Constitutive cholesterol-dependent endocytosis of melanocortin-4 receptor (MC4R) is essential to maintain receptor responsiveness to α-melanocyte-stimulating hormone (α-MSH).

Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus where it controls feeding behavior. MC4R cycles constitutively and is internalized at the same rate in the presence or absence of stimulation by the agonist, melanocyte-stimulating hormone (α-MSH). This is different from other G-protein-coupled receptors, such as ß(2)-adrenergic receptor (ß(2)AR), which internalizes more rapidly in response to agonist stimulation. Here, it is found that in immortalized neuronal Neuro2A cells expressing exogenous receptors, constitutive endocytosis of MC4R and agonist-dependent internalization of ß(2)AR were equally sensitive to clathrin depletion. Inhibition of MC4R endocytosis by clathrin depletion decreased the number of receptors at the cell surface that were responsive to the agonist, α-MSH, by 75%. Mild membrane cholesterol depletion also inhibited constitutive endocytosis of MC4R by ∼5-fold, while not affecting recycling of MC4R or agonist-dependent internalization of ß(2)AR. Reduced cholesterol did not change the MC4R dose-response curve to α-MSH, but it decreased the amount of cAMP generated per receptor number indicating that a population of MC4R at the cell surface becomes nonfunctional. The loss of MC4R function increased over time (25-50%) and was partially reversed by mutations at putative phosphorylation sites (T312A and S329A). This was reproduced in hypothalamic GT1-7 cells expressing endogenous MC4R. The data indicate that constitutive endocytosis of MC4R is clathrin- and cholesterol-dependent. MC4R endocytosis is required to maintain MC4R responsiveness to α-MSH by constantly eliminating from the plasma membrane a pool of receptors modified at Thr-312 and Ser-329 that have to be cycled to the endosomal compartment to regain function.

Sparc localizes to the blebs of hobit cells and human primary osteoblasts.

Secreted protein acidic and rich in cystein (SPARC) is a secreted glycoprotein involved in several biological processes such as tissue remodeling, embryonic development, cell/extracellular matrix interactions, and cell migration. In particular, SPARC affects bone remodeling through the regulation of both differentiation/survival of osteoblasts and bone extracellular matrix synthesis/turnover. Here, we investigated SPARC subcellular localization in the human osteoblastic HOBIT cell line by immunocytochemistry and western blot analysis. We show that, under normal exponential cell growth conditions, SPARC localized both to cell nucleus and to cytoplasm, with no co-localization on actin stress fibers. However, in colchicine-treated HOBIT cells and human primary osteoblasts undergoing blebs formation, SPARC showed a different cellular distribution, with an additional marked compartmentalization inside the blebs, where it co-localized with globular actin and actin-binding proteins such as alpha-actinin, cortactin, and vinculin. Moreover, we demonstrate by an in vitro assay that the addition of SPARC to actin and alpha-actinin inhibited the formation of cross-linked actin filaments and disrupted newly formed filaments, most likely due to a direct interaction between SPARC and alpha-actinin, as indicated by immunoprecipitation assay. The specific silencing of SPARC RNA expression markedly decreased the ability of colchicine-treated HOBIT cells to undergo blebbing, suggesting a direct role for SPARC in cell morphology dynamics during cytoskeletal reorganization.

Breast adenocarcinoma MCF-7 cell line induces spontaneous osteoclastogenesis via a RANK-ligand-dependent pathway.

The metastasis of breast cancer to the skeleton is a serious clinical problem resulting in hypercalcemia, bone fragility and insurmountable pain. The invasion of bony tissue by neoplastic cells usually very rapidly affects the balance between bone apposition and bone resorption. In order to elucidate a mechanism for cancer-induced osteoclastogenesis, cells from a human breast cancer line, MCF-7, were directly co-cultured with murine monocytes RAW 264.7 type CRL 2278. Compared with controls, co-culture of MCF-7 induced differentiation of multinucleated cells by membrane-bound and soluble receptor activator of NF-kB ligand (RANKL) as quantified by ELISA, Western blot analysis, transmission electron microscopy (TEM), and immunocytochemistry. The aim of this study was to determine an in vitro model system of MCF-7 human breast cancer cells grown together with monocytes to show that expression of RANKL promotes osteoclastogenesis, which may indicate a mechanism for the development of osteolytic lesions in breast cancer bone metastasis.

Effects of neridronic acid on osteoclasts derived by physiological dual-cell cultures.

Increased osteoclastic activity is observed in many osteopathic disorders - including postmenopausal osteoporosis, Paget's disease, primary bone tumours, lytic bone metastases, multiple myeloma and rheumatoid arthritis - that involve increased bone resorption and a loss of bone mass. Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect the osteoclasts. The aim of this study was to obtain more information about the mechanism of action of bisphosphonates such as neridronic acid using a dual-cell culture model. As a model of osteoclastogenesis we used a murine monocyte/macrophage cell line RAW 264.7 type CRL 2278 co-cultured with murine osteoblasts. The monocyte-osteoblast system allows physiological experimentation of bone anti-resorption drugs, simulating bone turnover in pathologies such as osteoporosis. The direct actions of neridronic acid on cell proliferation and functionality in the co-culture model were examined using tartrate-resistant acid phosphatase (TRAP) assay, immunohistochemical localization of actin, and transmission and scanning electron microscopy (SEM). Results showed that the percentage of TRAP-positive cells, an early marker of osteoclastic differentiation, was significantly higher in control cultures than in co-cultures treated with variable concentrations of neridronic acid. Neridronic acid induced dramatic morphological changes, characterized by the loss of the ruffled border. The actin ring associated with the plasma membrane of the cells treated with neridronic acid was shown to break down. The tissue-specific targeting of neridronic acid to bone mineral suggests that it may inhibit bone resorption by direct effects on osteoclasts or other bone cells in the immediate microenvironment of the osteoclasts. From our study, we conclude that structural alterations induced by neridronic acid in our co-culture system lead to decreased osteoclast function. This may encourage the use of neridronic acid to reduce bone resorption in the therapy of demineralizing metabolic bone disorders.

Morphological features of osteoclasts derived from a co-culture system.

The interaction between the receptor activator of NfKB (RANK) and its ligand receptor activator of NfKB ligand (RANKL) has recently been proven to be pivotal for osteoclast differentiation and activation. The influence of RANK-RANKL signaling on osteoclast formation was established by co-culturing murine osteoblasts (type CRL-12257) and murine mononuclear monocytes (RAW 264.7). The aim of the present study was to examine, by means of morphological techniques, the interaction between these two cell lines grown in the absolute absence of exogenous cytokines and other stimulating factors. Moreover, we wanted to show that our model could provide a system to analyze the bone resorption process. Mineralized matrix induced morphological changes of osteoclasts (OC) by the formation of organized ruffled-border and a large number of secondary lysosomal vesicles. On the contrary, OC grown on glass coverslips without dentin showed no organized ruffled border or secondary lysosomes. The study of the relationship between these two cell types could establish new approaches for a potential pharmacological control of these cell types and tissues in health and disease.

Immunolocalisation of bilitranslocase in mucosecretory and parietal cells of the rat gastric mucosa.

Bilitranslocase is a plasma membrane carrier localised at the vascular pole of the rat liver cell, where it mediates uptake of organic anions from the blood into the liver. This carrier is also present in the epithelium of the rat gastric mucosa, with similar molecular mass and functional properties. An immunohistochemical study reveals that both the mucus-secreting cells of the gastric pit and the H+-secreting parietal cells express bilitranslocase. These data point to a possible role of bilitranslocase and of its food-borne substrates (anthocyanins and nicotinic acid) in regulating the function and the permeability of the gastric mucosa.

Binding of elements of protein kinase C-alpha regulatory domain to lamin B1.

Previous results from our laboratory have demonstrated that lamin B1 is a protein kinase C (PKC)-binding protein. Here, we have identified the regions of PKC-alpha that are important for this binding. By means of overlay assays and fusion proteins made of glutathione-S-transferase (GST) fused to elements of the regulatory domain of rat PKC-alpha, we have established that binding occurs through both the V1 region and a portion of the C2 region (i.e., the calcium-dependent lipid binding [CaLB] domain) of the kinase. In particular, we have found that amino acids 200-217 of the CaLB domain are essential for binding lamin B1, as a synthetic peptide corresponding to this stretch of amino acids prevented the interaction between the CaLB domain of PKC-alpha and lamin B1. In agreement with the results of other investigators, we have determined that binding of regulatory elements of PKC-alpha to lamin B1 does not require the presence of cofactors such as PS and Ca(2+). We have also found that the binding site of lamin B1 for PKC-alpha is localized in the carboxyl-terminus of the lamin. Our findings may prove to be important in shedding more light on the mechanisms that regulate PKC functions within the nuclear compartment and may also lead to the synthesis of isozyme-specific pharmacological tools to attenuate or reverse PKC-dependent nuclear signalling pathways important for the pathogenesis of cancer.

Different levels of the neuronal nitric oxide synthase isoform modulate the rate of osteoclastic differentiation of TIB-71 and CRL-2278 RAW 264.7 murine cell clones.

It has been clearly established that osteoclasts, which play a crucial role in bone resorption, differentiate from hematopoietic cells belonging to the monocyte/macrophage lineage in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL). We have here investigated the M-CSF- and RANKL-induced osteoclastic differentiation of two distinct clones of the murine monocytic/macrophagic RAW 264.7 cell line, known as TIB-71 and CRL-2278, the latter cell clone being defective for the expression of the inducible nitric oxide synthase isoform in response to interferon-gamma or lipopolysaccharide. CRL-2278 cells demonstrated a more rapid osteoclastic differentiation than TIB-71 cells, as documented by morphology, tartrate-resistant acid phosphatase positivity, and bone resorption activity. The enhanced osteoclastic differentiation of CRL-2278 was accompanied by a higher rate of cells in the S/G2-M phases of cell cycle as compared to TIB-71. The analysis of nitric oxide synthase (NOS) isoforms clearly demonstrated that only neuronal NOS was detectable at high levels in CRL-2278 but not in TIB cells under all tested conditions. Moreover, the broad inhibitor of NOS activity L-NAME significantly inhibited osteoclastic differentiation of CRL-2278 cells. Altogether, these results demonstrate that a basal constitutive neuronal NOS activity positively affects the RANKL/M-CSF-related osteoclastic differentiation.

Receptor Activator for Nuclear Factor kappa B Ligand (RANKL) as an osteoimmune key regulator in bone physiology and pathology.

The strength and integrity of the human skeleton depends on a delicate equilibrium between bone resorption and bone formation. Bone resorption is an elementary cellular activity in the modelling of the skeleton during growth and development. Later in life a most important physiological process in the skeleton is bone remodelling, which is locally initiated by resorption. During remodelling bone resorption is coupled to new bone formation that ensures renewal of bone with only minor local and temporary bone loss. Cells responsible for bone resorption and subsequent bone formation are the osteoclasts and osteoblasts, respectively. The osteoclast is derived from the pluripotent hematopoietic stem cell, which gives rise to a myeloid stem cell that can further differentiate into megakaryocytes, granulocytes, monocytes/macrophages and osteoclasts. The respective bone resorbing and forming actions of osteoclasts and osteoblasts are finely coupled, so that bone mass remains remarkably stable in a healthy adult. Imbalance between osteoclast and osteoblast activities can arise from a wide variety of hormonal changes or perturbations of inflammatory and growth factors resulting in postmenopausal osteoporosis, Paget's disease, lytic bone metastases, or rheumatoid arthritis, leading to increased bone resorption and crippling bone damage. In view of the critical role of osteoclasts in diverse pathology, there has been immense effort aimed at understanding the biology of this unique cell. The present review is focused on the current knowledge of the mechanisms that regulate the functional links between bone turnover and the immune system helping us to understand the main factors that lead to bone loss observed in osteoporosis, cancer and in rheumatoid arthritis. The aim of this review paper is to consider the key molecular interactions involved in the formation of osteoclast cells in normal and pathological conditions.

Soluble TRAIL could enhance bone destruction acting on Rank-ligand in estrogen-independent human breast cancer cell line MDA-MB-231.

The development and spread of tumors is associated with the ability of malignant cells to avoid detection and subsequent elimination by the immune system, to grow in non-native sites and to avoid programmed or induced cell death. In addition to the well-described role of osteoprotegerin in the regulation of bone turnover, there is an emerging evidence that osteoprotegerin may have an additional function due to its ability to bind and inhibit the members of the tumor necrosis factor (TNF)-superfamily, such as TNF-alpha and TNF-related apoptosis inducing ligand (TRAIL). We have shown that the breast cancer cell line MDA-MB-231 produces a sufficient amount of osteoprotegerin to bind TRAIL, resulting in an upregulation of receptor activator factor kappa B ligand (RANKL) expression. In conclusion, the presence of osteoprotegerin, as secreted by this cell line, acting as a paracrine factor, could affect breast cancer RANKL production inducing an enhancement of osteolysis and the perpetuation of a vicious cycle. A better understanding of the complex tumor cell-host cell interactions in the bone microenvironment, and of the autocrine and paracrine effects of the secreted (from tumor cells) and released (from bone matrix) factors may facilitate development of effective strategies to inhibit disease progression.

Differentiation of activated monocytes into osteoclast-like cells on a hydroxyapatite substrate: an in vitro study.

BACKGROUND: Hydroxyapatite surface coatings of dental implants have been introduced to obtain more rapid and complete osteointegration. A possible complication associated with hydroxyapatite implant surface is the release of particles. Those particles may be phagocytosed by monocytes, the first cells to colonize the inflammatory sites. The activated monocytes produce cytokines that could cause osteoclast activation. METHODOLOGY: In order to establish the biological effect of particles released on monocyte differentiation to an osteoclast phenotype, we have used the murine monocyte/macrophage cell line, RAW 264.7 clone CRL-2278 cultured on a hydroxyapatite substrate. The direct action of hydroxyapatite on monocyte differentiation was examined using tartrate-resistant acid phosphatase (TRAP), immunohistochemistry and transmission electron microscopy (TEM) and Western Blot analysis. RESULTS: The present study demonstrated that hydroxyapatite substrate might be able to induce a self-production of RANKL cytokine that directly stimulates a different behaviour in terms of phenotype expression from monocyte/macrophage lineage to mature and functional osteoclasts without the addition of exogenous factors. CONCLUSIONS: These studies were designed to test a model in which osteoclasts could be formed from HA-activated monocytes via positive feedback elicited by RANKL, allowing for identification of innovative targets for therapeutic approaches.

Sequestration of mutated alpha1-antitrypsin into inclusion bodies is a cell-protective mechanism to maintain endoplasmic reticulum function.

A variant alpha1-antitrypsin with E342K mutation has a high tendency to form intracellular polymers, and it is associated with liver disease. In the hepatocytes of individuals carrying the mutation, alpha1-antitrypsin localizes both to the endoplasmic reticulum (ER) and to membrane-surrounded inclusion bodies (IBs). It is unclear whether the IBs contribute to cell toxicity or whether they are protective to the cell. We found that in hepatoma cells, mutated alpha1-antitrypsin exited the ER and accumulated in IBs that were negative for autophagosomal and lysosomal markers, and contained several ER components, but not calnexin. Mutated alpha1-antitrypsin induced IBs also in neuroendocrine cells, showing that formation of these organelles is not cell type specific. In the presence of IBs, ER function was largely maintained. Increased levels of calnexin, but not of protein disulfide isomerase, inhibited formation of IBs and lead to retention of mutated alpha1-antitrypsin in the ER. In hepatoma cells, shift of mutated alpha1-antitrypsin localization to the ER by calnexin overexpression lead to cell shrinkage, ER stress, and impairment of the secretory pathway at the ER level. We conclude that segregation of mutated alpha1-antitrypsin from the ER to the IBs is a protective cell response to maintain a functional secretory pathway.

Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons.

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.

Rabphilin localizes with the cell actin cytoskeleton and stimulates association of granules with F-actin cross-linked by {alpha}-actinin.

In endocrine cell, granules accumulate within an F-actin-rich region below the plasma membrane. The mechanisms involved in this process are largely unknown. Rabphilin is a cytosolic protein that is expressed in neurons and neuroendocrine cells and binds with high affinity to members of the Rab3 family of GTPases localized to synaptic vesicles and dense core granules. Rabphilin also interacts with alpha-actinin, a protein that cross-links F-actin into bundles and networks and associates with the granule membrane. Here we asked whether rabphilin, in addition to its granule localization, also interacts with the cell actin cytoskeleton. Immunofluorescence and immunoelectron microscopy show that rabphilin localizes to the sub-plasmalemmal actin cytoskeleton both in neuroendocrine and unspecialized cells. By using purified components, it is found that association of rabphilin with F-actin is dependent on added alpha-actinin. In an in vitro assay, granules, unlike endosomes or mitochondria, associate with F-actin cross-linked by alpha-actinin. Rabphilin is shown to stimulate this process. Rabphilin enhances by approximately 8-fold the granule ability to localize within regions of elevated concentration of cross-linked F-actin. These results suggest that rabphilin, by interacting with alpha-actinin, organizes the cell cytoskeleton to facilitate granule localization within F-actin-rich regions.

Morphological observations and morphometric analysis in three human fetuses with bilateral cervical cystic hygroma.

Three human fetuses (crown-rump length, CRL, ranging from 71 to 77 mm), presenting bilateral cervical cystic hygroma were examined. The specimens were cleared and double-stained with alcian blue and alizarin red S for detecting the ossification growth patterns in the vertebral column, ribs, ischium, limbs, and face. Longitudinal measurements of some long bones in the upper (humerus, ulna, radius) and lower (femur, tibia, fibula) limb were taken. The values of both the total length (TL) and the ossified part (OL) of each long bone, as well as the OL/TL per cent ratio were considered. Reference points were located on the mandible, i.e. condylar process (Pcl), coronoid process (Pco), gnathion (GN), gonion (GO), inferior interdental point (IDI) for measuring linear dimensions. All values obtained were related with those relative to a group of fetuses, without any detectable malformation and chromosomal abnormalities, with CRL mean value 75 mm, in order to assess the presence of further anomalies, besides the cystic hygroma, in the three fetuses considered.

Expression of protein kinase C (PKC) alpha, delta, epsilon, zeta in primary chick chondrocyte cultures: immunocytochemical study.

PKC is a family of 12 serine/threonine isoenzymes that plays a pivotal role in signal transduction in a large number of biological processes. In the present work we have investigated the expression of PKC (alpha, delta, epsilon, zeta) in chick chondrocyte primary cultures at different differentiation times, i.e. at 48, 55, 62 and 69 days after cell collection from tibiae of 6-day old chick embryos. We would also detect cell differentiation stages towards the osteoblast-like cell phenotype by observing the immunocytochemical expression of the specific osteoblast marker, type I collagen. At the considered culture times, cells exhibited immunocytochemical positivity for type I collagen, thus showing their differentiation towards the osteoblast-like phenotype. PKC-zeta was the isoenzyme that exhibited the most relevant immunocytochemical expression in all considered culture times, whereas PKC-epsilon always less expressed in comparison to the other PKC-isoforms. No relevant differences were observed for the immunocytochemical expressions of PKC-alpha and PKC-delta. On the basis of the immunocytochemical data obtained from the present investigation, we could affirm that PKC-alpha, -delta, -epsilon, and -zeta may play peculiar roles in the differentiation process of chick chondrocytes towards the osteoblast-like cell phenotype.

Tumour necrosis factor-related apoptosis-inducing ligand sequentially activates pro-survival and pro-apoptotic pathways in SK-N-MC neuronal cells.

The SK-N-MC neuroblastoma cell line, which expresses surface tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors TRAIL-R2 and TRAIL-R4, was used as a model system to examine the effect of TRAIL on key intracellular pathways involved in the control of neuronal cell survival and apoptosis. TRAIL induced distinct short-term (1-60 min) and long-term (3-24 h) effects on the protein kinase B (PKB)/Akt (Akt), extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), nuclear factor kappa B (NF-kappaB) and caspase pathways. TRAIL rapidly (from 20 min) induced the phosphorylation of Akt and ERK, but not of c-Jun NH2-terminal kinase (JNK). Moreover, TRAIL increased CREB phosphorylation and phospho-CREB DNA binding activity in a phosphatidylinositol 3-kinase (PI 3K)/Akt-dependent manner. At later time points (from 3 to 6 h onwards) TRAIL induced a progressive degradation of inhibitor of kappaB (IkappaB)beta and IkappaBepsilon, but not IkappaBalpha, coupled to the nuclear translocation of NF-kappaB and an increase in its DNA binding activity. In the same time frame, TRAIL started to activate caspase-8 and caspase-3, and to induce apoptosis. Remarkably, caspase-dependent cleavage of NF-kappaB family members as well as of Akt and CREB proteins, but not of ERK, became prominent at 24 h, a time point coincident with the peak of caspase-dependent apoptosis.

Immunochemical and immunocytochemical expression of protein kinase c isoenzymes alpha, delta, epsilon and zeta in primary adherent cultures of chick chondrocytes.

The family of protein kinase C (PKC) comprises serine/threonine isoenzymes involved in various biological processes, including cell proliferation and differentiation. On the bases of previous investigations performed by us on the expression of various PKC isoforms in the endochondral ossification process of the vertebral column, the aim of the present work was to investigate the expression of various PKC-isoenzymes in chick primary chondrocyte cultures i.e. the most used chondrocyte culture model in vitro. Immunochemical and immunocytochemical experiments were performed to detect the expression of PKC-alpha, -delta, -epsilon and -zeta. Chondrocyte cultures were examined two weeks after cell collection from tibiae of 6-day old chick embryos. By means of morphological observations associated with the immunocytochemical expression of type II collagen, two different cell phenotypes were identified, i.e. fibroblast-like and polygonal-roundish-shaped cells. As far as PKC-isoenzyme expression was concerned, PKC-zeta revealed a stronger immunochemical and immunocytochemical expression; PKC-alpha exhibited a positivity less marked than PKC-zeta, whereas PKC-delta and -epsilon were less expressed in this culture stage. It is reasonable that a major role could be played by PKC-alpha and -zeta in this phase of the chondrogenic process, whereas PKC-delta and -epsilon could be involved in different stages of chondrocyte differentiation in vitro.