Deficiency of Chemokine Receptor CCR1 Causes Osteopenia Due to Impaired Functions of Osteoclasts and Osteoblasts

Chemokines are characterized by the homing activity of leukocytes to targeted inflammation sites. Recent research indicates that chemokines play more divergent roles in various phases of pathogenesis as well as immune reactions. The chemokine receptor, CCR1, and its ligands are thought to be involved in inflammatory bone destruction, but their physiological roles in the bone metabolism in vivo have not yet been elucidated. In the present study, we investigated the roles of CCR1 in bone metabolism using CCR1-deficient mice. Ccr1^−/− mice have fewer and thinner trabecular bones and low mineral bone density in cancellous bones. The lack of CCR1 affects the differentiation and function of osteoblasts. Runx2, Atf4, Osteopontin, and Osteonectin were significantly up-regulated in Ccr1^−/− mice despite sustained expression of Osterix and reduced expression of Osteocalcin, suggesting a lower potential for differentiation into mature osteoblasts. In addition, mineralized nodule formation was markedly disrupted in cultured osteoblastic cells isolated from Ccr1^−/− mice. Osteoclastogenesis induced from cultured Ccr1^−/− bone marrow cells yielded fewer and smaller osteoclasts due to the abrogated cell-fusion. Ccr1^−/− osteoclasts exerted no osteolytic activity concomitant with reduced expressions of Rank and its downstream targets, implying that the defective osteoclastogenesis is involved in the bone phenotype in Ccr1^−/− mice. The co-culture of wild-type osteoclast precursors with Ccr1^−/− osteoblasts failed to facilitate osteoclastogenesis. This finding is most likely due to a reduction in Rankl expression. These observations suggest that the axis of CCR1 and its ligands are likely to be involved in cross-talk between osteoclasts and osteoblasts by modulating the RANK-RANKL-mediated interaction.     

Ultrastructural localization of phosphatases in the testes of the domestic fowl: Acid phosphatase and thiamine pyrophosphatase

Summary ACPase and TPPase activity has been examined in the germinal epithelium of the testes in the domestic fowl. ACPase activity in spermatogonia and spermatocytes was confined to the Golgi complex. In spermatids ACPase activity was seen in the endoplasmic reticulum and nuclear envelope in the phase I and especially in the phase II (the elongating phase). This activity gradually decreased during the next phase III, and had disappeared in the final phase IV. The membrane body showed ACPase reaction in the small peripheral vacuoles and cisternal structures surrounding large central vacuoles. ACPase was also present in vesicles surrounding the developing tail. Late spermatids showed an abundance of autophagic vacuoles which had a complex array of ACPase positive delimiting membranes. In Sertoli cells ACPase activity was predominant in the lysosomes. TPPase activity was seen in the cisternae of the Golgi complex in spermatogonia and spermatocytes. In spermatids activity was present in the endoplasmic reticulum during the phase II, but it is lost in later stages. The smaller vacuoles and cisternal structures in the membrane body also showed reaction products. According to the present results it is thought likely that the smaller vacuoles and cisternal structures of the membrane body are of endoplasmic reticulum origin. The autophagic vacuoles in spermatids and the lysosomes of Sertoli cells are considered responsible for the degradation of residual bodies cast off by spermatids.     

A new one-step method for the cytochemical localization of ouabain-sensitive,potassium-dependent p-nitrophenylphosphatase activity

Summary A new one-step method for the light and electron microscopic localization of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex is introduced. The incubation medium contains p-nitrophenylphosphate (NPP) as substrate, lead citrate as the capture reagent, and dimethylsulfoxide (DMSO) as an activator. It is usable at the optimal pH of the K-NPPase, which is about pH 9.0 in the presence of 25% DMSO. The effects of fixation, lead concentration, and DMSO on the enzyme activity were studied using rat kidney as a test tissue. The fixation of tissues in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde for 60 min at 0°–4° C preserved 45% of the enzyme activity. In the absence of DMSO, lead citrate (4.0 mM) caused 82% inhibition of the enzyme activity in fixed tissue. However, the addition of DMSO (25%) caused about 3-fold activation of the remaining activity. Cytochemical demonstration of the ouabain-sensitive K-NPPase activity was successfully made by this method at both light and electron microscopic levels.This study was supported partly by grants-in-aid for scientific reasearch from the Ministry of Education, the Japanese Government (Nos. 244016 and 337001)Part of this paper was presented at the 19th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Gifu, November 1–2, 1978 (Mayahara et al. 1979a), and the 35th Annual Meeting of the Japanese Society of Electron Microscopy, held at Takarazuka, May 23–25, 1979 (Mayahara et al. 1979b)     

Modification of calcium fluxes by dimethyl sulfoxide and 2-butoxyethanol in sarcoplasmic reticulum vesicles: a possible mechanism for skeletal muscle relaxation induced by dimethyl sulfoxide

In order to investigate the mechanism of skeletal muscle relaxation induced by dimethyl sulfoxide, 2-butoxyethanol and dimethyl sulfoxide were examined for their effects on 1) Ca2+ uptake into and efflux from sarcoplasmic reticulum vesicles prepared from rabbit fast skeletal muscle and crayfish tail muscle by the murexide method, 2) ATPase activities of rabbit reticulum vesicles, 3) the isolated phrenic nerve-diaphragm preparation of the rat and 4) crayfish opener muscle preparation. Ca2+ efflux rate from rabbit reticulum vesicles was markedly decreased with increasing concentrations (5-20% v/v) of dimethyl sulfoxide without affecting the maximum Ca2+ uptake by the reticulum. 2-Butoxyethanol showed quite contrary effects. Dimethyl sulfoxide strongly inhibited the activity of basal ATPase rather than of Ca2+-dependent ATPase. 2-Butoxyethanol did not significantly inhibit the activity of basal ATPase, but markedly increased Ca2+-dependent ATPase activity. Antagonisms between dimethyl sulfoxide and caffeine were demonstrated either in contractions of crayfish opener muscles or in the Ca2+ release from crayfish sarcoplasmic reticulum vesicles. These results indicate a possibility that dimethyl sulfoxide reversibly induces skeletal muscle relaxation mainly in the sarcoplasmic reticulum by means of decreasing the rate and the amount of Ca2+ release from the reticulum.     

Ultracytochemical localization of ouabain-sensitive,potassium-dependent p-nitrophenylphosphatase activity in the lacrimal gland of the rat

The electron-microscopic localization of ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na - K-ATPase complex was studied in the exorbital lacrimal gland of the untreated rat with the use of a newly developed one-step lead-citrate method (Mayahara and Ogawa 1980; Mayahara et al. 1980). In the rat lacrimal gland fixed for 15 min in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde, an electron-dense reaction product was observed on the plasma membrane of the basal infoldings and the lateral interdigitations of the ductal cells. The most intense reaction product - and thus the major site of the Na - K-ATPase activity - was evident on the basolateral membranes of the cells of the large interlobular ducts; a weak reaction was seen on the basolateral, extensively folded plasma membranes of the small intercalated ducts; no reaction product was observed on the plasma membranes of the acinar cells. Addition of 1) 10 mM ouabain, 2) p-chloromercuri-phenyl-sulfonic acid (PCMB-S), 3) elimination of K-ions from the incubation medium, or 4) preheating abolished completely the K-NPPase reaction. The activity was also substrate-dependent. Mg-ATPase-activity was observed not only in the basolateral membranes of all ductal cells but also in the basal part of the acinar cells and on the walls of blood vessels. This reaction was neither inhibited by ouabain nor activated by K-ions. The precipitate of the Mg-ATPase-activity was localized at the extracellular side of the plasma membrane, whereas the K-NPPase-reaction product was restricted to the cytoplasmic side of the plasmalemma. In contrast, non-specific alkaline-phosphatase (ALPase) activity was missing in cells of the large interlobular ducts, but obvious on the apical plasmalemma of cells lining the small intercalated ducts. With respect to its localization and reactivity pattern the activity of the K-NPPase (member of the Na - K-ATase complex) differs markedly from the Mg-ATPase- and ALPase-activity.     

An ultrastructural study of morphogenesis of fibrogranular complex and centriole in ductuli efferentes of Chinese hamster.

An ultrastructural study of ciliated epithelial cells in the ductuli efferentes of young and adult hamsters has revealed that these cells possess dense granules, dense granule clusters, dense bodies and fibrogranular complexes as reservoirs or precursors for ciliogenesis. The dense granules are first seen in the centrosomal region. Later, many dense granules and dense granule clusters appear in the apical portion of the epithelial cells where, subsequently, dense bodies are also found. Finally, the fibrogranular complexes are formed in adults. Morphological evidence strongly suggests that cilia are formed from diplosomal centrioles, de novo centrioles, dense body centrioles, and fibrogranular complex centrioles. Ciliogenesis begins in the fourth day after birth and increases rapidly in the fifth day. After the sixth day, cilia appear to be generated mostly from dense bodies and the total ciliogenesis activities gradually decrease as the animal ages.     

Ultracytochemical studies of capillary endothelial cells in the rat central nervous system

The ultracytochemical localization of eight hydrolytic enzymes (TMPase, 5'-NPase, TPPase, TTPase, Mg++-ATPase, Ca++-ATPase, ALPase and K+-NPPase) and one oxidative enzyme (MAO) was determined in rat brain capillary endothelial cells. In the somal plasma membrane, the enzymatic activity was mainly located in the antiluminal plasma membrane. This finding was appropriate for enzymes possessing the optimal pH at alkaline ranges, except for alkaline phosphatase. Most enzymes investigated showed a positive reaction on the pinocytotic vesicles of capillary endothelial cells. Differences in the intensity of the enzyme activities of the luminal and antiluminal plasma membranes may reflect the polarity in the capillary endothelial cells and relate to blood-brain barrier mechanisms.     

The new lead citrate method for the ultracytochemical demonstration of activity of non-specific alkaline phosphatase (orthophosphoric monoester phosphohydrolase)

Summary The new method, utilizing lead citrate as capture reagent, for the ultracytochemical demonstration of non-specific alkaline phosphatase activity at high alkaline ranges of pH was introduced. Results obtained by the new lead citrate method in various tissues of the rat were presented and discussed in connection with observations made by other methods. The method can be used at the light microscopic level also.Supported by grants from the U.S. Public Health Service MH 12269-01, the Jane Coffin Childs Memorial Fund for Medical Research (Project No. 196), the Anna Fuller Fund and the Department of Education, the Japanese Government (Sogokenkyu UDC 611.015.3:611-018:576.311).     

Optimal compressive force induces bone formation via increasing bone sialoprotein and prostaglandin E(2) production appropriately

Although orthodontic tooth movement can promote bone formation, the molecular mechanism that underlies this phenomenon is not fully understood. The purposes of this study were to determine how mechanical stress affects the osteogenic response of human osteoblastic cells (Saos-2), and also examine the optimal compression for osteogenesis in vitro. Saos-2 cells cultured with or without continuously compressive force (0.5 approximately 3.0 g/cm(2)). The expression of bone sialoprotein (BSP), osteopontin, and cyclooxygenase-2 (COX-2) were measured using real-time PCR, Western blot analysis and immunoassay. The calcium content in the mineralized nodules was determined using Calcium C-Test kit. Only one loading with 1.0 g/cm(2) of compressive force significantly increased the expression of BSP mRNA and protein, COX-2 mRNA expression and PGE(2) synthesis. Indomethacin, an inhibitor of PGE(2) synthesis, inhibited the compression-induced above phenomenon. Moreover, the conditioned medium from 1.0 g/cm(2) of compressive force apparently stimulated calcium content in mineralized nodules. This study demonstrates that an optimal compressive force stimulates in vitro mineralization by BSP synthesis through the autocrin action of PGE(2) production.