Alterations in osteoclast morphology following osteoprotegerin administration in the magnesium-deficient mouse

In the present study, we used osteoprotegerin (OPG), which blocks osteoclastogenesis, to correct and thus explain the hypercalcemia that is seen during dietary Mg deficiency in the mouse. Control and Mg-deficient mice received injections for 12 days of either OPG or vehicle only. Serum Ca was similar in Mg-deficient mice treated with OPG and in control mice receiving OPG (9.2±0.3 mg/dl vs. 9.2±0.5). Both groups had significantly higher serum Ca than controls or Mg-deficient animals receiving vehicle alone. Surprisingly, Mg-depleted mice that received OPG in doses that inhibit osteoclastic bone resorption remained hypercalcemic. Because mature osteoclasts still present in the marrow might be hyperactive, we examined osteoclast morphology at the light microscopic and ultrastructural level. Light microscopic examination of trabecular bone showed few osteoclasts in OPG-treated mice. Ultrastructural examination revealed that osteoclasts in OPG-treated mice have decreased contact with the endosteal bone surface and absence of a ruffled border. Because the morphology of the existing pool of mature osteoclasts did not enhance resorption, another mechanism, such as increased intestinal absorption of Ca in Mg-deficient mice, likely contributes to the hypercalcemia observed during Mg deficiency.     

Alterations in osteoclast morphology following long-term 17beta-estradiol administration in the mouse

Background  

Although the role of the osteoclast in bone resorption is becoming better understood, much remains to be learned about osteoclastogenesis and the exact mechanism of action of anti-resorbing agents such as 17β-estradiol. This study investigated bone and morphologic osteoclast alterations following long-term estrogen administration to the B6D2F1 mouse. B6D2F1 mice aged 4-5 weeks were exposed to high levels of estrogen via implanted silastic tubing for at least 12 weeks; controls received empty tubing. Femurs of control and treated mice were assessed with radiology, quantitative histomorphometry and transmission electron microscopy.     

Tissue manganese levels and liver pyruvate carboxylase activity in magnesium-deficient rats

To investigate the manganese status in magnesium deficiency, 40 male Wistar rats, 3 wk old, were divided into two groups and fed a magnesium deficient diet or a normal synthetic diet for 2 wk. Dietary magnesium depletion decreased magnesium levels in brain, spinal cord, lung, spleen, kidney, testis, bone, blood, and plasma, while it elevated the magnesium level in liver. In magnesium-depleted rats, calcium concentration was increased in lung, liver, spleen, kidney, and testis, while it was decreased in tibia. In magnesium-depleted rats, manganese concentration was decreased in plasma and all tissues except adrenal glands and blood. Dietary magnesium depletion diminished pyruvate carboxylase (EC 6.4.1.1) activity in the crude mitochondrial fraction of liver. Positive correlation was found between the liver manganese concentration and the pyruvate carboxylase activity. In the magnesium-depleted rats, glucose was decreased while plasma lipids (triglycerides, phospholipids, and total cholesterol) were increased. These results suggest that dietary magnesium deficiency changes manganese metabolism in rats.     

Alterations of tight and gap junctions in mouse hepatocytes following administration of colchicine

Summary Mature columnar cells of the midgut of Cubitermes contain a prominent secretion product observed at light- and electron-microscopic levels. At the ultrastructural level the product is resolved as an electron dense material contained in vesicles up to 1 m diameter that accumulate in the apical cytoplasm. The vesicles are composite, apparently formed by coalescence of at least two types of precursor vesicle both of which originate from the Golgi apparatus. Discharge of the product takes place by exocytosis into intercellular space at or in the vicinity of the apical septate junction complex. Augmentation of apical surface area by microvilli is less prominent in Cubitermes than in other termites for which data are available. This and other evidence suggests that absorptive functions are reduced in the midgut of this insect.     

Detection and characterization of RANK ligand and osteoprotegerin in the thyroid gland

Receptor activator of NF-kappaB (RANK) ligand (RANKL) and osteoprotegerin (OPG) play essential roles in bone metabolism and immune responses. RANKL activates RANK, which is expressed by osteoclasts and dendritic cells (DC), whereas OPG acts as its decoy receptor. The role of RANKL and OPG in thyroid physiology is unclear. Northern analysis revealed pronounced OPG mRNA levels in normal human thyroid. By contrast, RANKL mRNA levels were most abundant in lymph node and appendix, and low in the thyroid. In the human thyroid follicular cell line XTC and in primary human thyroid follicular cells, OPG mRNA levels and protein secretion were upregulated by interleukin (IL)-1beta (33-fold), tumor necrosis factor (TNF)-alpha (eightfold), and thyrotropin (TSH) (threefold). RANKL mRNA was stimulated in XTC by IL-1beta and TNF-alpha, but inhibited by TSH. Conditioned medium harvested from IL-1beta-treated XTC (containing high concentrations of OPG) inhibited RANKL-induced CD40 upregulation and cluster formation of DC. OPG mRNA levels were three times more abundant in surgical thyroid specimens of Graves' disease as compared to other thyroid diseases. Our data suggest that RANKL and OPG are produced in the thyroid gland by thyroid follicular cells, are regulated by cytokines and TSH, and are capable of modulating dendritic cell functions. Thus, these cytokines may represent important local immunoregulatory factors involved in the pathogenesis of autoimmune thyroid diseases.     

Oleanolic acid exerts inhibitory effects on the late stage of osteoclastogenesis and prevents bone loss in osteoprotegerin knockout mice

Postmenopausal women undergo rapid bone loss, which caused by the accelerated osteoclastic bone resorption. Receptor activator of nuclear factor kappa-B ligand (RANKL) plays critical and essential roles on varied stages of osteoclastogenesis. Oleanolic acid (OA), a naturally derived small compound, has been found suppress osteoclastogenesis in early stage of bone marrow macrophages (BMMs). However, whether OA also regulates the late stage of osteoclastogenesis remains unclear. Here, the regulatory effect of OA on the late stage of osteoclastogenesis was investigated in vitro using RANKL-pretreated BMMs and in vivo using osteoprotegerin (OPG) knockout mice. Our in vitro studies demonstrate that OA inhibits the late stage of osteoclastogenesis from RANKL-pretreated BMMs. For in vivo animal investigation, OA attenuates the bone loss phenotypes in OPG-knockout mice by decreasing the densities of osteoclast, which are in consistent with the finding with in vitro osteoclastogenesis. Mechanistic investigations found that OA largely inhibit the activity of c-Fos and Nuclear factor of activated T-cells c1 (NFATc1) with RANKL-pretreated BMMs and OPG-knockout mice. Furthermore, OA suppresses the activities of osteoclast genes, such as Tartrate resistant acid phosphatase (TRAP), CathepsinK (Ctsk), and Matrix metalloproteinase 9 (MMP9). Taken together these findings, they have not only defined an inhibitory effect of OA in the late stage of osteoclastogenesis but have also gained new molecular mechanisms underlying the process of osteoclast formation.     

Assessment of the relationship between hyperalgesia and peripheral inflammation in magnesium-deficient rats

Magnesium-deficient rats develop simultaneously a significant lowering of nociceptive threshold and a generalized inflammation. We investigated the relationship between these two phenomena by testing drugs that are able to suppress the inflammation in this model. In weaning rats fed a magnesium-depleted diet for ten days, the nociceptive threshold was assessed by the paw pressure test and the inflammation by a clinical score. A non-steroidal anti-inflammatory drug (piroxicam); antagonists of H1 and H2 receptors (astemizole and cimetidine. respectively); a glucocorticoid (dexamethasone); an inhibitor of mastocyte degranulation (cromoglycate); and estradiol benzoate were used to block the inflammatory response. Dexamethasone and estradiol significantly suppressed the inflammation (p < 0.001 vs control group). Cromoglycate showed a delayed anti-inflammatory effect (p < 0.01 vs control group on D10). The combination of astemizole and cimetidine partially blocked the inflammation process, whereas astemizole and piroxicam were without effect. Regardless of the effect of the test drugs on inflammation, no change in the time course of hyperalgesia was observed. These data support the view that hyperalgesia induced by the magnesium-depleted diet is not a consequence of the inflammatory process.     

Disruption of the Man-6-P targeting pathway in mice impairs osteoclast secretory lysosome biogenesis

Osteoclasts are specialized cells that secrete lysosomal acid hydrolases at the site of bone resorption, a process critical for skeletal formation and remodeling. However, the cellular mechanism underlying this secretion and the organization of the endo-lysosomal system of osteoclasts have remained unclear. We report that osteoclasts differentiated in vitro from murine bone marrow macrophages contain two types of lysosomes. The major species is a secretory lysosome containing cathepsin K and tartrate-resistant acid phosphatase (TRAP), two hydrolases critical for bone resorption. These secretory lysosomes are shown to fuse with the plasma membrane, allowing the regulated release of acid hydrolases at the site of bone resorption. The other type of lysosome contains cathepsin D, but little cathepsin K or TRAP. Osteoclasts from Gnptab(-/-) (gene encoding GlcNAc-1-phosphotransferase α, β-subunits) mice, which lack a functional mannose 6-phosphate (Man-6-P) targeting pathway, show increased secretion of cathepsin K and TRAP and impaired secretory lysosome formation. However, cathepsin D targeting was intact, showing that osteoclasts have a Man-6-P-independent pathway for selected acid hydrolases.     

Recent advances in understanding the mechanisms of osteoclast precursor fusion

Bone marrow macrophages fuse on the bone surface to form multinucleated osteoclasts that then organize to efficiently resorb bone. Many, if not all, of the stages of macrophage fusion involve cytoskeletal components that reorganize the cells. Recruitment may involve chemotactic responses to bone matrix protein and calcium ion gradients and/or chemokine production by bone forming osteoblasts. The roles of integrins vary, depending on the particular subunits with some interfering with fusion and others having a participatory role. RANKL is essential for fusion and many identified modulators of fusion influence RANKL signaling pathways. Tetraspanins have been implicated in fusion of macrophages and myoblasts, but differences in impacts exist between these two cell types. Macrophage recruitment to apoptotic cells prior to their engulfment is driven by the exposed phospholipids on the external surface of the apoptotic cells and there is evidence that this same identification mechanism is employed in macrophage fusion. Because loss of cadherin or ADAM family members suppresses macrophage fusion, a crucial role for these membrane glycoproteins is evident. The Ig membrane glycoprotein superfamily members CD200 and MFR/SIRPα are involved in macrophage fusion, although their influences are unresolved. Differential screenings have identified the structurally related membrane proteins DC‐STAMP and OC‐STAMP as required components for fusion and the contributions to fusion remain active areas of investigation. While many of the key components involved in these processes have been identified, a great deal of work remains in resolving the precise processes involved and the interactions between key contributors to multinucleated osteoclast formation. J. Cell. Biochem. 110: 1058–1062, 2010. Published 2010 Wiley‐Liss, Inc.     

Convergent metatarsal fusion in jerboas and chickens is mediated by similarities and differences in the patterns of osteoblast and osteoclast activities

In many vertebrate animals that run or leap, the metatarsals and/or metacarpals of the distal limb are fused into a single larger element, likely to resist fracture due to high ground‐reaction forces during locomotion. Although metapodial fusion evolved independently in modern birds, ungulates, and jerboas, the developmental basis has only been explored in chickens, which diverged from the mammalian lineage approximately 300 million years ago. Here, we use a bipedal rodent, the lesser Egyptian jerboa (Jaculus jaculus), to understand the cellular processes of metatarsal fusion in a mammal, and we revisit the developing chicken to assess similarities and differences in the localization of osteoblast and osteoclast activities. In both species, adjacent metatarsals align along flat surfaces, osteoblasts cross the periosteal membrane to unite the three elements in a single circumference, and osteoclasts resorb bone at the interfaces leaving a single marrow cavity. However, the pattern of osteoclast activity differs in each species; osteoclasts are highly localized to resorb bone at the interfaces of neighboring jerboa metatarsals and are distributed throughout the endosteum of chicken metatarsals. Each species, therefore, provides an opportunity to understand mechanisms that pattern osteoblast and osteoclast activities to alter bone shape during development and evolution.     

Alterations of PLCbeta1 in mouse eggs change calcium oscillatory behavior following fertilization

Inositol 1,4,5-trisphosphate generated by the action of a phospholipase C (PLC) mediates release of intracellular Ca²⁺ that is essential for sperm-induced activation of mammalian eggs. Much attention currently focuses on the role of sperm-derived PLCζ in generating changes in egg intracellular Ca²⁺ despite the fact that PLCζ constitutes a very small fraction of the total amount of PLC in a fertilized egg. Eggs express several isoforms of PLC, but a role for an egg-derived PLC in sperm-induced Ca²⁺ oscillations has not been examined. Reducing egg PLCβ1 by a transgenic RNAi approach resulted in a significant decrease in Ca²⁺ transient amplitude, but not duration or frequency, following insemination. Furthermore, overexpressing PLCβ1 by microinjecting a Plcb1 cRNA significantly perturbed the duration and frequency of Ca²⁺ transients and disrupted the characteristic shape of the first transient. These results provide the first evidence for a role of an egg-derived PLC acting in conjunction with a sperm-derived PLCζ in egg activation.     

Changes in the H-alloantigen-recognizing function of mouse lymphoid cells following hydrocortisone administration]

The capacity of spleen, thymus, and bone marrow cells of intact (control) and of hydrocortisone-treated mice CBA to induce the lymph node type of graft-v-host reaction (GVHR) in hybrids F1 (CBA X c57bl) was studied. After hydrocortisone injection (2.5 mg per mouse) the donor spleen cells became more active in GVHR, considering the value of lymph node indices and immunoblast content in the regional lymph node as compared with a control group. Following transplantation of thymus cells taken from the hydrocortison-treated donors the immunoblast count was higher, although the lymph node weight remained the same as in the control group. On the contrary, following the transfer of the bone marrow cells from the hydrocortisone-treated mice the lymph nodes enlarged, while the immunoblast count remained as low as in control. Consequently, exogenously conditioned increase in the hydrocortisone level was accompanied by an enrichment of the spleen and thymus cell populations with T-lymphocytes, proliferating in response to H-alloantigens.     

Proliferative processes in the epithelium of mouse skin allotransplants following cortisone administration]

Proliferative processes were studied in the epithelium of skin grafts from mice of strain CC57 Brown and that of the skin surrounding the grafts in mice of CBA strain. The chart (Fig 1) illustrates cortisone action on normalization of DNA synthesis in the epithelial cells of the allotransplant. Throughout the experiment in operated animals which received no cortisone (Fig. 2, a and 3, a) a high proliferative activity was observed in the recipient's skin epithelium while it was sharply decreased in the epithelium of the allograft. Repeated cortisone injections gave a reverse picture: inhibition of proliferative processes in the recipient's epithelium and their intensification in the epithelium of the allotransplant.     

Alterations in the onset of ovulatory failure and gonadotropin secretion following steroid administration to lightly androgenized female rats

The present studies were designed to characterize the gonadotropin response to exogenous steroids in neonatally androgenized female rats in various states of reproductive decline. Female rats were androgenized by the administration of a single injection of testosterone propionate (TP) (10 or 100 micrograms) at 5 days of age. Control rats received sesame oil. Treatment with 100 micrograms TP resulted in persistent vaginal estrus (PVE) from the onset of vaginal introitus. Treatment with 10 micrograms TP resulted in a period of regular estrous cyclicity followed by PVE. In the first experiment, all animals were ovariectomized between the ages of 60-85 days and the gonadotropin response to exogenously administered estradiol benzoate (EB) (10 micrograms/100 g BW) and progesterone (P) (2 mg/animal) was determined. When testing began 3 days following ovariectomy, control females exhibited significant (P less than 0.01) afternoon elevations of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) following EB, which were further amplified following P. When ovariectomy occurred prior to the onset of PVE (PRE PVE), lightly androgenized females (10 micrograms TP) showed no significant afternoon gonadotropin increase following EB. Following P, phasic LH secretion was present but significantly (P less than 0.01) decreased in amplitude and delayed in onset versus that of control females. When ovariectomy occurred 3 to 4 wk following the onset of PVE, lightly androgenized females (PVE group) as well as fully androgenized females (FAS) (100 micrograms TP) showed no gonadotropin response to steroid priming.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in the interleukin-1/interleukin-1 receptor antagonist balance modulate cardiac remodeling following myocardial infarction in the mouse

Background

Healing after acute myocardial infarction (AMI) is characterized by an intense inflammatory response and increased Interleukin-1 (IL-1) tissue activity. Genetically engineered mice lacking the IL-1 receptor (IL-1R1-/-, not responsive to IL-1) or the IL-1 receptor antagonist (IL-1Ra, enhanced response to IL-1) have an altered IL-1/IL-1Ra balance that we hypothesize modulates infarct healing and cardiac remodeling after AMI.

Methods

IL-1R1-/- and IL-1Ra-/- male mice and their correspondent wild-types (WT) were subjected to permanent coronary artery ligation or sham surgery. Infarct size (trichrome scar size), apoptotic cell death (TUNEL) and left ventricular (LV) dimensions and function (echocardiography) were measured prior to and 7 days after surgery.

Results

When compared with the corresponding WT, IL-1R1-/- mice had significantly smaller infarcts (−25%), less cardiomyocyte apoptosis (−50%), and reduced LV enlargement (LV end-diastolic diameter increase [LVEDD], −20%) and dysfunction (LV ejection fraction [LVEF] decrease, −50%), whereas IL-1Ra-/- mice had significantly larger infarcts (+75%), more apoptosis (5-fold increase), and more severe LV enlargement (LVEDD increase,+30%) and dysfunction (LVEF decrease, +70%)(all P values <0.05).

Conclusions

An imbalance in IL-1/IL-1Ra signaling at the IL-1R1 level modulates the severity of cardiac remodeling after AMI in the mouse, with reduced IL-1R1 signaling providing protection and unopposed IL-1R1 signaling providing harm.