Molecular characterization of two novel isoforms of the human calcitonin receptor.

Calcitonin inhibits bone resorption by acting on osteoclasts via a specific receptor. The calcitonin receptor (CTR) is also found in many other normal and malignant tissues and cell lines. It has been cloned and sequenced in several species including humans. It belongs to a subclass of seven-transmembrane G protein-coupled receptors. Four human CTR (H-CTR) isoforms generated by alternatively spliced mRNA have previously been described. Two H-CTR encoding DNAs containing an unidentified 50-bp insert are now reported from T47D cells. The 50-bp insert corresponds to a DNA region located between exon 9 and exon 10, and appears to originate from an alternative splicing process. The two H-CTR cDNAs encode 274 and 290 aa long isoforms. Both are deleted from the putative fourth transmembrane domain to C-tail. They differ by the presence (H-CTR5) or absence (H-CTR6) of a previously known 16-aa insert in the putative first intracellular loop. Cell- and tissue-distribution analysis using RT-PCR demonstrates that the shorter one, HCTR6, is more prevalent. The mRNA of both isoforms was detected in giant cell tumor, whereas only H-CTR6 mRNA was detected in TT cells and kidney tissue. Neither H-CTR5 nor H-CTR6 could be detected in peripheral blood mononuclear cells cultured in the presence of RANKL, in MCF7 cells, and in cortical brain and ovarian tissues. When H-CTR6 was transiently expressed in HEK293 cells, CT failed to induce production of cAMP or to bind to the receptor. These suggest either an intrinsic loss of ligand binding function, or an altered intracellular trafficking. Our findings therefore indicate the existence of two novel splice variants of the H-CTR and confirm that multiple splicing patterns could be involved in the post-transcriptional regulation of the gene.

The increased bone mass in deltaFosB transgenic mice is independent of circulating leptin levels.

Transgenic mice overexpressing deltaFosB, a naturally occurring splice variant of FosB, develop an osteosclerotic phenotype. The increased bone formation has been shown to be due, at least in part, to autonomous effects of deltaFosB isoforms on cells of the osteoblast lineage. However, abdominal fat and marrow adipocytes are also markedly decreased in deltaFosB mice, leading to low serum leptin levels. Increased bone mass has been linked to the absence of leptin and leptin receptor signaling in ob/ob and db/db mice. Thus, in addition to affecting directly osteoblastogenesis and bone formation, deltaFosB isoforms might increase bone mass indirectly via a decrease in leptin. To test this hypothesis, we restored normal circulating levels of leptin in deltaFosB mice via sc implanted osmotic pumps. Complete histomorphometric analysis demonstrated that trabecular bone volume as well as dynamic parameters of bone formation was unchanged by this treatment in both deltaFosB transgenic mice and control littermates. This demonstration that restoring circulating levels of leptin in deltaFosB transgenic mice failed to rescue the bone phenotype further indicates that the marked increase in bone formation is autonomous to the osteoblast lineage.

Calcitonin induces dephosphorylation of Pyk2 and phosphorylation of focal adhesion kinase in osteoclasts.

Calcitonin induces the association and tyrosine phosphorylation of focal adhesion kinase (FAK), paxillin, and HEF1 in HEK-293 cells that overexpress the calcitonin receptor (C1a-HEK), but the hormone's effect on these adhesion-related proteins in osteoclasts is not known. We therefore studied the effect of calcitonin on the tyrosine phosphorylation and subcellular distribution of paxillin, HEF1, FAK, and Pyk2, a FAK-related tyrosine kinase, in osteoclasts. Osteoclasts expressed both Pyk2 and FAK, with Pyk2 much more highly expressed. The two tyrosine kinases and paxillin were prominently associated with small punctate structures that were most densely clustered in the region of the peripheral F-actin-rich ring. Some of the punctate structures stained either for Pyk2 alone or FAK alone. Treatment with calcitonin disrupted the actin ring and induced the loss of the peripheral staining of paxillin, Pyk2, and FAK. In calcitonin-treated osteoclast-like cells, the tyrosine phosphorylation of paxillin and FAK increased, whereas the tyrosine phosphorylation of Pyk2 decreased. Calcitonin also induced increased phosphorylation of Erk1 and Erk2 in osteoclasts, as it did in the C1a-HEK cells. The unexpected dephosphorylation of Pyk2 correlated with decreased phosphorylation of Tyr(402), the autophosphorylation site of Pyk2. The calcitonin-induced dephosphorylation of Pyk2 was not observed in C1a-HEK cells transfected with Pyk2, suggesting that the reduced phosphorylation seen in osteoclasts may be specific to these cells. Treatment of osteoclast-like cells with 12-phorbol 13-myristate acetate increased the tyrosine phosphorylation of both Pyk2 and FAK, and calphostin C, an inhibitor of protein kinase C, blocked calcitonin-stimulated FAK phosphorylation. Increasing intracellular calcium with ionomycin caused a decrease in the tyrosine phosphorylation of Pyk2 and the loss of the actin ring in a manner similar to the effect of calcitonin. Ionomycin had no effect on FAK tyrosine phosphorylation. Calcitonin (CT)-induced changes in Pyk2, FAK, and Erk1/2 phosphorylation were independent of c-Src.

The Cbl family: ubiquitin ligases regulating signaling by tyrosine kinases.

The Cbl proteins compose a family of ubiquitin ligases that play a central role in the down-regulation of signaling cascades involving receptor and nonreceptor tyrosine kinases. Analysis of the activity of these proteins suggests that they can regulate the signaling process through ubiquitination of the plasma membrane receptors and various downstream signaling components, including the Cbl proteins themselves. Structural analysis of the Cbl proteins shows that, in many instances, they interact with phosphorylated tyrosine residues on their targets. Furthermore, phosphorylation of specific tyrosine residues on the Cbl proteins may provide an additional level of control on the ubiquitinating activity of these proteins.

Calcitonin (CT) rapidly increases NA(+)/H(+) exchange and metabolic acid production: effects mediated selectively by the C1A CT receptor isoform.

Two isoforms of the calcitonin receptor are expressed in rabbit: the common C1a isoform and the calcitonin receptor Delta e13 isoform, which has a deletion in the seventh transmembrane domain. Using microphysiometry, we investigated the effects of calcitonin on proton efflux from HEK293 cells stably transfected with C1a, calcitonin receptor Delta e13, or empty vector. In C1a-expressing cells only, calcitonin rapidly induced a biphasic elevation in proton efflux consisting of an initial transient and a sustained plateau, accompanied by an increase in lactate efflux. Inhibitors of Na(+)/H(+) exchange abolished only the initial transient, whereas removal of extracellular glucose abolished only the sustained plateau. These data suggest that activation of Na(+)/H(+) exchange mediates the initial transient, whereas increased glucose metabolism underlies the sustained plateau. Because both receptor isoforms activate adenylyl cyclase, the lack of effect of calcitonin on proton efflux from calcitonin receptor Delta e13-expressing cells argued against involvement of cAMP in activating proton efflux. Similarly, studies involving elevation or buffering of cytosolic free Ca(2+) concentration argued against involvement of Ca(2+). Activation of PKC mimicked the plateau phase of calcitonin-induced proton efflux from C1a cells, whereas inhibition or depletion of PKC suppressed it. Activation of proton transport and production are novel cellular responses to calcitonin, mediated selectively by the C1a receptor isoform via a mechanism involving PKC.

Carbonic anhydrase II is an AP-1 target gene in osteoclasts.

c-Fos, a member of the AP-1 family of transcription factors, is necessary for osteoclast differentiation but to date, none of the osteoclast-phenotypic markers have been identified as AP-1 target genes. Here, we demonstrate that carbonic anhydrase II (CA II), an enzyme necessary for osteoclast activity, is transcriptionally upregulated by c-Fos/AP-1. A functional AP-1 binding site is present in the CA II promoter and is necessary for this regulation. Furthermore, we show that AP-1 binding activity, mainly composed of Fra-2 and JunD, is induced by treatment of bone marrow cultures with the osteoclastogenic hormone 1,25 dihydroxyvitamin D(3). Fra-2 and JunD are found in mature osteoclasts as well. Thus, our data demonstrate that cFos/AP-1 can directly regulate the expression of this osteoclast marker and that AP-1 activity is upregulated in osteoclast progenitors in response to osteoclastogenic signals.

Cbl associates with Pyk2 and Src to regulate Src kinase activity, alpha(v)beta(3) integrin-mediated signaling, cell adhesion, and osteoclast motility.

The signaling events downstream of integrins that regulate cell attachment and motility are only partially understood. Using osteoclasts and transfected 293 cells, we find that a molecular complex comprising Src, Pyk2, and Cbl functions to regulate cell adhesion and motility. The activation of integrin alpha(v)beta(3) induces the [Ca(2+)](i)-dependent phosphorylation of Pyk2 Y402, its association with Src SH2, Src activation, and the Src SH3-dependent recruitment and phosphorylation of c-Cbl. Furthermore, the PTB domain of Cbl is shown to bind to phosphorylated Tyr-416 in the activation loop of Src, the autophosphorylation site of Src, inhibiting Src kinase activity and integrin-mediated adhesion. Finally, we show that deletion of c Src or c-Cbl leads to a decrease in osteoclast migration. Thus, binding of alpha(v)beta(3) integrin induces the formation of a Pyk2/Src/Cbl complex in which Cbl is a key regulator of Src kinase activity and of cell adhesion and migration. These findings may explain the osteopetrotic phenotype in the Src(-/-) mice.

Integrin engagement, the actin cytoskeleton, and c-Src are required for the calcitonin-induced tyrosine phosphorylation of paxillin and HEF1, but not for calcitonin-induced Erk1/2 phosphorylation.

We have previously shown that in a HEK-293 cell line that overexpresses the C1a isoform of the calcitonin receptor (C1a-HEK), calcitonin induces the tyrosine phosphorylation of the focal adhesion-associated proteins HEF1 (a p130(Cas)-like docking protein), paxillin, and focal adhesion kinase and that it also stimulates the phosphorylation and activation of Erk1 and Erk2. We report here that cell attachment to the extracellular matrix, an intact actin cytoskeleton, and c-Src are absolutely required for the calcitonin-induced phosphorylation of focal adhesion-associated proteins. In contrast to the phosphorylation of paxillin and HEF1 in cells attached to fibronectin-coated dishes, calcitonin failed to stimulate the phosphorylation of paxillin and HEF1 in suspended cells, in cells attached to poly-d-lysine-coated dishes, and in attached cells pretreated with the RGD-containing peptide GRGDS. Overexpression of wild-type c-Src increased calcitonin-induced paxillin and HEF1 phosphorylation, whereas overexpression of kinase-dead Src or Src lacking a functional SH2 domain inhibited the calcitonin-stimulated tyrosine phosphorylation of these proteins. Overexpression of Src lacking the SH3 domain did not affect the calcitonin-induced phosphorylation of paxillin and HEF1. In contrast to the regulation of paxillin and HEF1 phosphorylation, the calcitonin-induced phosphorylation of Erk1 and Erk2 did not appear to involve c-Src and was only partially dependent on cell adhesion to the extracellular matrix and an intact actin cytoskeleton. Furthermore, inhibition of Erk1 and Erk2 phosphorylation had no effect on the calcitonin-induced phosphorylation of paxillin and HEF1. Thus, in C1a-HEK cells, the calcitonin receptor is coupled to the tyrosine phosphorylation of focal adhesion-associated proteins and to Erk1/2 phosphorylation by mechanisms that are in large part independent.

Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7.

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

The presence of the alternatively spliced A2 cassette in the vacuolar H+-ATPase subunit A prevents assembly of the V1 catalytic domain.

Vacuolar ATPases (V-ATPases) are multisubunit enzymes that couple the hydrolysis of ATP to the transport of H+ across membranes, and thus acidify several intracellular compartments and some extracellular spaces. Despite the high degree of genetic and pharmacological homogeneity of V-ATPases, cells differentially modulate the lumenal pH of organelles and, in some cells, V-ATPases are selectively targetted to the plasma membrane. Although the mechanisms underlying such differences are not known, the subunit isoform composition of V-ATPases could contribute to altered assembly, targeting or activity. We previously identified an alternatively spliced variant of the chicken A subunit in which a 30 amino acid cassette (A1) containing the Walker consensus sequence for ATP binding is replaced by a 24 amino acid cassette (A2) that lacks this feature. We have examined the ability of chimeric yeast/chicken A subunits containing either the A1 or the A2 cassette to restore the V-ATPase activity of yeast that lack the A subunit. The A1-containing chimeric subunit, but not the chimera that contains the A2 cassette, partially restores the ability of the mutated yeast to grow at neutral pH. Both chimeric proteins are expressed, although at lower levels than the similarly transfected yeast A subunit. The A2-containing subunit fails to associate with the vacuolar membrane or support the assembly of V-ATPase complexes. Thus, the substitution of the A1 sequence by A2 not only removes the Walker nucleotide binding sequence but also compromises the ability of the A subunit to assemble with other V-ATPase subunits.

The tyrosine phosphatase SHP-1 is a negative regulator of osteoclastogenesis and osteoclast resorbing activity: increased resorption and osteopenia in me(v)/me(v) mutant mice.

Naturally occuring inactivating mutations of the Src homology 2 (SH2) domain-containing tyrosine phosphatase 1 (SHP-1) in mice give rise to the motheaten (me) phenotype. me/me mice have multiple hematopoietic abnormalities, suggesting that this phosphatase plays an important role in hematopoiesis. SHP-1 binds to and is activated by several hematopoietic surface receptors, including the colony-stimulating factor type 1 receptor. We have examined the role of SHP-1 in osteoclastogenesis and osteoclast function using mice with the viable motheaten (me(v)/me(v)) mutation, which has markedly decreased SHP-1 activity. Histomorphometric analysis of 6-week-old me(v)/me(v) mice and control littermates showed a marked osteopenia with an increase in bone resorption indices. The number of formed osteoclast-like cells (OCLs) in cocultures of me(v)/me(v) hematopoietic cells with normal osteoblasts was significantly increased. In contrast, the number of OCLs formed in the coculture of normal bone marrow cells with the me(v)/me(v) osteoblasts was not significantly different from controls. The bone-resorbing activity of me(v)me(v) OCLs and authentic osteoclasts was also found to be increased. Finally, Western blotting of proteins from me(v)/me(v) and control OCLs revealed an overall increase in tyrosine phosphorylation in the me(v)/me(v) lysates. These in vivo and in vitro results suggest that SHP-1 is a negative regulator of bone resorption, affecting both the formation and the function of osteoclasts.

Cytoskeleton-dependent tyrosine phosphorylation of the p130(Cas) family member HEF1 downstream of the G protein-coupled calcitonin receptor. Calcitonin induces the association of HEF1, paxillin, and focal adhesion kinase.

HEF1 is a recently described p130(Cas)-like docking protein that contains one SH3 domain and multiple SH2 binding motifs. In B cells, HEF1 is phosphorylated by a cytoskeleton-dependent mechanism that is triggered by integrin ligation. However, the induction of HEF1 phosphorylation by G protein-coupled receptors has not been reported. We found that HEF1, but not p130(Cas), is tyrosine-phosphorylated following stimulation of the rabbit C1a calcitonin receptor stably expressed in HEK-293 cells. The calcitonin-induced tyrosine phosphorylation of HEF1 increased in a time- and dose-dependent manner. Dibutyryl cAMP and forskolin had little or no effect on HEF1 phosphorylation, and the protein kinase A inhibitor H89 failed to detectably inhibit the response to calcitonin, indicating that the G(s)/cAMP/protein kinase A pathway does not mediate the calcitonin effect. Pertussis toxin, which selectively blocks G(i/o) signaling, also had no effect. Increasing cytosolic Ca(2+) with ionomycin stimulated HEF1 phosphorylation and preventing any calcitonin-induced change in cytosolic calcium by a combination of BAPTA and extracellular EGTA completely blocked the calcitonin-induced tyrosine phosphorylation of HEF1. Phorbol 12-myristate 13-acetate also induced HEF1 tyrosine phosphorylation, and the protein kinase C inhibitor calphostin C completely inhibited both calcitonin- and phorbol 12-myristate 13-acetate-stimulated HEF1 phosphorylation. Calcitonin also induced the tyrosine phosphorylation of paxillin and focal adhesion kinase, and the association of these two proteins with HEF1. Pretreatment with cytochalasin D, which disrupts actin microfilaments, prevented the calcitonin-induced HEF1 and paxillin phosphorylation. In conclusion, the calcitonin-stimulated tyrosine phosphorylation of HEF1 is mediated by calcium- and protein kinase C-dependent mechanisms and requires the integrity of the actin cytoskeleton.

Biological properties of salmon calcitonin IV.

In this study we characterized the biological activity of the recently identified salmon calcitonin (sCT) IV, in order to evaluate its potential therapeutic value. In the rat bioassay, sCT IV exhibited a 30% higher hypocalcemic activity than sCT I. The capacity of the molecule to inhibit bone resorption was assessed in vitro by the bone resorbing assay and the pit assay. An inhibitory effect, similar to that of sCT I, was observed in both assays. The interaction of sCT IV with the rabbit CT receptor was also studied. The affinity of sCT IV for the receptor was similar to that of sCT I, as was the potency for stimulating cAMP production. The antigenicity of the two molecules was not identical. Thus, this new CT could represent a useful novel therapeutic agent for the treatment of bone disorders.

Protein kinase C antagonizes pertussis-toxin-sensitive coupling of the calcitonin receptor to adenylyl cyclase.

The calcitonin receptor is known to couple to Gs and Gq, activating adenylyl cyclase and phospholipase C, respectively. The observation of pertussis-toxin-sensitive responses to calcitonin suggests that the receptor is capable of coupling to Gi/o as well. However, the calcitonin-dependent activation of adenylyl cyclase in HEK-293 cells that stably express the cloned rabbit calcitonin receptor, as in many other cells that express calcitonin receptors, shows little pertussis toxin sensitivity. Calcitonin treatment of these cells stimulates protein kinase C, which is reported to antagonize the receptor-dependent activation of Gi. The possibility that protein kinase C could be antagonizing Galphai-adenylyl cyclase coupling was tested by examining the effects of protein kinase C inhibitors (chelerythrine chloride and sphingosine) or of chronic treatment with phorbol ester to deplete protein kinase C. All three treatments led to a reduction of calcitonin-induced adenylyl cyclase activity that was reversed by pertussis toxin. Inhibiting or depleting protein kinase C had no effect on the activation of adenylyl cyclase by cholera toxin, indicating that Gs and adenylyl cyclase were not affected by these treatments. Calcitonin treatment of HEK-293 cells, that stably express a myc-tagged rabbit calcitonin receptor, induced the formation of complexes of the receptor and Galphai subunits, confirming that the calcitonin receptor interacts with Gi. Thus, the calcitonin receptor can couple to Gi, but the inhibition of adenylyl cyclase by Galphai is negatively regulated by protein kinase C.

Calcitonin-dependent down-regulation of the mouse C1a calcitonin receptor in cells of the osteoclast lineage involves a transcriptional mechanism.

Although expression of the calcitonin (CT) receptor (CTR) decreases after CT binding, there has been no evidence that it occurs at the transcriptional level. In the present study we investigated the mechanism of CTR messenger RNA (mRNA) down-regulation by CT in mouse cocultures of bone marrow and osteoblasts. Ribonuclease protection analysis revealed that osteoclast-like cells purified from cocultures predominantly express the C1a isoform and do not express an appreciable amount of the brain-specific C1b mRNA (< 1% of C1a). Treatment of day 5 cocultures with CT caused a dose- and time-dependent decrease in the steady state level of C1a mRNA. This CT effect was mimicked by the cAMP agonists forskolin and (Bu)2cAMP. Prolonged suppression of C1a mRNA was observed after short treatment with CT, but not with (Bu)2cAMP, suggesting that persistent intracellular cAMP elevation is necessary for the prolonged CT effect. The half-life of the C1a mRNA in cocultures was 4-6 h and was not altered by CT or (Bu)2cAMP. Moreover, competitive RT-PCR analysis revealed that 1-h treatment with CT reduced the level of CTR heterogeneous nuclear RNA to 10% in a cycloheximide-independent manner. These results suggest that CT down-regulates C1a-CTR mRNA expression at least in part by a transcriptional mechanism, thereby contributing to the ligand-induced desensitization in cells of the osteoclast lineage.

A new method to isolate large numbers of rabbit osteoclasts and osteoclast-like cells: application to the characterization of serum response element binding proteins during osteoclast differentiation.

We have developed a new method that allows the purification of large numbers of both authentic osteoclasts (OCs) and in vitro differentiated osteoclast-like cells (OCLs) from rabbits. We characterized the OCLs in terms of the expression of different phenotypic markers of OC differentiation and their ability to resorb bone. The method provides a system for performing biochemical and molecular studies of OC differentiation and function in a single species. We used this system to characterize the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the expression of proteins that bind to the serum response element (SRE) of the c-fos promoter. We found that OCLs and OCs displayed similar SRE-binding activities, including the serum response factor (SRF). This pattern is established in a time-dependent and cell-specific manner in response to long-term treatment of rabbit bone marrow by 1,25(OH)2D3. Thus, 1,25(OH)2D3 can modulate SRF and/or SRF-related protein. This finding may contribute to understanding the role of c-Fos in the regulation of OC differentiation.

The calcitonin receptor stimulates Shc tyrosine phosphorylation and Erk1/2 activation. Involvement of Gi, protein kinase C, and calcium.

While it is well established that adenylyl cyclase and phospholipase C-beta are two proximal signal effectors for the calcitonin receptor, the more distal signaling pathways are less well characterized. G protein-coupled receptors can activate Erk1/2 by Gs-, Gi-, or Gq-dependent signaling pathways, depending on the specific receptor and cell type examined. Since the calcitonin receptor can couple to all three of these G proteins, the ability of calcitonin to activate Erk1/2 was investigated. Calcitonin induced time- and concentration-dependent increases in Shc tyrosine phosphorylation, Shc-Grb2 association and Erk1/2 phosphorylation and activation in a HEK 293 cell line that stably expresses the rabbit calcitonin receptor C1a isoform. Pertussis toxin, which inactivates Gi, and calphostin C, a protein kinase C inhibitor, each partially inhibited calcitonin-induced Shc tyrosine phosphorylation, Shc-Grb2 association, and Erk1/2 phosphorylation. In contrast, neither forskolin nor H89, a protein kinase A inhibitor, had a significant effect on basal or calcitonin-stimulated Erk1/2 phosphorylation. Our results suggest that the calcitonin receptor induces Shc phosphorylation and Erk1/2 activation in HEK293 cells by parallel Gi- and PKC-dependent mechanisms. The calcitonin-induced elevation of cytosolic free Ca2+ was required for Erk1/2 phosphorylation, since preventing any change in cytosolic free Ca2+ by chelating both cytosolic and extracellular Ca2+ abolished the response. However, the change in Ca2+ that is induced by calcitonin is not sufficient to account for the calcitonin-induced Erk1/2 phosphorylation, since treatment with 100 nM ionomycin or 10 microM thapsigargin, each of which induced elevations of Ca2+ comparable to those induced by calcitonin, induced significantly less Erk1/2 phosphorylation than that induced by calcitonin. Erk1/2 may have important roles as downstream effectors mediating cellular responses to calcitonin stimulation.

Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts.

Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered.

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

1,25-Dihydroxyvitamin D3 selectively induces increased expression of the Na,K-ATPase beta 1 subunit in avian myelomonocytic cells without a concomitant change in Na,K-ATPase activity.

Treatment of avian myelomonocytic cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) results in an approximately two fold increase in levels of Na,K-ATPase beta 1 subunit mRNA and protein (both total and plasma membrane-associated). The changes in beta 1 subunit expression occur in the absence of a detectable increase in expression of any of the three alpha subunit isoforms or in Na,K-ATPase activity. The selective induction of the expression of the beta subunit in avian myelomonocytic cells by 1,25(OH)2D3 reveals a previously unobserved feature of the regulation of Na,K-ATPase expression, while the targeting of beta subunit polypeptides to the plasma membrane in the absence of a corresponding increase in active Na,K-ATPase suggests that, in these cells, transport of the beta subunit to the plasma membrane may be independent of its binding to the alpha subunit.