Elevated levels of active Transforming Growth Factor ß1 in the subchondral bone relate spatially to cartilage loss and impaired bone quality in human knee osteoarthritis.

OBJECTIVE: The association between the spatially distributed level of active TGFß1 in human subchondral bone, and the characteristic structural and cellular parameters of human knee OA, was assessed. DESIGN: Paired subchondral bone samples from 35 OA arthroplasty patients, (15 men and 20 women, aged 69 ± 9 years) were obtained from beneath macroscopically present (CA+) or denuded cartilage (CA-) to determine the concentration of active TGFß1 (ELISA) and its relationship to bone quality (synchrotron micro-CT), cellularity, and vascularization (histology). RESULTS: Bone samples beneath (CA-) regions had significantly increased concentrations of active TGFß1 protein (mean difference: 26.4; 95% CI: [3.2, 49.7]), when compared to bone in CA + regions. Trabecular Bone below (CA-) regions had increased bone volume (median difference: 4.3; 96.49% CI: [-1.7, 17.8]), increased trabecular number (1.5 [0.006, 2.6], decreased trabecular separation (-0.05 [-0.1,-0.005]), and increased bone mineral density (394.5 [65.7, 723.3]) comparing to (CA+) regions. Further, (CA-) bone regions showed increased osteocyte density (0.012 [0.006, 0.018]), with larger osteocyte lacunae (39.8 [7.8, 71.7]) that were less spherical (-0.02 [-0.04, -0.003]), and increased bone matrix vascularity (12.4 [0.3, 24.5]) compared to (CA+). In addition, increased levels of active TGFß1 related to increased bone volume (0.04 [-0.11, 0.9]), while increased OARSI grade associated with lacunar volume (-44.1 [-71.1, -17.2]), and orientation (2.7 [0.8, 4.6]). CONCLUSION: Increased concentration of active TGFß1 in the subchondral bone of human knee OA associates spatially with impaired bone quality and disease severity, suggesting that TGFß1 is a potential therapeutic target to prevent or reduce human OA disease progression.

Bone marrow lesions in knee osteoarthritis: regional differences in tibial subchondral bone microstructure and their association with cartilage degeneration.

OBJECTIVE: The aim of this study was to investigate how bone microstructure within bone marrow lesions (BMLs) relates to the bone and cartilage across the whole human tibial plateau. DESIGN: Thirty-two tibial plateaus from patients with osteoarthritis (OA) at total knee arthroplasty and eleven age-matched non-OA controls, were scanned ex vivo by MRI to identify BMLs and by micro CT to quantitate the subchondral (plate and trabecular) bone microstructure. For cartilage evaluation, specimens were processed histologically. RESULTS: BMLs were detected in 75% of the OA samples (OA-BML), located predominantly in the anterior-medial (AM) region. In contrast to non-OA control and OA-no BML, in OA-BML differences in microstructure were significantly more evident between subregions. In OA-BML, the AM region contained the most prominent structural alterations. Between-group comparisons showed that the AM region of the OA-BML group had significantly higher histological degeneration (OARSI grade) (P < .0001, P < .05), thicker subchondral plate (P < .05, P < .05), trabeculae that are more anisotropic (P < .0001, P < .05), well connected (P < .05, P = n.s), and more plate-like (P < 0.05, P < 0.05), compared to controls and OA-no BML at this site. Compared to controls, OA-no BML had significantly higher OARSI grade (P < .0001), and lower trabecular number (P < .05). CONCLUSION: In established knee OA, both the extent of cartilage damage and microstructural degeneration of the subchondral bone were dependent on the presence of a BML. In OA-no BML, bone microstructural alterations are consistent with a bone attrition phase of the disease. Thus, the use of BMLs as MRI image-based biomarkers appear to inform on the degenerative state within the osteochondral unit.

Recombinant sclerostin antagonizes effects of ex vivo mechanical loading in trabecular bone and increases osteocyte lacunar size.

Sclerostin has emerged as an important regulator of bone mass. We have shown that sclerostin can act by targeting late osteoblasts/osteocytes to inhibit bone mineralization and to upregulate osteocyte expression of catabolic factors, resulting in osteocytic osteolysis. Here we sought to examine the effect of exogenous sclerostin on osteocytes in trabecular bone mechanically loaded ex vivo. Bovine trabecular bone cores, with bone marrow removed, were inserted into individual chambers and subjected to daily episodes of dynamic loading. Cores were perfused with either osteogenic media alone or media containing human recombinant sclerostin (rhSCL) (50 ng/ml). Loaded control bone increased in apparent stiffness over time compared with unloaded bone, and this was abrogated in the presence of rhSCL. Loaded bone showed an increase in calcein uptake as a surrogate of mineral accretion, compared with unloaded bone, in which this was substantially inhibited by rhSCL treatment. Sclerostin treatment induced a significant increase in the ionized calcium concentration in the perfusate and the release of ß-CTX at several time points, an increased mean osteocyte lacunar size, indicative of osteocytic osteolysis, and the expression of catabolism-related genes. Human primary osteocyte-like cultures treated with rhSCL also released ß-CTX from their matrix. These results suggest that osteocytes contribute directly to bone mineral accretion, and to the mechanical properties of bone. Moreover, it appears that sclerostin, acting on osteocytes, can negate this effect by modulating the dimensions of the lacunocanalicular porosity and the composition of the periosteocyte matrix.

Osteocyte regulation of bone mineral: a little give and take.

Osteocytes actively participate in almost every phase of mineral handling by bone. They regulate the mineralisation of osteoid during bone formation, and they are also a major RANKL-producing cell. Osteocytes are thus able to liberate bone mineral by regulating osteoclast differentiation and activity in response to a range of stimuli, including bone matrix damage, bone disuse and mechanical unloading, oestrogen deficiency, high-dose glucocorticoid and chemotherapeutic agents. At least some of these activities may be regulated by the osteocyte-secreted product, sclerostin. There is also mounting evidence that in addition to regulating phosphate homeostasis systemically, osteocytes contribute directly to calcium homeostasis in the mature skeleton. Osteocyte cell death and the local loss of control of bone mineralisation may be the cause of focal hypermineralisation of bone and osteopetrosis, as seen in aging and pathology. The sheer number of osteocytes in bone means that "a little give and take" in terms of regulation of bone mineral content translates into a powerful whole organism effect.

Relationship between serum RANKL and RANKL in bone.

It is now well accepted that the molecule receptor activator of NFκB ligand (RANKL) and osteoprotegerin play key roles in regulating physiological and pathological bone turnover. There are a large number of published reports of circulating RANKL levels in both health and pathology. However, interpretation of these data has been elusive, and the relationship between circulating RANKL and RANKL levels in bone is still not clear. This review explores this subject, documenting the possible origins of circulating RANKL and suggesting additional information that is required before serum RANKL levels can provide useful diagnostic or research information.

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.

Strontium ranelate treatment of human primary osteoblasts promotes an osteocyte-like phenotype while eliciting an osteoprotegerin response.

SUMMARY: The effect of strontium ranelate (SR) on human osteoblast differentiation was tested. SR induced osteoblastic proliferation, in vitro mineralization, and increased the expression of osteocyte markers. SR also elicited an osteoprotegerin (OPG) secretory response. We conclude that SR promotes the osteoblast maturation and osteocyte differentiation while promoting an additional antiresorptive effect. INTRODUCTION: SR is a new treatment for osteoporosis that reduces the risk of hip and vertebral fractures in postmenopausal women. This study sought to investigate the extent, to which SR modulates human osteoblast differentiation. METHODS: Adult human primary osteoblasts (NHBC) were exposed to SR under mineralizing conditions in long-term cultures. Osteoblast differentiation status was investigated by cell-surface phenotypic analysis. Expression of genes associated with osteoblast/osteocyte differentiation was examined using real-time RT-PCR. Secreted OPG was assayed by enzyme-linked immunosorbent assay. RESULTS: SR significantly increased osteoblast replication. SR time- and dose-dependently induced an osteocyte-like phenotype, as determined by cell surface alkaline phosphatase and STRO-1 expression. SR at 5 mM or greater dramatically increased in vitro mineralization. In parallel, mRNA levels of dentin matrix protein (DMP)-1 and sclerostin were higher under SR treatment, strongly suggestive of the presence of osteocytes. SR also increased the OPG/RANKL ratio throughout the culture period, consistent with an effect to inhibit osteoblast-induced osteoclastogenesis. CONCLUSIONS: This study suggests that SR can promote osteoblast maturation and an osteocyte-like phenotype. Coupled with its effect on the OPG/RANKL system, these findings are consistent with in vivo effects in patients receiving SR for the treatment of osteoporosis.

Vascular pathology and osteoarthritis.

There is mounting evidence that vascular pathology plays a role in the initiation and/or progression of the major disease of joints: osteoarthritis (OA). Potential mechanisms are: episodically reduced blood flow through the small vessels in the subchondral bone at the ends of long bones, and related to this, reduced interstitial fluid flow in subchondral bone. Blood flow may be reduced by venous occlusion and stasis or by the development of microemboli in the subchondral vessels. There are several likely effects of subchondral ischaemia: the first of these is compromised nutrient and gas exchange into the articular cartilage, a potential initiator of degradative changes in the cartilage. The second is apoptosis of osteocytes in regions of the subchondral bone, which would initiate osteoclastic resorption of that bone and at least temporarily reduce the bony support for the overlying cartilage. It may be important to recognize these potential aetiological factors in order to develop more effective treatments to inhibit the progression of OA.

RNAi-mediated silencing of CYP27B1 abolishes 1,25(OH)2D3 synthesis and reduces osteocalcin and CYP24 mRNA expression in human osteosarcoma (HOS) cells.

Although local synthesis of 1,25D has been postulated to regulate parameters of cell growth and differentiation in non-renal cells, the physiological role of 1,25D production in bone cells remains unclear. We used the technique of RNA interference to inhibit the mRNA encoding the enzyme responsible for 1,25D synthesis, 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1). Human osteosarcoma (HOS) cells were transfected with siRNA for CYP27B1 or non-silencing RNA before being treated with 25D for 48h under normal growth conditions. De novo synthesis of 1,25D was measured in the media as well as mRNA levels for CYP27B1, osteocalcin (OCN) and 25-hydroxyvitamin D 24-hydroxylase (CYP24). We demonstrated that HOS cells express CYP27B1 mRNA, metabolize 25D and secrete detectable levels of de novo synthesized 1,25D. CYP27B1 mRNA silencing by RNAi, resulted in the suppression of 1,25D production and subsequent reduction of OCN and CYP24 mRNA expression. Our findings suggest that local 1,25D synthesis has paracrine effects in the bone microenvironment implying that vitamin D metabolism in human osteoblasts represents a physiologically important pathway, possibly regulating the maturation of osteoblasts.

Regulation of cell growth mediated by the calcitonin receptor.

Calcitonin (CT) is a 32 amino acid peptide hormone of thyroidal origin, whose main recognised physiological role is the inhibition of osteoclast--mediated bone resorption. There is also evidence that CT might modulate bone formation. However, both CT and its receptors (CTR) have also been identified in a large number of other cell types and tissue sites, suggesting roles for the CT/CTR system distinct from those involving calcium homeostasis. Evidence has accumulated consistent with the involvement of CT in cell growth and differentiation and in tissue development and remodelling. The close proximity of cells expressing CT, or CT receptors (CTR), during development, and during pregnancy and lactation, is consistent with important roles for CT in morphogenesis. It thus appears that, in tissues such as the uterus, breast and pituitary, CT acts in a paracrine manner to Influence cell proliferation and function, as distinct from its endocrine actions to regulate calcium stores in the skeleton. In vitro studies have shown that CT can be either mitogenic or can inhibit cell proliferation, depending on the cell type and the conditions of the experiment. More recently, evidence has also been obtained for a role for CT in cell survival, in cells as diverse as osteoblast--like and osteocyte--like cells, osteoclasts and neurons.

Properties of a calcitonin receptor and adenylate cyclase in BEN cells, a human cancer cell line.

A human lung cancer cell line (BEN cells) was found to have a calcitonin-responsive adenylate cyclase. Various calcitonins and synthetic analogs stimulated adenylate cyclase activity withe same relative potency as they show in lowering blood calcium in the rat. Preincubation of the cells with calcitonin, followed by washing, led to loss of subsequent adenylate cyclase response to hormone. This was a dose-dependent phenomenon. The binding of [125I]salmon calcitonin to freshly subcultured cells was studied. The ability of calcitonins and analogs to compete for binding paralleled their efficacies in stimulating adenylate cyclase. Binding was saturable, reversible, and consisted of a single class of noninteracting sites with a mean Kd of 10.75 X 10(-10) M, K of 0.93 X 10(9)/M, and mean receptor number of 2.71 X 10(4)/cell. It is not known whether the calcitonin receptor is inappropriate to the cell of origin of the tumor. The BEN cells provide a means of isolating and studying the properties of the calcitonin receptor and of evaluating the significance for the tumor of a hormone-responsive adenylate cyclase.

Calcitonin and 1,25-dihydroxyvitamin D3 receptors in human breast cancer cell lines.

Five human breast cancer cell lines (MCF 7, T 47D, BT 20, MDA 157, and MDA 231) and a human breast epithelial cell line (HBL 100) have been found to contain specific high-affinity receptors for 1,25-dihydroxyvitamin D3, Kd values ranged from 0.6 to 2.0 X 10(-11) M and receptor concentration from 31 to 150 fmol/mg cytosol protein. Two of the breast cancer lines (MCF 7 and T 47D) contain specific high-affinity receptors for calcitonin and a calcitonin-responsive adenylate cyclase, which have been characterized with the aid of salmon, eel, and human calcitonins and in several substituted analogues of human calcitonin. The 1,25-dihydroxyvitamin D3 receptor may reflect a normal property of the breast cell. Breast cancer cell lines provide a useful source of 1,25-dihydroxyvitamin D3 receptors. Their coexistence with a calcitonin receptor and biological response in some breast cancers offers the opportunity to investigate new aspects of breast cancer endocrinology.

Presence of 1,25-dihydroxyvitamin D3 receptors in established human cancer cell lines in culture.

Receptors for 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] have been described in several human breast cancer cell lines and more recently in human melanoma. The presence of 1,25-(OH)2D3 receptor (1,25-DR) in two cultured breast cancer cell lines was associated with receptors for calcitonin, another hormone thought to have effects on calcium handling. Therefore, it seemed important to examine a range of established human cancer cell lines for the presence of receptors for 1,25-(OH)2D3 and calcitonin. Thirty-three cancer cell lines were examined. 1,25-DR was found to be present in 23 lines, while calcitonin receptors were not detected in any of them. The 1,25-DR from several cell lines sedimented at about 3.5S in sucrose density gradients, had the appropriate specificity for vitamin D metabolites, had Kds of 0.8 to 2.2 x 10(-11) M, and had receptor concentrations of 12 to 99 fmol/mg protein. Ten malignant melanoma and nine colonic carcinoma lines constituted the largest groups of carcinoma cell lines, and seven and eight, respectively, of these were 1,25-DR positive. The high frequency of 1,25-DR positivity in the cultured colonic carcinoma cells is quite different from the low frequency of 1,25-DR in primary colonic carcinomas. It was also interesting that both of two cell lines derived from patients who had had both bone metastases and malignant hypercalcemia were 1,25-DR positive. These various cell lines may provide useful models for the examination of 1,25-(OH)2D3 action in vitro.

Semaphorin-3a, neuropilin-1 and plexin-A1 in prosthetic-particle induced bone loss.

Peri-prosthetic osteolysis (PPO) occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. Semaphorin-3a (SEM3A), neuropilin-1 (NRP1) and plexin-A1 (PLEXA1) are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This study investigated SEM3A, NRP1 and PLEXA1 protein and mRNA expression in human PPO tissue and polyethylene (PE) particle-stimulated human peripheral blood mononuclear cell (PBMC)-derived osteoclasts in vitro. In addition, the effects of tumour necrosis factor alpha (TNFα) on cultured osteoclasts was assessed. In PPO tissues, a granular staining pattern of SEM3A and NRP1 was observed within large multi-nucleated cells that contained prosthetic wear particles. Immunofluorescent staining confirmed the expression of SEM3A, NRP1 and PLEXA1 in large multi-nucleated human osteoclasts in vitro. Furthermore, SEM3A, NRP1 and PLEXA1 mRNA levels progressively increased throughout osteoclast differentiation induced by receptor activator of nuclear factor κB ligand (RANKL), and the presence of PE particles further increased mRNA expression of all three molecules. Soluble SEM3A was detected in human osteoclast culture supernatant at days 7 and 17 of culture, as assessed by ELISA. TNFα treatment for 72h markedly decreased the mRNA expression of SEM3A, NRP1 and PLEXA1 by human osteoclasts in vitro. Our findings suggest that SEM3A, NRP1 and PLEXA1 may have important roles in PPO, and their interactions, alone or as a complex, may have a role in pathological bone loss progression. STATEMENT OF SIGNIFICANCE: Peri-prosthetic osteolysis occurs in response to prosthetic wear particles causing an inflammatory reaction in the surrounding tissue that leads to subsequent bone loss. The rate of hip and knee arthroplasty is increasing by at least 5% per year. However, these joint replacements have a finite lifespan, with data from the National Joint Replacement Registry (Australia) showing that the major cause of failure of total hip replacements is aseptic loosening. In aseptic loosening, wear particles liberated from prostheses are phagocytosed by macrophages, leading to release of inflammatory cytokines and up-regulation of osteoclast formation and activity. Semaphorin-3a, neuropilin-1 and plexin-A1 are axonal guidance molecules that have been recently implicated in regulating bone metabolism. This is the first report to show that these molecules may be involved in the implant failure.

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+.

Identification of differentially expressed genes between osteoarthritic and normal trabecular bone from the intertrochanteric region of the proximal femur using cDNA microarray analysis.

Osteoarthritis (OA) is a common age-related joint disease resulting in progressive degenerative damage to articular cartilage. The etiology of primary OA has not yet been determined. However, there is evidence supporting the hypothesis that primary OA is a disease affecting bone remodeling in addition to articular cartilage. In this study, we have used cDNA microarray analysis to compare gene expression in bone between normal (CTL) and OA individuals. Trabecular bone was sampled from the intertrochanteric region of the proximal femur, a site distal to the diseased hip joint. Total RNA was extracted from three pairs of age- and sex-matched CTL and OA bone samples, reverse-transcribed and radioactively labeled to generate cDNA probes, before hybridization with the Research Genetics GF211 human gene microarray filter. The CTL and OA samples were found to have similar levels of gene expression for more than 4000 known human genes. However, forty-one genes were identified that were differentially expressed, twofold or more, between all three CTL-OA sample pairs. Using semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis, three genes, fms-like tyrosine kinase 1 (FLT1), plexin B1 (PLXNB1), and small inducible cytokine A2 (SCYA2), were confirmed to be consistently expressed at lower levels in OA, in a majority of twenty age- and sex-matched CTL-OA bone sample pairs tested. FLT1, PLXNB1, and SCYA2 have known or potential roles in angiogenesis and bone remodeling. Down-regulation of these genes is consistent with a role for bone in the pathogenesis of OA.

Calcitonin receptor-mediated growth suppression of HEK-293 cells is accompanied by induction of p21WAF1/CIP1 and G2/M arrest.

We investigated the mechanisms by which calcitonin (CT) suppresses cellular proliferation, using HEK-293 cells stably transfected with either the rat C1a CT receptor (CTR) or the insert-negative form of the human CTR. CT treatment of clonal cell lines expressing either receptor type, but not untransfected HEK-293 cells, strongly suppressed cell growth in a concentration-dependent manner. The reduction in cell growth with CT treatment could not be attributed to cellular necrosis or apoptotic cell death, the latter assessed by both DNA fragmentation analysis and caspase 3 (CPP-32) assay. Growth inhibition was associated with an accumulation of cells in the G2 phase of the cell cycle. CT treatment of the human and rat CTR-expressing cell lines resulted in a rapid and sustained induction of mRNA encoding the cyclin-dependent kinase inhibitor, p21WAF1/CIP1, increased levels of which were maintained at least 48 h after initiation of treatment. Western blot analysis showed a rapid corresponding increase in p21WAF1/CIP1 protein, whereas protein levels of another member of the cyclin-dependent kinase inhibitor family, p27kip1, were unchanged. In parallel with the induction of p21, CT treatment reduced levels of p53 mRNA and protein. CT treatment resulted in a specific cell cycle block in G2, which was associated with inhibition of Cdc2/cyclin B kinase activity as measured by histone H1 phosphorylation. There was no evidence for p21 association with this complex despite the inhibition of Cdc2 activity. Evidence that p21 induction was causative of cell growth suppression was obtained from p21 antisense oligonucleotide experiments. Treatment with a p21 antisense oligonucleotide blocked induction of p21 expression and significantly reduced the CT-mediated growth inhibition. These observations suggest that p21 is required for the G2 arrest in response to CT, but argue against a direct role of p21 in the inhibition of Cdc2 activity. These studies suggest a novel regulation of cell cycle progression by CT and will provide a basis for detailed examination of the molecular mechanisms involved.

Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts.

UMR 201 is a nontransformed rat clonal cell line derived from neonatal calvaria with phenotypic characteristics of preosteoblasts. Retinoic acid strongly induces expression of alkaline phosphatase and its mRNA in these cells. Dexamethasone substantially reduced the retinoic acid-induced expression of alkaline phosphatase. This apparent interaction between dexamethasone and retinoic acid effects raised the possibility that interactions may extend to other osteoblast-related phenotypic characteristics in UMR 201 cells. Treatment with dexamethasone resulted in a decrease in the expression of mRNA for pro-alpha 1(I) collagen, but upon coincubation with 1 microM retinoic acid for 24 h, the decrease in mRNA for pro-alpha 1(I) collagen was abrogated. Dexamethasone (Dex) treatment caused a dose-dependent increase in osteonectin mRNA, half maximally effective between 1 nM and 10 nM Dex. One micromolar of retinoic acid alone led to a small increase in expression of osteonectin mRNA but prevented any further increase when Dex was added to retinoic acid-treated cells. To study transcriptional control, osteonectin genomic fragments were linked to the bacterial reporter gene, chloramphenicol acetyltransferase, and introduced by transfection into UMR 201 cells. Dexamethasone increased the transcriptional activity of an osteonectin-chloramphenicol acetyltransferase construct; 100 nM Dex resulted in a 3-fold increase over control cells which was attenuated when 1 microM retinoic acid was added to the incubation, while retinoic acid alone resulted in a 2-fold increase in transcriptional activity. Finally, it was noted that coincubation with retinoic acid and Dex stimulated the proliferation of UMR 201 cells when compared with either treatment alone. This study shows the potential importance of hormonal interactions in the expression of osteoblast function.

Truncation of the porcine calcitonin receptor cytoplasmic tail inhibits internalization and signal transduction but increases receptor affinity.

A series of mutant porcine calcitonin receptors with progressively truncated carboxy termini have been expressed in COS and HEK 293 cells. All forms of the receptor, including those totally lacking the cytoplasmic tail, were able to bind 125I-labeled salmon calcitonin. However, removal of C-terminal domains resulted in multiple functional changes in the receptor. First, compared with the wild type receptor, affinity of binding of salmon calcitonin was increased for truncated receptors, whether determined in intact transfected cells or in cell membranes. Second, internalization of the ligand-receptor complex was greatly attenuated for mutants truncated by 44 or 83 amino acids but not for an intermediate form truncated by 63 amino acids. Third, truncation affected signal transduction, which for the porcine calcitonin receptor occurs by generation of intracellular cAMP and Ca2+. The magnitude of adenylate cyclase responses was much reduced for the same mutants defective in internalization. Under conditions where expression of each receptor form was approximately equal, the magnitude of intracellular Ca2+ responses was decreased by C-terminal truncation. These results draw attention to the functional significance of the cytoplasmic tail of the porcine calcitonin receptor and suggest intramolecular interactions between the carboxy terminus and other receptor domains and/or cellular regulatory elements.

Identification of brain isoforms of the rat calcitonin receptor.

Two rat calcitonin receptor isoforms have been identified by cDNA cloning from a hypothalamic library. The clones, C1a and C1b, specified proteins of 478 and 515 amino acids, respectively. The clones were identical, except that the C1b sequence encoded a 37-amino acid insert in the second extracellular domain, which conferred altered ligand recognition. Compared to the C1a receptor, expressed C1b receptors exhibited decreased affinity for porcine CT, relative to salmon CT, and negligible affinity for human CT. Clone C1b mRNA was predominately expressed in the brain, whereas mRNA for the C1a clone was present in both brain and peripheral tissues. Both receptors were able to couple functionally to adenylate cyclase. Thus, clone C1b represents a novel brain isoform of the CT receptor with different affinity for CT analogs resulting from an altered second extracellular domain.