans. These were found in over 80% of individuals with red hair and/or fair skin that tans poorly but in fewer than 20% of individuals with brown or black hair and in less than 4% of those who showed a good tanning response. Our findings suggest that in humans, as in other mammals, the MC1R is a control point in the regulation of pigmentation phenotype and, more importantly, that variations in this protein are associated with a poor tanning response.

B-16 mouse melanoma melanosomes contain two forms of tyrosinase that can be resolved by SDS/PAGE. These forms interact to different extents with the ion-exchanger DEAE-Sephadex and with hydroxyapatite, and have different affinity for the melanosomal membrane and/or the intraorganular matrix. After partial purification and complete separation of the two tyrosinases, several kinetic parameters were analyzed. The form of lower electrophoretic mobility displayed a higher Km for 3,4-dihydroxy-L-phenylalanine (L-dopa) and L-tyrosine, an absolute requirement for the cofactor L-dopa in its tyrosine hydroxylase activity, and a lower ratio of tyrosine hydroxylation to Dopa oxidation. The form of higher electrophoretic mobility displayed lower values of Km for both substrates and was able to exhibit tyrosine hydroxylase activity after a lag period even in the absence of L-dopa. Both forms were stereospecific for the L isomers and sensitive to the specific tyrosinase inhibitor 2-phenylthiourea. These forms do not appear to result from different degrees of glycosylation, nor from limited proteolysis and are also present in the microsomal fraction of B16 mouse melanoma. They might correspond to different gene products, most likely derived from the b and c loci.

In mouse melanoma melanocytes, alpha-melanocyte-stimulating hormone (MSH) stimulates differentiation, melanin synthesis and tyrosinase activity. However, the molecular mechanisms underlying these events have not yet been characterized. We have studied the activation of tyrosinase by MSH. Treatment of B16 melanoma cells with either theophylline, MSH, or its superpotent analog [Ahx4, DPhe7]MSH promotes a larger induction of tyrosine hydroxylase than of dopa oxidase activity in whole cell extracts. This higher activation of tyrosine hydroxylation was found not only in the melanosomal but also in the microsomal fraction; it appears to be dependent on continued transcription and translation since it can be blocked by actinomycin and cycloheximide. The tyrosinase activity of control and theophylline-treated extracts displayed several kinetic differences, including different Km values for both substrates and requirements for the cofactor L-dopa. SDS/PAGE, followed by a sensitive specific activity stain, demonstrated that melanosomes of control cells contain one lower-electrophoretic-mobility form of tyrosinase, whereas melanosomes of cells treated with either theophylline or MSH display, in addition to the lower-mobility form, a faster-migrating activity band. These tyrosinase forms are not interconvertible by proteolysis or deglycosylation. Their nature is discussed as related to the properties of the previously described low- and high-electrophoretic-mobility tyrosinases (LEMT and HEMT), as well as of the proteins encoded by the c and b loci.

In spite of the central role of tyrosinase in mammalian pigmentation, few data are available on its structure and structure-function relationships based on direct analysis of the protein. A number of reasons have been invoked to account for this situation, including the problems for its purification and its resistance to proteases. However, no study on the effects of proteases on purified tyrosinase has been reported. We have purified the melanosomal and cytosolic tyrosinases from B16 mouse melanoma and analyzed their susceptibility to trypsin digestion. Both isoforms are sensitive to trypsin, and display similar peptide maps and kinetics of proteolysis, suggesting that they are products of the same gene. The peptide maps and the kinetics of appearance of the fragments were consistent with the sequential removal of N-terminal peptides, leading to a core of 55.3 kDa for the melanosomal form and 48.6 kDa for the cytosolic enzyme. This core was apparently resistant to further proteolysis and catalytically inactive. The difference in molecular weight for the core of the cytosolic and melanosomal forms is the same as that calculated for the native isoforms. The kinetics of enzyme inactivation indicate that the tyrosine hydroxylase and Dopa oxidase activities of tyrosinase are lost at the same rate, and should therefore display similar if not identical structural requirements. The results are discussed in terms of the relationship of both isoforms and of the putative protein sequences deduced from the cDNA clones proposed for tyrosinase.

Tyrosinase induction in murine malignant melanocytes by alpha MSH is well known, but its molecular basis has not been characterized. Treatment of B16 melanoma cells with theophylline or alpha MSH mediates a larger induction of tyrosine hydroxylase than of dopa oxidase activity in total cell extracts, and in the melanosomal and microsomal fractions. No evidence for the modulation of a tyrosinase effector was found. SDS-PAGE and specific activity stain demonstrated two forms of tyrosinase, with different degrees of induction by theophylline. These results agree with the recent proposal that two tyrosinases, encoded by different genes, are present in murine melanocytes.