Biomedical subjects

D M Findlay

Publications and source records attributed to D M Findlay.

At least 55 records · Page 3Linked to original sources

PubMed1996

Effects of continuous calcitonin treatment on osteoclast-like cell development and calcitonin receptor expression in mouse bone marrow cultures.

Continuous treatment with calcitonin (CT) to inhibit osteoclastic bone resorption results in acquired resistance. The mechanisms of this "escape" phenomenon are not yet established. The aim of this study was to examine the effects of continuous treatment with CT on the generation of osteoclasts and calcitonin receptor (CTR) expression in mouse bone marrow cultures. This was done by daily CT treatment of mouse bone marrow cultures from day 0, when only undifferentiated mononuclear precursors of osteoclast-like cells were present, or commencing from day 6, when differentiated osteoclast-like cells were abundant. The response to CT treatment was determined by quantitation of cells positive for tartrate-resistant acid phosphatase (TRAP) and binding of 125I-salmon CT. Calcitonin receptor and TRAP mRNA levels were determined using semi-quantitative reverse transcription/polymerase chain reaction. When cultures were treated with CT from day 0, TRAP-positive multinucleated cells appeared. These cells expressed only very low levels of CTR or CTR mRNA and were morphologically indistinguishable from osteoclast-like cells formed in control cultures. They also displayed the ability to resorb bone. Continuous CT treatment of cultures from day 6 rapidly reduced the CTR mRNA levels, with a t1/2 of 6 to 12 h, and these levels remained low thereafter. 125I-salmon CT binding capacity, as determined by autoradiography, was lost in parallel. These effects were specific for the CTR since there was no consistent effect on TRAP mRNA levels. Based on these data, we suggest that the "escape" phenomenon may result from a prolonged CT-induced loss of CT responsiveness due, at least in part, both to reduced synthesis of CTR, and to the appearance in bone of CTR-deficient osteoclasts.

Acid Phosphatase↗

PubMed1996

c-fos antisense DNA inhibits proliferation of osteoclast progenitors in osteoclast development but not macrophage differentiation in vitro.

We previously reported that osteoclast formation in vitro, by coculture of mouse bone marrow and primary osteoblastic cells, occurs in two phases: proliferation of osteoclast progenitors followed by terminal differentiation into mature osteoclasts. Using this coculture system, we examined the effects of c-fos antisense and sense phosphorothioate oligonucleotides on osteoclast development and macrophage differentiation. Treatment with c-fos antisense for the first 4 days of coculture inhibited osteoclast formation in a dose-dependent fashion. However, when c-fos antisense was added during the second phase of coculture (4-6 days), osteoclast formation was unaffected. In contrast, c-fos antisense treatment had no effect on the appearance of F4/80 antigen-positive cells of the macrophage lineage in these cultures or on the induction by colony stimulating factor-1 of macrophage colony formation in cultures of mouse bone marrow cells in agar. Neither osteoclast differentiation nor macrophage appearance was inhibited by adding control c-fos sense in the cocultures. When c-fos antisense was added into an assay of bone resorption by mature osteoclasts, pit formation on dentine slices was unaffected. These results indicate that c-fos plays an important role in the proliferative phase of osteoclast progenitors in osteoclast development, but not in the terminal differentiation phase or in the bone resorbing activity of mature osteoclasts. c-fos antisense specifically inhibited osteoclast formation but had no effect on macrophage development.

Analysis of Variance↗

PubMed1996

Physiological levels of calcitonin regulate the mouse osteoclast calcitonin receptor by a protein kinase Alpha-mediated mechanism.

We have reported that calcitonin (CT) treatment induced downregulation of the CT receptor (CTR) in mouse osteoclast-like cells (OCLs). Here, we studied the features of homologous down-regulation of the CTR in mature mouse OCLs. Treatment with salmon CT (sCT) and human CT (hCT) reduced [125I]sCT specific binding. The decreased binding after 24 h of CT treatment was associated with a decrease in the cell surface receptor concentration. The extent of CT-induced down-regulation in 24 h was dose-dependent, and the ED50 value was 3.6 +/- 4.1 (mean +/- SD; n = 3) x 10(-13 M for sCT and 4.9 +/- 3.3 x 10(-11) M for hCT. These values were very similar to those for the CT inhibition of the bone-resorbing activity of OCLs. The data suggest that these two distinct actions of CT may be mediated by a common intracellular pathway. Treatment of OCLs with activators of protein kinase A (PKA) mimicked the effect of CT on CTR downregulation, whereas neither activation of protein kinase C nor elevation of intracellular Ca2+ did so. Attenuation of CT-induced CTR down-regulation by the competitive cAMP antagonist, RpcAMP, and high concentrations of H-7, but not by protein kinase C-specific inhibitors (sphingosine, staurosporine, and a lower concentration of H-7), suggested that the PKA pathway is primarily involved in homologous regulation of the CTR. The changes in CTR messenger RNA confirm the findings in binding studies and demonstrate that CT treatment of OCLs results in decreased CTR synthesis through the PKA pathway. The low concentrations of hCT that result in CTR regulation are very close to the physiological range, providing new insights into a dynamic relationship between circulating levels of CT and CTR expression in osteoclasts.

Animals↗

PubMed1996

Homologous regulation of the rat C1a calcitonin receptor (CTR) in nonosteoclastic cells is independent of CTR messenger ribonucleic acid changes and cyclic adenosine 3',5'-monophosphate-dependent protein kinase activation.

UMR106-06 cells predominantly express the C1a isoform of the rat calcitonin (CT) receptor (CTR). We have compared the homologous regulation of the C1a CTR endogenously expressed in UMR106-06 cells with the cloned C1a CTR in transfected HEK 293 cells, in which expression is driven by a heterologous promoter. It was found that treatment of both cell lines with either salmon CT or human CT reduced the density of cell surface CTR in a dose- and time-dependent manner. However, the magnitude of the response was greater in UMR106-06 cells, and salmon CT was more potent than human CT in both cell lines. Recovery from down-regulation was rapid in transfected cells (< 2 h), but was comparatively delayed in UMR106-06 cells, where less than 70% of receptor-binding capacity had returned by 24 h. In both cell lines, treatment with either agonist increased the basal activity of CT-sensitive adenylate cyclase and caused a time-dependent reduction in the responsiveness of adenylate cyclase to a second challenge with CT. Reduced responsiveness occurred under conditions of minimal loss of CTR from the cell surface, consistent with an uncoupling of the receptor from the signal transduction apparatus. Recovery of CT-sensitive adenylate cyclase was complete in transfected cells by 24 h, but was delayed in UMR106-06 cells, paralleling the slow recovery of receptor binding. CT-induced down-regulation of the CTR was not mimicked by receptor-independent activation of protein kinase A or protein kinase C. However, treatment of cells for 24 h, but not for 4 h, with phorbol ester caused a partial loss of CTR binding in UMR106-06 cells and resulted in an approximately 200% increase in CTR binding in transfected HEK 293 cells. CTR messenger RNA levels, as assessed by reverse transcription-PCR, were not changed by any of the above treatments. These results suggest that CT-induced receptor down-regulation and modulation of the ability of CT to activate adenylate cyclase are inherent properties of the receptor, as they can be recapitulated in an otherwise CTR-naive cell line, in which receptor expression is driven by a heterologous gene promoter. Moreover, and in contrast with CTR regulation in osteoclasts, activation of protein kinase A is insufficient for ligand-induced regulation of the CTR in these nonosteoclastic cell lines, and receptor regulation does not appear to involve altered messenger RNA levels.

1-Methyl-3-isobutylxanthine↗

PubMed1996

Calcitonin receptor down-regulation relates to calcitonin resistance in mature mouse osteoclasts.

We have reported that short calcitonin (CT) treatment of mature mouse osteoclast-like cells (OCLs) in culture induced prolonged down-regulation of the CT receptor (CTR) and desensitization to CT rechallenge, at the level of adenylate cyclase activity. In this study, we have extended those studies to examine the bone resorbing activity of OCLs pretreated with CT. OCLs, which formed on gelled type I collagen, were pretreated with salmon CT (sCT)(10(-9)M, 1 h) and 24 h later were replated onto plastic dishes or dentine slices after removal from the gel by collagenase digestion. The number and population of either mononuclear or multinuclear OCLs that adhere to either surface was not affected by sCT pretreatment. It was found that OCLs pretreated with sCT regained reduced but significant bone resorbing capacity, which was quantitated as the surface area resorbed by OCLs on dentine slices. However, compared with control, the number of resorption pits produced by sCT- pretreated OCLs was slightly reduced, and the total pit area was decreased by approximately 40-50%. The distribution of individual pit sizes was altered by sCT-pretreatment so that the number of larger pits was predominantly reduced, suggesting that short sCT treatment may produce a long lasting decrease in osteoclast mobility. sCT was able to inhibit bone resorption activity of CT-pretreated OCLs (ED50:10(-13)-10(-12)M). Importantly, the ED50 of sCT inhibition of bone resorption in sCT-pretreated OCLs was approximately 100-fold greater than for control, indicating resistance of the OCLs to CT rechallenge. Consistent with these results, treatment of OCLs with sCT greatly decreased the expression of CTR messenger RNA, whereas no significant effect was observed on the tartrate-resistant acid phosphatase messenger RNA expression, a marker of resorptive capacity of osteoclasts. These results indicate, therefore, that an important component of escape of osteoclastic resorption from CT inhibition is CT resistance of mature osteoclasts, which regain bone resorbing function.

Animals↗

PubMed1996

A type I collagen substrate increases PTH/PTHrP receptor mRNA expression and suppresses PTHrP mRNA expression in UMR106-06 osteoblast-like cells.

We have previously shown that the response of osteoblasts to parathyroid hormone (PTH) can be influenced at the receptor level by growth on the physiological substrate, type I collagen, or by treatment with retinoic acid. We have also shown differential expression of genes when cells of the osteoblast lineage are grown on type I collagen. The aim of this study was therefore to examine the effect of retinoic acid and growth on type I collagen on PTH/PTH-related protein (PTHrP) receptor mRNA expression in the osteosarcoma osteoblast-like cell line UMR 106-06. PTH/PTHrP receptor mRNA levels, as assessed by Northern blot, of cells grown on collagen were increased up to 2-fold compared with cells on plastic and in a concentration-dependent manner with respect to collagen. An increase was seen as early as 6 h and was maintained over a 24 h period. This was not due to increased mRNA stability. Retinoic acid decreased the level of receptor mRNA on both plastic and collagen at each time but did not alter mRNA stability. For all treatments PTH/PTHrP receptor mRNA abundance, relative to glyceraldehyde-3-phosphate dehydrogenase, increased steadily over 24 h after subculture of cells. In contrast, PTHrP mRNA levels were reduced in cells on collagen, compared with plastic. PTH-stimulated cAMP levels of cells grown on collagen were increased compared with plastic at 24 h, but not earlier. Consistent with the mRNA data, retinoic acid decreased the amplitude of cAMP responses in cells on plastic and collagen. There was no evidence for changes in adenylate cyclase per se, since forskolin-induced cAMP levels did not change with either treatment. This study shows that known modulators of osteoblast maturation also affect signal transduction in these cells by regulating gene expression of the PTH/PTHrP receptor as well as the PTHrP ligand.

Animals↗

PubMed1996

Regulation of osteopontin expression by type I collagen in preosteoblastic UMR201 cells.

When UMR201 cells, phenotypically preosteoblastic, were placed onto a type I collagen gel, expression of osteopontin (OP) mRNA and protein were strongly upregulated, compared to cells plated onto plastic. This upregulation was dose-dependent, with respect to the concentration of collagen gel, and was observable within hours of cells having attached and spread on the substrate. Retinoic acid (RA) acted synergistically with type I collagen at each concentration to induce a much greater increase in OP mRNA than in cells on plastic. In addition, RA increased the phosphorylation of secreted OP. The exogenous collagen substrate inhibited the growth of UMR201 cells, with the extent and duration of inhibition dependent on the collagen concentration. The effect of type I collagen was specific; plating cells on fibronectin, laminin or vitronectin did not upregulate OP expression. In contrast to the effects on OP expression, the strong RA induction of alkaline phosphatase (ALP) mRNA in cells on plastic was attenuated in cells plated on type I collagen. Growth on type I collagen did not change OP mRNA stability or transcription rate, although there was decreased stability of the ALP mRNA in cells on collagen.

Alkaline Phosphatase↗

PubMed1995

Interleukin (IL)-6 induction of osteoclast differentiation depends on IL-6 receptors expressed on osteoblastic cells but not on osteoclast progenitors.

We reported that interleukin (IL) 6 alone cannot induce osteoclast formation in cocultures of mouse bone marrow and osteoblastic cells, but soluble IL-6 receptor (IL-6R) strikingly triggered osteoclast formation induced by IL-6. In this study, we examined the mechanism of osteoclast formation by IL-6 and related cytokines through the interaction between osteoblastic cells and osteoclast progenitors. When dexamethasone was added to the cocultures, IL-6 could stimulate osteoclast formation without the help of soluble IL-6R. Osteoblastic cells expressed a very low level of IL-6R mRNA, whereas fresh mouse spleen and bone marrow cells, both of which are considered to be osteoclast progenitors, constitutively expressed relatively high levels of IL-6R mRNA. Treatment of osteoblastic cells with dexamethasone induced a marked increase in the expression of IL-6R mRNA. By immunoblotting with antiphosphotyrosine antibody, IL-6 did not tyrosine-phosphorylate a protein with a molecular mass of 130 kD in osteoblastic cells but did so in dexamethasone-pretreated osteoblastic cells. Osteoblastic cells from transgenic mice constitutively expressing human IL-6R could support osteoclast development in the presence of human IL-6 alone in cocultures with normal spleen cells. In contrast, osteoclast progenitors in spleen cells from transgenic mice overexpressing human IL-6R were not able to differentiate into osteoclasts in response to IL-6 in cocultures with normal osteoblastic cells. These results clearly indicate that the ability of IL-6 to induce osteoclast differentiation depends on signal transduction mediated by IL-6R expressed on osteoblastic cells but not on osteoclast progenitors.

Animals↗

PubMed1995

Modulation of the signal recognition particle 54-kDa subunit (SRP54) in rat preosteoblasts by the extracellular matrix.

Rat preosteoblastic cells, UMR201, develop a more mature phenotype when subcultured onto a type I collagen gel when compared with their growth on plastic. Basal osteopontin mRNA expression is up-regulated, whereas retinoic acid-induced alkaline phosphatase expression is reduced in cells on collagen when compared with cells plated onto plastic. We have used differential display polymerase chain reaction (PCR) of mRNA to identify other mRNA species that are regulated by collagen and/or retinoic acid in UMR201 cells. A number of differentially expressed PCR products were isolated, whose sequences did not correspond to known sequences in the data bank. However, one species which was up-regulated by growth on collagen showed 95 and 94% homology to the murine and canine 54-kDa subunit of the signal recognition particle (SRP54), respectively. In time course experiments, using reverse transcription PCR, it was found that SRP54 mRNA was up-regulated in UMR201 cells as early as 1 h after subculture onto collagen, when compared with cells subcultured onto plastic, and levels remained elevated after 48 h. The increased expression of SRP54 paralleled the increased expression of a known secreted protein, osteopontin. SRP54 recognizes signal sequences of proteins destined for secretion and retards them for further elongation in the endoplasmic reticulum. The increased expression may correlate with the synthesis of specific extracellular matrix molecules in differentiating osteoblasts.

Amino Acid Sequence↗

PubMed1995

Tumor necrosis factor alpha facilitates nuclear actions of retinoic acid to regulate expression of the alkaline phosphatase gene in preosteoblasts.

This study examines the molecular mechanisms of interaction between tumor necrosis factor alpha (TNF alpha) and retinoic acid on the expression of the alkaline phosphatase gene by rat clonal preosteoblastic cells. In this cell line, alkaline phosphatase mRNA was not constitutively expressed but was progressively induced by treatment with 1 microM retinoic acid, detectable by 6 h. Combining retinoic acid with 0.6 nM TNF alpha resulted in alkaline phosphatase mRNA appearing by 2 h, as well as enhanced expression above that observed with retinoic acid alone at 6, 12, and 24 h. Nuclear run-on analysis showed constitutive transcription of the alkaline phosphatase gene in control and TNF alpha-treated cells. At 4 h, retinoic acid, alone or combined with TNF alpha, increased alkaline phosphatase gene transcriptional rate by 2-fold. However, at 24 h, while no retinoic acid effect was retained, retinoic acid plus TNF alpha resulted in a 5-fold increase in alkaline phosphatase transcriptional rate. Examination of the distribution of nuclear alkaline phosphatase mRNA demonstrated that pre-spliced precursor mRNA was localized to the nuclear matrix in control and all treatment groups. Retinoic acid caused a time-dependent accumulation of mature, spliced alkaline phosphatase mRNA located in the non-matrix and cytoplasmic fractions, implying a post-transcriptional action of retinoic acid in nuclear processing and nucleocytoplasmic transport. Adding TNF alpha with retinoic acid greatly enhanced this effect, which was observed after 4 h, prior to any detectable interaction between TNF alpha and retinoic acid on gene transcription. In sharp contrast, only a negligible amount of nuclear processing occurred in control and TNF alpha-treated cells. This study reveals distinct interactions between TNF alpha and retinoic acid at post-transcriptional as well as transcriptional levels to regulate expression of the alkaline phosphatase gene in preosteoblasts.

Alkaline Phosphatase↗

PubMed1995

Calcium inflow in cells transfected with cloned rat and porcine calcitonin receptors.

Ca2+ fluxes were examined in HEK 293 cells stably expressing the rat or porcine calcitonin receptors (CTRs). Calcitonin (CT) rapidly increased cytosolic Ca2+ ([Ca2+]i) concentrations in these cells in a manner which was sustained in the presence of extracellular Ca2+ ([Ca2+]e). In cells pretreated with CT, elevation of the [Ca2+]e concentration resulted in a further increase in [Ca2+]i which was concentration-dependent with respect to both the concentration of CT and the increment of [Ca2+]e. Untransfected cells, cells transfected with vector alone, and CTR-transfected cells not treated with CT, were unresponsive to [Ca2+]e. The microsomal Ca(2+)-ATPase inhibitor thapsigargin was able to mimic both the acute [Ca2+]i fluxes and responsiveness to [Ca2+]e mediated by CT in these cells. The CT-induced responsiveness to [Ca2+]e was neither mimicked by, nor affected by, activators of the cAMP or protein kinase C pathways. Treatment of cells with pertussis toxin influenced neither the primary Ca2+ fluxes in response to CT or thapsigargin nor the agonist-induced [Ca2+]e influx. Nifedipine failed to block responses to either CT or thapsigargin. These results lead to the important conclusion that the CTR participates in receptor-activated Ca2+ inflow, in which depletion of intracellular Ca2+ pools leads secondarily to influx of extracellular Ca2+.

Animals↗

PubMed1995

Regulation of membrane-associated tyrosine phosphatases in UMR 106.06 osteoblast-like cells.

Protein tyrosine phosphatases play an important role in cell metabolism. Three distinct protein tyrosine phosphatase activities have been identified in an osteoblast-like cell line, UMR 106.06. These activities comprised two membrane-associated phosphatases and one cytosolic phosphatase of apparent molecular mass > 153 kDa, 80 kDa and 40 kDa respectively, estimated by gel filtration. On the basis of differences in apparent molecular mass, proteolytic-digestion profiles, substrate specificities and responses to a range of extracellular influences and inhibitor molecules, the two membrane-associated tyrosine phosphatases are distinct proteins. Tyrosine phosphatase activity in UMR 106.06 cells was sensitive to cell density. Cells at confluence contained membrane protein tyrosine phosphatase with specific activity 9-fold higher than cells at medium or low cell density. This elevation in membrane tyrosine phosphatase activity was due specifically to an increase in the high-molecular-mass enzyme. This phosphatase was also responsive to extracellular matrix components. This activity was elevated in cells grown on a collagen type-I matrix independently of cell density. Membrane and cytosolic protein tyrosine phosphatases were differentially regulated by a variety of agents including phorbol 12-myristate 13-acetate, parathyroid hormone, epidermal growth factor, okadaic acid and transforming growth factor beta. These observations suggest that regulatory influences control tyrosine phosphorylation in UMR 106.06 cells including cell-cell contact, cell-matrix contact and signal transduction involving tyrosine and serine/threonine phosphorylation events.

Animals↗

PubMed1995

Calcitonin receptor isoforms in mouse and rat osteoclasts.

Calcitonin receptors (CTRs) from several species have recently been cloned and shown to belong to the 7 transmembrane domain class of receptor. We have identified two CTR isoforms in the rat, termed C1a and C1b, identical except for a 37-amino-acid insert in the putative second extracellular domain of C1b. To examine the CTR isoforms expressed in rat and mouse osteoclasts and the time course of their appearance in culture, bone marrow cells were cultured from C57/Bl6J mice and osteoclasts were isolated from newborn rat long bones. CTR-bearing cells were detected by autoradiography of 125I-salmon CT binding, and cultures were stained for tartrate-resistant acid phosphatase (TRAP). RNA was extracted from parallel cultures, and CTR mRNA was detected by Northern blot analysis, using a rat digoxigenin-labeled riboprobe. Characterization of mRNA for the CTR isoforms was by reverse transcription-polymerase chain reaction (RT-PCR) using primer sets and oligonucleotide probes specific for the two rat receptor isoforms. In mouse marrow cultures, TRAP positive mononucleated cells were present by day 2 of culture at which time CTR positive cells were few. Multinucleated cells with both these markers were seen only from day 4 and later. By Northern analysis of total RNA, a band of approximately 4 kb could be detected in day 4 and later cultures. RT-PCR showed that mouse homologs of both C1a and C1b mRNA species were expressed early in cultures of mouse osteoclasts, although at each time C1a appeared to predominate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Short treatment of osteoclasts in bone marrow culture with calcitonin causes prolonged suppression of calcitonin receptor mRNA.

Cells exhibiting osteoclast characteristics of calcitonin receptors (CTRs) and tartrate-resistant acid phosphatase (TRAP) histochemistry are formed in murine bone marrow cultures treated with 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. We have previously demonstrated that CTR mRNA is highly expressed in these cultures. The aim of this study was to investigate homologous regulation of the CTR, and regulation of TRAP expression in osteoclast-like cells after brief treatment with salmon CT (sCT). Murine bone marrow cells were cultured in 9 cm dishes in the presence of 10 nmol/L 1,25-(OH)2D3. On day 6 of culture, when multinucleated cells were abundant, the cells were treated with 1 nmol/L sCT for 1 h. Both control and treated cells were then harvested at intervals up to 72 h posttreatment, and both CTR and TRAP mRNA levels assessed by reverse-transcription PCR (RT-PCR). In parallel cultures, cells with CTR expression detectable by autoradiography, and TRAP positivity by histochemistry, were counted. The effects of brief sCT treatment could be seen 6 h after treatment when the CTR RT-PCR product was markedly reduced. Total recovery of CTR mRNA levels had not occurred even after 72 h. Calcitonin treatment had little effect on TRAP mRNA levels. There was no difference in the numbers of multinucleated TRAP(+) osteoclast-like cells between treated and control cells. These results indicate that brief sCT treatment, while not influencing multinucleated osteoclast-like cell number, causes specific, acute reduction of CTR mRNA in bone marrow culture-derived osteoclasts. The prolonged decrease in CTR mRNA levels suggests that recovery may require new osteoclast formation, and indicates that regulation of the CTR in cells of the osteoclast lineage is different from that in nonosteoclastic cells and tissues.

Acid Phosphatase↗

PubMed1995

Heterogeneity of the calcitonin receptor: functional aspects in osteoclasts and other sites.

The recent cloning of the calcitonin receptor reveals it as a member of a new family of 7-transmembrane, G protein-linked receptors. Data from rat, mouse and human receptor cloning reveal that in each of these species the receptor exists in more than one form, most likely the result of alternate, splicing. In the rat, the two forms are C1a and C1b, the latter differing from C1a in that it contains a 37-amino acid insert in the second extracellular domain. The two receptor isoforms differ in their distribution in vivo, with the C1b predominantly in the central nervous system and C1a in other tissues. Both forms have been shown to be present in mature and developing osteoclasts, with the C1a isoform predominating. The two isoforms differ in their kinetics and pharmacological properties, with C1b being virtually unable to bind the human and rat calcitonins but readily binding salmon calcitonin. It will be important to elucidate the physiological significance of the structural heterogeneity of the calcitonin receptor.

Animals↗

PubMed1995

Homologous regulation of the calcitonin receptor in mouse osteoclast-like cells and human breast cancer T47D cells.

Calcitonin (CT) down-regulates its receptor in several cancer cell lines, including T47D human breast cancer cells. Removal of CT results in the recovery of CT receptor (CTR) binding. However, homologous regulation of the CTR in osteoclasts is not well understood. To elucidate these phenomena in cells of the osteoclast lineage, mouse osteoblasts and bone marrow cells were cocultured on type 1 collagen gels. For the experiments, osteoclast-like cell (OCL)-enriched populations were subcultured from the collagen gels into multiwell dishes on days 7-8, and CT regulation of the CTR was determined and compared with that in T47D cells. When cells of either type were treated with CT for 1 h and then washed, binding capacity for [125I] salmon CT ([125I]sCT) was decreased dependent upon the preincubating concentration of CT. After removal of CT, the binding capacity in OCLs recovered toward the control level over 12 h. However, in contrast to that in T47D cells, recovery was transient, so that 24 h after removal of CT, the binding capacity in preincubated cells was strikingly reduced compared with that in control cells. This occurred even when the preincubating concentration of CT was too low to cause down-regulation of binding in 1 h. Scatchard analysis showed a decrease in receptor number in CT-treated compared with control OCLs 24 h after CT removal, with unchanged receptor affinity. By autoradiography, decreased CTR density on multinuclear OCLs was indicated. Preexposure of either OCLs or T47D cells to CT caused elevation of intracellular cAMP, which persisted for 6-12 h after removal of CT. In addition, there was desensitization to a rechallenge with CT, which, in T47D cells, recovered by 24-36 h. In contrast, OCLs showed incomplete recovery of desensitization. These data correlated with the results of semiquantitative reverse transcription-polymerase chain reaction studies; the CTR messenger RNA level was increased to about 150% of the control level in sCT-treated T47D cells 18-36 h after sCT removal; the level was markedly decreased to about 20% of the control value in sCT-treated OCLs 12-48 h after sCT removal and remained suppressed. This study suggests that CT-induced homologous down-regulation is a potential cause of the "escape" phenomenon, by producing a population of CT-resistant osteoclasts.

Animals↗

PubMed1995

Functionally different isoforms of the human calcitonin receptor result from alternative splicing of the gene transcript.

Two subtypes of the human calcitonin receptor (hCTR) have been described which differ from one another by the presence or absence of a 16-amino acid insert in the first intracellular loop. Both isoforms were stably expressed in baby hamster kidney cells to compare their ligand binding and second messenger coupling. The binding affinity and the on/off rate of binding for salmon CT were identical for the two receptor isoforms. However, the presence of the insert significantly reduced the ability of the receptor to couple to both adenylate cyclase and phospholipase C. Stimulation of a transient calcium response was only observed with the insert-negative receptor. Similarly, the ED50 for the cAMP response is 100-fold higher for the insert-positive form compared with the insert-negative form of the receptor. However, the maximal cAMP response was equivalent for both receptor isoforms. The rate of internalization of the insert-positive form of the receptor is significantly impaired relative to the insert-negative receptor, which suggests that this process may be dependent on the stimulation of a second messenger pathway. Cloning and characterization of the relevant portion of the hCTR gene revealed that these isoforms are generated by alternative splicing. We also discovered a third isoform of the hCTR, which can be generated by alternative splicing at the same position. The presence of a stop codon in this newly described alternative exon would lead to premature termination of the receptor at the C-terminal end of the first transmembrane domain.

Alternative Splicing↗

PubMed1995

Divergent structural requirements exist for calcitonin receptor binding specificity and adenylate cyclase activation.

The basis of the high potency of salmon calcitonin (sCT) in radioligand binding competition and cAMP accumulation studies with cloned calcitonin (CT) receptors from rats, pigs, and humans was examined using two sets of CT analogues, i.e., chimeric sCT/human CT (hCT) analogues and analogues of sCT with differing capacities to form an amphipathic alpha-helix. In competition for 125I-sCT binding the following relative specificities were observed for the chimeric peptides: rat C1a CT receptor, sCT > or = (1-16)hCT/(17-32)sCT (ACT-15) > (1-16)sCT/(17-32)hCT (ACT-27); rat C1b CT receptor, sCT >> ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > ACT-27; porcine CT receptor, sCT > ACT-27 > ACT-15. In contrast, in ligand-induced cAMP accumulation studies the relative efficacies were as follows: rat C1a CT receptor, sCT = ACT-15 > ACT-27; rat C1b CT receptor, sCT = ACT-15 > ACT-27; hCT receptor, sCT = ACT-15 > or = to ACT-27; porcine CT receptor, sCT = ACT-15 = ACT-27. The data demonstrate that residues present in the carboxyl-terminal half of sCT are more important for binding competition with the rat C1a, rat C1b, and human CT receptors, whereas residues in the amino-terminal half of sCT are more important for binding competition with the porcine CT receptor. Carboxyl-terminal sCT residues are also important for full potency in adenylate cyclase activation with the rat C1a and rat C1b CT receptors but are less important for activation via the hCT receptor. The disparity in the relative potencies of the peptides in studies of binding competition and cAMP accumulation is suggestive of significant differences in the relative affinities of the peptides for active and inactive conformations of the CT receptor. The use of sCT analogues with varying capacities to form alpha-helices also revealed divergence in the responses of different receptors. This was most apparent for the stimulation of cAMP production by the rat receptor isoforms C1a and C1b. In cells expressing the C1a receptor, the helical analogues sCT and des-Ser2-sCT were equipotent with [Gly8]-des-Leu19-sCT and des-1-amino-[Ala1,7,Gly8]-des-Leu19 sCT, analogues that have reduced or absent helical structure, respectively. In contrast, the nonhelical analogues were 100-1000-fold less potent than sCT and des-Ser2-sCT at the C1b receptor. In general, reduction in the ability of sCT analogues to form helix structures had a greater impact on the potency of the analogues in competition for 125I-sCT binding than in cAMP accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylyl Cyclases↗