Differentiation of rat lens epithelial cells in tissue culture. II. Effects of cytochalasins B and D on actin organization and differentiation

Cytochalasins B and D were used to investigate the involvement of microfilaments in the differentiation of rat lens epithelial cells in tissue culture. Two questions were asked: (1) Does the organization of microfilaments change upon morphological differentiation of the lens epithelial cell? (2) Is the change in the organization of microfilaments required for the production of the differentiation-specific protein, γ-crystallin? Cytochalasin B arborized differentiating lens epithelial cells and had no effect on the undifferentiated cells. Immunofluorescent staining of these two types of cells revealed significant differences in the organization of actin. Actin appeared as longitudinal filaments in the differentiating cells, while it appeared in a diffuse nonfibrillar form in the undifferentiated cells. This indicated changes in the organization of actin during differentiation. Cytochalasin B caused a decline in cell number at 10^?6–10^?5M. However, only that concentration which caused arborization of cells and disruption of microfilaments (10^?5M) inhibited morphological differentiation and production of γ-crystallin. Cytochalasin D (10^?7–10^?5M) did not cause a dramatic decrease in cell number; nevertheless, it induced the arborization of cells and disruption of microfilaments at lower concentrations (10^?7–10^?6M) and inhibited morphological differentiation and production of γ-crystallin at lower concentrations (10^?7–10^?6M) than did cytochalasin B. Thus, only those concentrations of cytochalasins which disrupt microfilaments and prevent their organization into filamentous form seem to inhibit differentiation. This suggests that the organization of actin is required for the program of differentiation of the lens epithelial cells.     

Human articular cartilage and chondrocytes produce hemopoietic colony-stimulating factors in culture in response to IL-1.

The hemopoietic CSF, granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF), are cytokines that mediate the clonal proliferation and differentiation of progenitor cells into mature macrophages and/or granulocytes. We have employed an all-human cell culture system, specific ELISA for GM-CSF and G-CSF, and Northern analysis to investigate whether chondrocytes are a potential source of CSF in rheumatoid disease. We report that human rIL-1 stimulated in a dose-dependent manner the production of GM-CSF and G-CSF by human articular cartilage and chondrocyte monolayers in organ and cell culture, respectively. Increased levels of the CSF Ag were detected after 2 to 8 h stimulation with IL-1, and the optimum dose of IL-1 was 10 to 100 U/ml (0.06 to 0.6 nM IL-1 alpha; 0.02 to 0.2 nM IL-1 beta); neither CSF was detectable in nonstimulated cultures nor in IL-1-stimulated cultures treated with actinomycin D or cycloheximide, indicating the requirement for de novo RNA and protein synthesis. The IL-1-mediated increase in GM-CSF could also be inhibited by the corticosteroid, dexamethasone, but not by the cyclo-oxygenase inhibitor, indomethacin. Although having little effect when tested alone, TNF-alpha and lymphotoxin (TNF-beta) could synergize with IL-1 for the production of GM-CSF. Basic fibroblast growth factor, platelet-derived growth factor, and IFN-alpha and IFN-gamma each had no effect on GM-CSF levels. Results obtained by Northern analysis of chondrocyte total RNA reflected those found for the CSF Ag, namely that CSF mRNA levels were elevated in response to IL-1, but not TNF, and that there was synergy between these two cytokines. We propose that chondrocyte CSF production in response to IL-1, and the concurrent destruction of cartilage by IL-1, could provide a mechanism for the chronic nature of rheumatoid disease.     

Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro

We studied the effect of vitamin A and its analogues (retinoids) on the clonal growth in vitro of normal human myeloid progenitor cells. Normal human bone marrow cells were cultured in soft gel in the presence of a source of colony-stimulating factor (CSF), and various retinoids, and the number of granulocyte-macrophage colonies (CFU-GM) were scored. The addition of 3 × 10^?8 to 3 × 10^?6 M retinoic acid to culture plates containing CSF markedly increased the number of myeloid colonies as compared with culture plates containing CSF alone. Maximal stimulation occurred at a concentration of 3 × 10^?7 M retinoic acid which increased the mean number of colonies by 213 ± 8 % (±S.E.) over plates containing CSF alone. Retinal or retinyl acetate was less potent than retinoic acid, and retinol (vitamin A) had no effect on colony growth. Retinoic acid had no direct CSF activity nor did it stimulate CSF production by the cultured bone marrow cells. Our studies show for the first time that retinoids can stimulate granulopoiesis in vitro and we suggest that this stimulation may be mediated by increased responsiveness of the granulocyte-macrophage progenitors to the action of CSF.     

Inhibition of glucose utilization for acetylcholine synthesis by cerebrospinal fluid.

Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [¹⁴C]ACh formation from [U-¹⁴C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [¹⁴C]ACh synthesis and ¹⁴CO₂ production from [U-¹⁴C]glucose but not from [2-¹⁴C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting ¹⁴CO₂ production from [U-¹⁴C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [¹⁴C]ACh synthesis and ¹⁴CO₂ production.     

Stimulation of oxidative metabolism of granulocytes by recombinant granulocyte-macrophage-colony-stimulating-factor and a conditioned medium of a urinary bladder carcinoma cell line

Neutrophils (PMN) are the major host defence cells protecting the body against invasion by microorganisms. Products of oxidative metabolism mediate PMN microbicidal and tumoricidal activity, but the mechanisms by which these pathways become activated are not well understood. The colony stimulating factors (CSF) are known to stimulate proliferation and differentiation of committed bone marrow stem cells. These regulators may probably play an important role in non specific resistance to infections. We studied the oxidative metabolism of neutrophils after stimulation with recombinant GM-CSF (r.GM-CSF) and the concentrated conditioned medium of the UBC-5637 cell line (UBC-CM) showing CSF activity. It could be demonstrated that the r.GM-CSF, as well as the UBC-CM, induce an activation of the neutrophil respiratory burst without any cofactors such as f-MLP, PMA, or zymosan. In addition, we observed an increase of the response to those stimulants in the presence of either r.GM-CSF or UBC-CM. These effects were not endotoxin-induced, since stimulation persisted after addition of Polymyxin B, which is known to inhibit the action of endotoxins.     

Granulocyte‐Macrophage Colony‐Stimulating Factor (GM‐CSF) Controls the Proliferation and Differentiation of Mouse Epidermal Melanocytes from Pigmented Spots Induced by Ultraviolet Radiation B

Repeated exposure of ultraviolet radiation B (UVB) on the dorsal skin of hairless mice induces the development of pigmented spots long after its cessation. The proliferation and differentiation of epidermal melanocytes in UVB‐induced pigmented spots are greatly increased, and those effects are regulated by keratinocytes rather than by melanocytes. However, it remains to be resolved what factor(s) derived from keratinocytes are involved in regulating the proliferation and differentiation of epidermal melanocytes. In this study, primary melanoblasts (c. 80%) and melanocytes (c. 20%) derived from epidermal cell suspensions of mouse skin were cultured in a basic fibroblast growth factor‐free medium supplemented with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF induced the proliferation and differentiation of melanocytes in those keratinocyte‐depleted cultures. Moreover, an antibody to GM‐CSF inhibited the proliferation of melanoblasts and melanocytes from epidermal cell suspensions derived from the pigmented spots of UV‐irradiated mice, but not from control mice. Further, the GM‐CSF antibody inhibited the proliferation and differentiation of melanocytes co‐cultured with keratinocytes derived from UV‐irradiated mice, but not from control mice. The quantity of GM‐CSF secreted from keratinocytes derived from the pigmented spots of UV‐irradiated mice was much greater than that secreted from keratinocytes derived from control mice. Moreover, immunohistochemistry revealed the expression of GM‐CSF in keratinocytes derived from the pigmented spots of skin in UV‐irradiated mice, but not from normal skin in control mice. These results suggest that GM‐CSF is one of the keratinocyte‐derived factors involved in regulating the proliferation and differentiation of mouse epidermal melanocytes from UVB‐induced pigmented spots.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

Activation of macrophage adenylate cyclase by stimulants of the oxidative burst and by arachidonic acid--two distinct mechanisms

The effect of agents stimulating the oxidative burst (OB) in oil-elicited guinea pig peritoneal macrophages (MPs) on cyclic adenosine 3′,5′-monophosphate (cAMP) levels was examined. We found that: (i) Phorbol myristate acetate (PMA), the Ca²⁺ ionophore A23187, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and opsonized zymosan, elevated cAMP levels two- to fivefold; (ii) the biologically inactive PMA analog, 4-O-methyl-PMA, was proportionally less effective than PMA in stimulating cAMP accumulation; (iii) increased levels of cAMP were evident after 10 min of incubation with the stimulants, in the presence of the phosphodiesterase inhibitor 3-isobutyl methylxanthine (IBX); (iv) basal cAMP levels in MPs increased proportionally with the extracellular Ca²⁺ concentration; (v) the cAMP-elevating effect of all stimulants (with the exception of A23187) was more pronounced in low Ca²⁺ media, associated with lower basal cAMP levels. A23187 did not elevate cAMP levels in the absence of extracellular Ca²⁺; (vi) short-term incubation of MPs with arachidonic acid and with the arachidonic acid precursor, linoleic acid, induced an increase in the level of cAMP; (vii) the elevations in cAMP levels induced by OB stimulants were enhanced, not blocked, by mepacrine, 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin or aspirin, demonstrating that prostaglandin (PG) synthesis was not involved; (viii) the cAMP-elevating effect of arachidonic and linoleic acids was blocked by ETYA and indomethacin, indicating that it was mediated by PGs. The mechanism by which OB stimulants elevate cAMP levels could not be determined but changes in the cellular level of Ca²⁺ seem to play a pivotal role.     

Inhibition of Macrophage DNA Synthesis by Immunomodulators

Oil-induced guinea pig peritoneal exudate macrophages were found to incorporate actively [³H]thymidine without any tissue fluids such as conditioned medium, lymphokines or inflammatory tissue exudates. The [³H]thymidine incorporation was markedly suppressed by macrophage stimulants such as muramyl dipeptide (MDP) or bacterial lipopolysaccharide (LPS), while glucosamine incorporation was simultaneously increased by these stimulants. The degree of suppression of thymidine incorporation depended on the cell density, the concentrations of the stimulants, and sera or culture media used. The exposure of macrophages to MDP for 30 min was sufficient to cause significant suppression.     

The regulation of haemopoiesis in long-term bone marrow cultures: I. Role of L-cell CSF

The effects of L-cell conditioned medium which contains granulocyte/macrophage colony stimulating factor (CSF); of highly purified L-cell CSF; and the antiserum directed against L-cell CSF, have been investigated in long-term murine bone marrow cultures. Treatment of cultures with CSF containing conditioned medium led to a rapid decline in haemopoiesis. However, this inhibition of in vitro haemopoiesis is probably caused by materials other than CSF, since the addition of highly purified L-cell CSF had no appreciable effect upon long-term haemopoietic cell proliferation or differentiation. Furthermore, the inhibitory activity of L-cell conditioned medium was not abrogated following neutralization of the CSF activity by CSF antiserum. The direct addition of CSF antiserum did not inhibit granulocyte or macrophage formation. These results suggest that long-term cultures of murine marrow cells may show extensive interactions with stromal cells which are not influenced by exogenous stimulatory or inhibitory factors.     

Stem cell‐mediated natural tissue engineering

Recently, we demonstrated that a fully differentiated tissue developed on a ventricular septal occluder that had been implanted due to infarct‐related septum rupture. We suggested that this tissue originated from circulating stem cells. The aim of the present study was to evaluate this hypothesis and to investigate the physiological differentiation and transdifferentiation potential of circulating stem cells. We developed an animal model in which a freely floating membrane was inserted into each the left ventricle and the descending aorta. Membranes were removed after pre‐specified intervals of 3 days, and 2, 6 and 12 weeks; the newly developed tissue was evaluated using quantitative RT‐PCR, immunohistochemistry and in situ hybridization. The contribution of stem cells was directly evaluated in another group of animals that were by treated with granulocyte macrophage colony‐stimulating factor (GM‐CSF) early after implantation. We demonstrated the time‐dependent generation of a fully differentiated tissue composed of fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells and new blood vessels. Cells differentiated into early cardiomyocytes on membranes implanted in the left ventricles but not on those implanted in the aortas. Stem cell mobilization with GM‐CSF led to more rapid tissue growth and differentiation. The GM‐CSF effect on cell proliferation outlasted the treat ment period by several weeks. Circulating stem cells contributed to the development of a fully differentiated tissue on membranes placed within the left ventricle or descending aorta under physiological conditions. Early cardiomyocyte generation was identified only on membranes positioned within the left ventricle.     

Decrease of intracellular fluorescein fluorescence polarization (IFFP) in human peripheral blood lymphocytes undergoing stimulation with phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody

In the present study we describe the induction of changes in intracellular fluorescein fluorescence polarization (IFFP) in lymphocytes undergoing activation with a variety of stimulants. These stimulants included the lectins phytohaemagglutinin (PHA), concanavalin (ConA), pokeweed mitogen (PWM) and anti-CD3 antibody. Changes in IFFP were detected in individual cells using the Cellscan apparatus. Our results show that by employing mitogenic concentrations of PHA, as revealed in a [³H]-thymidine incorporation assay, a decrease in the IFFP in human peripheral blood lymphocytes (PBL) occurred within 40 min. ConA and anti-CD3 affected similarly IFFP, whereas PWM, a B lymphocyte lectin, had no effect on IFFP at the concentrations employed. Kinetic analysis revealed that changes in IFFP occurred within 20–40 min after exposure to the stimulants and lasted for 24 h. Our results show that stimulants which activate CD3⁺ lymphocytes caused immediate changes in IFFP, in an enriched population of human PBL. The possible mechanisms involved in IFFP modulation following exposure to selected stimulants are discussed.     

ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages

Differentiation to different types of macrophages determines their distinct functions. Tumor-associated macrophages (TAMs) promote tumorigenesis owing to their proangiogenic and immune-suppressive functions similar to those of alternatively activated (M2) macrophages. We report that reactive oxygen species (ROS) production is critical for macrophage differentiation and that inhibition of superoxide (O²⁻) production specifically blocks the differentiation of M2 macrophages. We found that when monocytes are triggered to differentiate, O²⁻ is generated and is needed for the biphasic ERK activation, which is critical for macrophage differentiation. We demonstrated that ROS elimination by butylated hydroxyanisole (BHA) and other ROS inhibitors blocks macrophage differentiation. However, the inhibitory effect of ROS elimination on macrophage differentiation is overcome when cells are polarized to classically activated (M1), but not M2, macrophages. More importantly, the continuous administration of the ROS inhibitor BHA efficiently blocked the occurrence of TAMs and markedly suppressed tumorigenesis in mouse cancer models. Targeting TAMs by blocking ROS can be a potentially effective method for cancer treatment.     

T-cell hybridoma secreting hemopoietic regulatory molecules: granulocyte-macrophage and eosinophil colony-stimulating factors.

Murine spleen cells stimulated in vitro with pokeweed mitogen were fused with a HAT-sensitive AKR thymoma (BW5147) to produce T-cell hybridomas secreting hemopoietic colony-stimulating factors (CSFs). A stable cloned T-cell hybridoma has been isolated which expressed the H-2 antigens of both fusion parents, has a median chromosome number of 56 and secretes a factor(s) which stimulates the growth of granulocyte-macrophage and eosinophil colonies. The CSF-secreting hybridoma exhibited only the Thy 1.1 associated with the parent tumor, but no markers normally associated with normal T-cells or macrophages were detected. No CSF was secreted by the parent tumor line, but the hybridoma-conditioned medium, when used at 10% (v/v), contained sufficient CSF to stimulate 10–30 colonies per 10⁵ bone marrow cells. Lipopolysaccharide (1 μg/ml) stimulated the production of CSF by the hybridoma cells 3 fold. CSF production also increased when the cells were held at high density in serum-free medium. The colony-stimulating factor(s) secreted by the hybridoma exhibited similar molecular properties to those produced by pokeweed mitogen-stimulated spleen cells, and both the GM- and EO-CSFs had an apparent molecular weight by gel filtration of approximately 35,000.     

Chromosome condensation activity in Rana pipiens eggs matured in vivo and in blastomeres arrested by cytostatic factor (CSF)

The ability of brain nuclei to give rise to condensed chromosomes was studied inRana pipiens eggs which had undergone meiotic maturation in vivo, in blastomeres of two-cell embryos which had been arrested at metaphase by the injection of cytostatic factor (CSF) from mature eggs, and in immature fully grown ovarian oocytes with and without prior CSF injection. Chromosomes from brain nuclei were found to condense within 4 h in mature eggs and this chromosome condensation activity was enhanced by the chelation of free Ca²⁺ in the nuclear isolation medium. Chromosomes also condensed in CSF-arrested blastomeres whether they were placed in the blastomere 30 min before the CSF injection or as long as 22 h after the CSF. Both the Ca²⁺-sensitive CSF, 1CSF, and the Ca²⁺-insensitive CSF, 2CSF, resulted in chromosome condensation within arrested blastomeres. The condensation was accompanied by the formation of multipolar spindles and asters. However, it was found that cytoplasm in CSF-arrested blastomeres does not arrest mitosis at metaphase when transferred into a cleaving blastomere. Other experiments demonstrated that chromosome condensation does not occur in ovarian oocytes even when supplied with CSF. The results are interpreted as indicating that CSF does not directly bring about chromosome condensation, but arrests the cell cycle at metaphase and stabilizes the cytoplasmic conditions of metaphase which, in turn, induce chromosome condensation in foreign nuclei as well as spindle and aster formation.     

Production of Strigolactones by Arabidopsis thaliana responsible for Orobanche aegyptiaca seed germination

The germination stimulants produced by Arabidopsis thaliana, a host of root parasitic plants Orobanche spp. but not of arbuscular mycorrhizal (AM) fungi were examined. Root exudates from the hydroponically grown A. thaliana plants were subjected to reverse phase high performance liquid chromatography (HPLC) and retention times of germination stimulants inducing O. aegyptiaca seed germination were compared with those of strigolactone standards. In addition, the root exudates were analyzed by using HPLC linked with tandem mass spectrometry (LC/MS/MS). A. thaliana was found to exude at least three different germination stimulants of which one was identified as orobanchol. This is the first report of strigolactone production by a non-mycotrophic plant. These results together with recent knowledge imply that strigolactones have other unrevealed functions in plant growth and development.     

Differential effect of hybrid resistance on the localization of virus-immune effector T cells to spleen and brain

The hybrid resistance (Hr) effect operates in the lymphocytic choriomeningitis (LCM) in vivo transfer model to inhibit both the level of cytotoxicity T lymphocyte (CTL) generation in spleen and the induction of inflammation in cerebrospinal fluid (CSF). The effect is seen when LCM virus-immune T cells that are homozygous for H-2D ^b are injected into virus-infected, immunosuppressed recipients that are heterozygous for this allele, or into radiation chimeras that express an appropriate F₁ phenotype. Evidence that Hr to T -cell transfer is cell-dose-dependent and tends to diminish with age was found in both chimeric and normal F₁ mice. Inhibition of the capacity of injected T cells to cause meningitis is a more sensitive measure of Hr than is the further stimulation of CTL effectors in recipient lymphoid tissue. The injection of large numbers of H-2^b virus-immune T cells into (H-2 ^k X H-2 ^bF₁H-2 ^k) virus-infected recipients did not induce any cellular extravasation into CSF, though potent H-2^b-restricted CTL effectors were generated in recipient spleen. Evidence of minimal inflammatory process was found in one experiment where these chimeras were given a comparable dose of (H-2 ^b X H-2 ^d)F₁ immune spleen cells. Development of this Tcell-mediated immunopathological process depends essentially on the expression of the appropriate H-2 restriction element on radiation-resistant host cells which, in this case, presumably constitute part of the physiological barrier between blood and CSF.     

Automated cerebrospinal fluid cytology

OBJECTIVE: To evaluate the Abbott CELL-DYN Sapphire cytometer for cerebrospinal fluid (CSF) cell count and differentiation. METHODS: One hundred three analyses of CSF cells by the CELL-DYN Sapphire were compared with routine cell count and microscopic differentiation and correlation coefficients calculated. RESULTS: The total cell count of both methods correlated well. The detection of erythrocytes was good (0.898), and a higher content of erythrocytes >100/microL had little effect on total leukocyte count. The correlation between both methods was best with higher leukocyte counts >25/microL (r=0.987), whereas at cell counts <25/microL, the correlation was considerably less precise (r=0.613). For the differentiation of cells, lymphocytes and neutrophils showed moderate correlation. The results for monocytes and eosinophils did not correlate. CONCLUSION: The results for the total cell count in this study are comparable with those achieved with the Bayer Advia 120. While the Abbott CELL-DYN Sapphire yielded slightly better results for erythrocytes and total cell count with a higher erythrocyte content, the Advia 120 achieved slightly better results of lymphocyte and neutrophil count in a previous study.     

The role of calcium release activated calcium channels in osteoclast differentiation

Osteoclasts are specialized macrophage derivatives that secrete acid and proteinases to mobilize bone for mineral homeostasis, growth, and replacement or repair. Osteoclast differentiation generally requires the monocyte growth factor m‐CSF and the TNF‐family cytokine RANKL, although differentiation is regulated by many other cytokines and by intracellular signals, including Ca²⁺. Studies of osteoclast differentiation in vitro were performed using human monocytic precursors stimulated with m‐CSF and RANKL, revealing significant loss in both the expression and function of the required components of store‐operated Ca²⁺ entry over the course of osteoclast differentiation. However, inhibition of CRAC using either the pharmacological agent 3,4‐dichloropropioanilide (DCPA) or by knockdown of Orai1 severely inhibited formation of multinucleated osteoclasts. In contrast, no effect of CRAC channel inhibition was observed on expression of the osteoclast protein tartrate resistant acid phosphatase (TRAP). Our findings suggest that despite the fact that they are down‐regulated during osteoclast differentiation, CRAC channels are required for cell fusion, a late event in osteoclast differentiation. Since osteoclasts cannot function properly without multinucleation, selective CRAC inhibitors may have utility in management of hyperresorptive states. J. Cell. Physiol. 226: 1082–1089, 2011. © 2010 Wiley‐Liss, Inc.