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Recently, we cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines, embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. Mouse A-C1 has homology with a ras-responsive gene, rat Ha-rev107 (Hrasls ), and modulates a Ha-ras-mediated signaling pathway. Here, we report a cDNA encoding a human homolog of mouse A-C1. The deduced amino acid sequence of human A-C1 consists of 168 amino acids, and shows 83% identity with that of mouse A-C1. Human A-C1 mRNA was expressed in skeletal muscle, testis, heart, brain, and thyroid in vivo. Moreover, expression of human A-C1 mRNA was detected at a high level in human osteosarcoma-derived U2OS cells in vitro. By FISH analysis the human A-C1 gene (HRASLS) was mapped to human chromosome 3q28→ q29. Copyright © 2001 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We examined 17 patients with primary hyperparathyroidism for their serum 1,25 dihydroxyvitamin D levels and for their fractional intestinal calcium absorption rates using a whole body counter and calcium-47. As controls, 10 normal volunteers were examined both before and after administration of 1 alpha-hydroxyvitamin D to increase serum 1,25 dihydroxyvitamin D. Values of serum 1,25 dihydroxyvitamin D were 71.6 +/- 37.6 pg/ml (mean +/- SD) in patients with primary hyperparathyroidism and 75.3 +/- 27.7 pg/ml (mean +/- SD) in normal volunteers after administration of 1 alpha-hydroxyvitamin D, while values of intestinal calcium absorption rate were 61.5 +/- 16.5% (mean +/- SD) in patients with primary hyperparathyroidism and 34.1 +/- 5.1% (mean +/- SD) in normal controls, respectively. There was a positive correlation between values of serum 1,25 dihydroxyvitamin D and intestinal calcium absorption in both groups. However, in patients with primary hyperparathyroidism, intestinal calcium absorption was more increased than that in normal volunteers when compared to their serum values of 1,25 dihydroxyvitamin D. This suggests that another factor than 1,25 dihydroxyvitamin D plays an important role in the intestinal calcium absorption in patients with primary hyperparathyroidism. [ABSTRACT FROM AUTHOR]

Synthetic human calcitonin gene-related peptide (CGRP) was examined for the action on the bone in vitro. Human CGRP inhibited bone resorption stimulated by both human parathyroid hormone and basal. The mode of inhibitory action of human CGRP seemed to be similar to that of calcitonin and the relative potency of human CGRP to inhibit bone resorption is one five-hundredth of that of human calcitonin. Thus, a novel pharmacological action of CGRP was demonstrated. [ABSTRACT FROM AUTHOR]

A 67-year-old woman who presented with hypocalcemia compatible with idiopathic hypoparathyroidism gradually changed into a state of primary hyperparathyroidism. The left upper parathyroid gland, which was larger and harder than other glands, was resected. Despite the operation, hypercalcemia and high levels of intact PTH persisted. Six weeks later total parathyroidectomy was done to induce remission. The resected gland in the first operation had clusters of lymphoid follicles with germinal centers indicating a chronic autoimmune inflammation. This case suggests a transition from hypoparathyroidim to hyperparathyroidism associated with chronic parathyroiditis, possibly by a mechanism analogous to that observed in chronic thyroiditis.

In children with end-stage liver disease, little is known regarding the long-term effects of liver transplantation on bone. In this 2-year prospective study, we evaluated the effects of liver transplantation on bone mineral density (BMD) and on other parameters of bone metabolism in 30 consecutive children with biliary atresia who underwent liver transplantation after failed Kasai operation. Tacrolimus and steroids were used as immunosuppressants. BMD of the first through fourth lumbar spine (L1-4) was measured by dual-energy radiographic absorptiometry (QDR-2000, Hologic) and expressed in Z-values. Before transplantation, Z-values of BMD, height, and weight were low in all patients (-3.4 +/- 0.34, -2.0 +/- 0.28, and -1.67 +/- O.16 [mean +/- SEM], respectively). Low BMD states were associated with low levels of serum 25-hydroxyvitamin D and serum levels of insulin-like growth factor-I (IGF-I), one of the most potent anabolic skeletal growth factors. Liver transplantation resulted in marked improvement in BMD values as well as in height and body weight: 24 months after transplantation, recovery was achieved in each parameter (0.16 +/- 0.30, -0.29 +/- 0.23, and 0.42 +/- 0.18, respectively). BMD recovery first was observed 3 months after transplantation. Moreover, these favorable effects of transplantation were accompanied by increases in serum levels of 25-hydroxyvitamin D and IGF-I. We conclude that liver transplantation effectively reverses the low bone mass status and growth retardation of children with end-stage liver disease.

The bone morphogenetic proteins (BMPs) play crucial roles in chondrogenic differentiation. Little is known, however, regarding the regulation of BMP gene expression. Here we examined the effect of parathyroid hormone-related peptide (PTHrP) (1-141), a full-length form of PTHrP molecules, on the expression of BMP-4 mRNA in clonal mouse chondrogenic EC cells, ATDC5. In differentiated ATDC5 cells, the expression of BMP-4 mRNA was inhibited by PTHrP (1-141), which stimulated cAMP accumulation and protein kinase A (PKA) activity in these cells. Dibutyryl cAMP, a permeable analog of cAMP, mimicked and H-89, a selective PKA inhibitor, blocked this effect of PTHrP (1-141). Moreover, actinomycin D attenuated the inhibition of BMP-4 mRNA expression by PTHrP (1-141). These results indicate that PTHrP (1-141) transcriptionally inhibits the expression of BMP-4 mRNA through a cAMP/PKA pathway in ATDC5 cells.

Tumour necrosis factor (TNF)-alpha causes the degradation of articular cartilage in arthritis via direct actions on chondrocytes. However, it remains unknown whether TNF-alpha affects chondrogenesis in chondroprogenitors. In the present study, we assessed the effects of TNF-alpha in vitro on chondrogenesis using mouse clonal chondrogenic EC cells, ATDC5. TNF-alpha (10 ng/ml) stimulated [3H] thymidine incorporation in undifferentiated ATDC5 cells, and suppressed cartilaginous nodule formation and the accumulation of cartilage-specific proteoglycan. We recently showed that undifferentiated ATDC5 cells express BMP-4 and that exogenously administered BMP-4 promotes chondrogenesis in these cells. Interestingly, TNF-alpha up-regulated the expression of BMP-4 mRNA in undifferentiated ATDC5 cells in time- and dose-dependent manners. However, exogenously administered BMP-4 was not capable of reversing the inhibitory action of TNF-alpha on chondrogenesis in ATDC5 cells. These results indicate that TNF-alpha stimulates both cell proliferation and BMP-4 expression but inhibits chondrogenesis in chondroprogenitor-like ATDC5 cells.

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.

We evaluated the roles of bone morphogenetic protein (BMP)-6, BMP-4 and parathyroid hormone-related protein (PTHrP) in the hypertrophic conversion using mouse chondrogenic EC cells, ATDC5. In ATDC5 cells, the expression of BMP-6 and PTHrP receptor mRNAs increased in parallel with the progression of chondrogenic differentiation of these cells, exhibiting a time course similar to that of type II collagen, a phenotypic marker of proliferating chondrocytes, while BMP-4 mRNA was continuously expressed throughout the differentiation processes. The expression of type X collagen mRNA, a phenotypic marker of hypertrophic chondrocytes, was upregulated by BMP-6 and BMP-4, and downregulated by PTHrP(1-141). The expression of BMP-6 mRNA was upregulated while that of BMP-4 mRNA was downregulated by both BMP-6 and BMP-4. Moreover, the expression of BMP-6 mRNA was downregulated by PTHrP(1-141). Furthermore, even in the presence of PTHrP(1-141), BMP-6 increased the transcript level of type X collagen in a dose-dependent manner. These results indicate that transiently expressed BMP-6 promotes the hypertrophic conversion in association with the augmentation of BMP-6 gene expression by BMP signals and that both BMP-6 and PTHrP coordinately regulate the rate of the hypertrophic conversion of ATDC5 cells.

Ihh is a secreted protein expressed in chondrocytes in cartilaginous soft callus and thought to be involved in regulation of chondrogenic differentiation in fracture repair processes. However, gene expression and function of Ihh and its signaling molecules, Ptc and Smo, at the initial stage of fracture repair remain unknown. In the present study, we showed by RT-PCR of mouse rib fractures that the upregulation of Ihh mRNA occurred within hours after fracture, immediately followed by that of Ptc mRNA, and that both Ihh and Ptc mRNAs exhibited the time course similar to those of OP and OC mRNAs at the initial stage of fracture repair. The transcript level of Smo mRNA gradually increased within hours after fracture and was continuously maintained throughout the subsequent fracture repair processes. By in situ hybridization analysis, the transcripts of Ptc and Smo genes localized in bone marrow of unfractured ribs, and those of Ihh, Ptc, and Smo were expressed in the vicinity of the fracture site at 8 h after fracture. Furthermore, in adherent bone marrow cells in culture, mrIhh-N upregulated the gene expression of TGF-beta(1) as well as OPGL, a potent stimulator of osteoclastogenesis and osteoclast activity. These observations suggest that Ihh may play roles in the initial stage of fracture repair via TGF-beta(1) and OPGL., (Copyright 1999 Academic Press.)

TAK-778, a novel synthetic 3-benzothiepin derivative, stimulates the formation of cartilaginous nodules in mouse chondroprogenitor-like ATDC5 cells in vitro in association with upregulation of the gene expression of transforming growth factor-beta(2), but not bone morphogenetic protein-4 and insulin-like growth factor-I. One-shot injection of the TAK-778-containing sustained-release microcapsules accelerated the repair process of the full thickness defects of articular cartilage in rabbit knees. Our in vitro and in vivo results indicate that TAK-778 may be a therapeutically useful synthetic agent for articular cartilage repair., (Copyright 1999 Academic Press.)

Here we report the gene expression and regulation and the function of noggin in clonal mouse chondrogenic EC cells, ATDC5. In ATDC5 cells, the expression of Noggin mRNA increased in parallel with the progression of chondrogenic differentiation. The treatment with conditioned medium of noggin-transfected COS-7 cells decreased the levels of type II and type X collagen gene transcripts of differentiated ATDC5 cells in a dose-dependent manner, and this inhibitory action was reversed by exogenously administered BMP-4 in a dose-dependent manner. The steady-state level of noggin gene transcripts was markedly upregulated by exogenously administered BMP-4 in time- and dose-dependent manners. Furthermore, this stimulatory effect of BMP-4 was attenuated by treatment with actinomycin D, but not with cycloheximide. These results indicate that noggin and BMP-4 coordinately regulate the progression of chondrogenic differentiation in ATDC5 cells., (Copyright 1999 Academic Press.)

Endochondral bone formation includes a cascade of cellular events such as proliferation, maturation, hypertrophic conversion and calcification of chondrocytes and the cartilage replacement by bone. During these processes, hypertrophic conversion and calcification of chondrocytes (the late-phase differentiation) is a crucial process of chondrogenic differentiation. Indian hedgehog (Ihh), a secreted protein expressed in early hypertrophic chondrocytes, is thought to be involved in regulation of hypertrophic conversion via a feedback loop through the perichondrium. In the present study, we showed by Northern analysis and in situ hybridization that Smoothened (Smo), a key component in hedgehog signal transduction, was expressed in chondrocytes in both adult mice and mouse embryos at 16 days post-coitum in vivo, suggesting that Ihh directly acts on chondrocytes. We previously reported that Ihh, Patched and Smo were all expressed in differentiated ATDC5 cells. Exogenously administered mouse recombinant N-terminal protein of Ihh (mrIhh-N) upregulated the gene expression of type X collagen, a phenotypic marker of hypertrophic chondrocytes, as well as osteoprotegerin ligand (OPGL), a potent stimulator of osteoclastogenesis and osteoclast activity, while it did not modulate the expression of Ihh itself, bone morphogenetic protein (BMP)-4, BMP-6, transforming growth factor (TGF)-beta1 and TGF-beta2 in differentiated ATDC5 cells. Moreover, when added to the osteoclast cultures, mrIhh-N markedly stimulated the formation of resorption pits on dentine slices. Our data support the hypothesis that Ihh stimulated the late-phase chondrogenic differentiation in differentiated ATDC5 cells and upregulated the gene expression of OPGL in these cells., (Copyright 1999 Academic Press.)

We cloned a full-length cDNA encoding a novel mouse protein, A-C2, by differential display method using mouse embryonic fibroblast C3H10T1/2 cells and mouse chondroprogenitor-like EC cells, ATDC5. The deduced amino acid sequence of A-C2 consisted of 106 amino acids with no significant homology to the sequences previously reported. Northern blot analysis showed two major bands of 2.1 and 1.8 kb sizes. Expression of A-C2 mRNA was exclusive to ATDC5 cells at their undifferentiated stage. None of ATDC5 cells at their differentiated stage and adult mice tissues examined expressed A-C2 gene.

Background: We retrospectively studied the results of diagnostic imaging using 3 different modalities to determine their usefulness for preoperative localization of the parathyroid, and whether accurate preoperative localization information could be used to modify the surgical approach for parathyroidectomy in patients with primary hyperparathyroidism., Methods: Images of 37 parathyroid adenomas or hyperplasias in 35 patients with primary hyperparathyroidism were obtained using ultrasonography, computed tomography, and subtraction scintigraphy (using thallium 201 [thallous chloride] and either iodine 123 or technetium 99m pertechnetate [99mTcO4-])., Results: Approximately three fourths of the adenomas or hyperplasias were successfully identified by ultrasound (76.7%) and computed tomography (76.4%), even when the weight of the tumor was less than 500 mg. However, subtraction scintigraphy was of limited use (61.3% successfully identified). A combination of these modalities gave excellent results for detecting adenomas and hyperplasias, leading to an accurate prediction rate of 96.0%., Conclusion: We conclude that using the combination of these 3 imaging modalities is very useful for the detection of parathyroid adenomas and hyperplasias, and that with such accurate localization information, the unilateral approach alone, or even simple excision of the parathyroid tumors might be feasible, enabling less invasive surgical treatment.

Hedgehog (hh) family proteins appear to use the conserved targets in their signalling pathway including Patched (Ptc), Smoothened (Smo), and Gli. Although Indian hedgehog (Ihh) plays an important role in endochondral bone formation, the involvement of hh signalling molecules in skeletogenesis is unknown. We cloned a mouse (m) Smo cDNA and studied the expression patterns of Ihh, Ptc, Smo, and Gli mRNAs in mouse chondrogenic EC cells, ATDC5. The deduced amino acid sequence of mSmo consisted of 793 amino acids and was 98 and 93% homologous to the rat (r) Smo and human (h) Smo, respectively. In ATDC5 cells, the expression of Ihh mRNA paralleled that of type X collagen mRNA. Smo, Ptc, and Gli mRNAs were constitutively expressed throughout chondrogenesis and the subsequent cartilage differentiation processes except for the transient decrease in Ptc mRNA at the cellular condensation stage. Our data suggest that hh signalling molecules may be involved in chondrogenesis and cartilage differentiation in ATDC5 cells.

High levels of immunoreactive and biologically active parathyroid hormone-related peptide (PTHrP) were detected in synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The levels of PTHrP immunoreactivity in synovial fluids, measured by a two-site immunoradiometric assay (IRMA) which detects hPTHrP(1-72) or longer peptides and a radioimmunoassay (RIA) specific to the carboxy-terminal portion of hPTHrP, were 3.2 +/- 0.3 pmol of hPTHrP(1-86)/l and 61 +/- 7.0 pmol of hPTHrP(109-141)/l in OA patients (mean +/- SE, n = 23), and 4.8 +/- 0.8 pmol of hPTHrP(1-86)/l and 164 +/- 30 pmol of hPTHrP(109-141)/l in RA patients (n = 26). Synovial fluid PTHrP levels distributed above the normal plasma reference ranges in each assay (0.7-2.6 pmol of hPTHrP(1-86)/l; 16-60.6 pmol of hPTHrP(109-141)/l). After concentration using sequential cation-exchange and reverse-phase chromatography, synovial fluid exhibited the activity that stimulated cyclic adenosine monophosphate (cAMP) accumulation in osteoblastic ROS 17/2.8 cells expressing PTH/PTHrP receptors. The cAMP accumulation activity in synovial fluid was sensitive to coincubation with excess hPTHrP(3-40), a PTH/PTHrP receptor antagonist, and was completely neutralized by preincubation with a monoclonal antibody specific to hPTHrP but not PTH. Immunohistochemical analysis of RA synovium revealed that PTHrP was localized in fibroblast-like cells in the synovial pannus invading articular cartilage. Our data show that PTHrP is produced locally by the diseased synovial tissue and released into synovial fluid at high concentrations, allowing us to hypothesize that PTHrP plays a novel role as a paracrine/autocrine factor in the pathology of OA and RA.

We conducted a retrospective analysis to evaluate the risk factors associated with the occurrence of acute hepatic failure following transcatheter arterial embolization (TAE) for hepatocellular carcinoma. From 1984 to 1993 we performed a total of 623 embolization procedures in 369 patients with both hepatocellular carcinoma and chronic liver disease. Within 2 weeks after TAE, 13 patients (2.1%) experienced hepatic failure as characterized by a rapid increase in serum bilirubin levels and the development of hepatic encephalopathy of grade 2 or higher. These results indicated that the following are risk factors for acute hepatic failure after TAE: poor hepatic functional reserve; high-dose infusion of chemotherapeutic agents, and a history of multiple embolization procedures.

Purpose: We investigated the acid-base balance and bone mineral status in patients with 3 types of urinary intestinal diversion., Materials and Methods: Of 46 men with urinary intestinal diversions 20 had a Kock pouch, 15 had an Indiana pouch and 11 had an ileal conduit. Acid-base balance was assessed by arterial blood gas analysis. Bone mineral status was measured by urinary pyridinium cross-links and dual energy x-ray absorptiometry. In addition, urinary deoxypyridinoline was measured in 79 patients., Results: Of the 46 patients 7 (15%) with the Kock pouch (1), Indiana pouch (5) and ileal conduit (1) had metabolic acidosis associated with significantly lower bone mineral densities (p < 0.05) and higher urinary pyridinium cross-links (p < 0.005) than did those with normal acid-base status. No difference was found in metabolic acidosis and bone demineralization among the 3 groups. Additionally, in 79 patients urinary deoxypyridinoline reached the highest level immediately postoperatively and then gradually decreased to the stable level within 1 or 2 years., Conclusions: Metabolic acidosis following urinary intestinal diversion results in bone demineralization. The types of diversion did not cause differences in metabolic acidosis and bone resorption. Bone has a major role in buffering acid overload in the early postoperative period.

A rare case of functioning oxyphil parathyroid adenoma associated with primary hyperparathyroidism and marked hungry bone syndrome was revealed in a 29-year-old man with hypercalcemia and elevated circulating parathyroid hormone (PTH) level. A large parathyroid tumor weighing 8.4 g was resected and proved to be an oxyphil adenoma. Hypocalcemia was sustained after the operation, despite intensive calcium supplementation. During the postoperative 8 months, bone mineral density at the lumbar spine increased dramatically from 0.892 g/cm2 to 1.244 g/cm2, and whole body bone mineral content increased from 1,913.4 g to 2,419.2 g. This case gives insight to the reversibility of bone loss in this disorder.

Here we report the effects of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vitro on the growth and chondrogenesis of a chondroprogenitor-like clonal mouse EC cell line, 10(-10) to 10(-7) M ATDC5. 1,25(OH)2D3 inhibited [3H]thymidine incorporation in undifferentiated chondroprogenitor-like ATDC5 cells in time- and dose-dependent manners. 1,25(OH)2D3 suppressed cartilage-nodule formation and the accumulation of cartilage-specific proteoglycan in ATDC5 cells in a dose-dependent manner. The 1,25(OH)2D3-induced inhibition of cartilage-nodule formation was reversible and direct, unrelated to the antiproliferative action of the hormone on the undifferentiated ATDC5 cells. ATDC5 cells even in the precartilaginous stage expressed 4.4 kb vitamin D receptor (VDR) mRNA as assessed by northern blot analysis. The equilibrium saturation binding experiment revealed the presence of a single class of saturable and high-affinity binding sites for 1,25(OH)2D3 in the cytosols. These results provide evidence for the hypothesis that both recruitment and chondrogenesis of chondroprogenitors are negatively regulated by 1,25(OH)2D3 via a VDR-mediated process in vivo.

Urinary metabolites of N-telopeptide of type 1 collagen cross-links (NTx) are known as a bone resorption marker. We performed a multi-center trial of NTx measurement in evaluation of bone metastasis. In total, 251 patients with or without bone metastasis from various malignancies were studied. Comparing with other bone markers such as urinary total deoxypyridinoline, osteocalcin, and bone specific alkaline phosphatase, NTx was the most sensitive one to detect bone metastasis and its levels correlated well with the extensiveness of bone metastasis. Measurement of NTx will be useful to determine to order bone scan in patients with malignancy and to monitor the clinical course in patients with bone metastasis.

We report a case of histologically proven intraosseous hemangiomatosis in which marked accumulation of pentavalent technetium-99m-dimercaptosuccinic acid (99mTc(V)DMSA) and technetium-99m-hydroxymethylene diphosphonate (99mTc-HMDP) was observed in the osteolytic hemangiomatous lesions.

Here we report that bone morphogenetic proteins 2 and 3 (BMP-2 and BMP-3) induced marked expression of c-fos mRNA in a biphasic manner, i.e. the late phase (48 to 60 h) as well as the immediate-early phase (0.5 h), in murine osteoblastic MC3T3-E1 cells in vitro. The BMP-induced late phase c-fos gene expression was temporally associated with the onset of marked expression of the genes for osteocalcin and alkaline phosphatase, differentiation markers of mature osteoblasts. In contrast, none of TGF-beta 1, 10% FBS, IGF-I and IGF-II, which induced only the immediate-early c-fos mRNA expression, stimulated the expression of osteocalcin and alkaline phosphatase genes. These data suggest that in osteoblasts BMP-2 and BMP-3 induce the late phase expression of c-fos, which may play a role in transcriptional activation of the genes involved in differentiation of osteoblasts.

We tested the effects of 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3), 2 beta-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 (ED-71) and dexamethasone on osteocalcin mRNA levels in rat tibiae in vivo. Northern blot analysis showed that both 1,25-(OH)2D3 and ED-71 caused an increase in osteocalcin mRNA levels in bone: 1,25-(OH)2D3 induced a transient increase in the mRNA levels followed by a decrease in the control level by 12 h post administration. In contrast, ED-71 caused a persistent increase in osteocalcin mRNA level for seven days post administration. Serum osteocalcin levels paralleled the osteocalcin mRNA level in bone in both groups. Dexamethasone caused a marked reduction in both osteocalcin mRNA and serum osteocalcin levels. Suppressive effect of dexamethasone on osteocalcin expression was persistent for seven days at higher dose. Our results represent the first demonstration of the effect of active vitamin D and corticosteroid on the expression of osteocalcin mRNA in bone in vivo.

We studied the effects of highly purified bone morphogenetic protein 2 and 3 (BMP-2 and -3) on growth plate chondrocytes and osteoblastic cells in vitro and compared to TGF-beta. A mixture of BMP-2 and 3 (BMPs) strongly stimulated DNA synthesis of chondrocytes in the presence of fibroblast growth factor (FGF). BMPs induced rapid maturation of chondrocytes at a growing stage: BMPs transformed the cells into rounded cells and induced marked accumulation of cartilage matrix; TGF-beta slightly reduced matrix accumulation and changed cell morphology into spindle-like in the presence of FGF. Moreover, exposure of chondrocytes to BMPs resulted in a dramatic increase of the putative approximately 80 kD PTH receptors expressed on the cell surface. In multilayered chondrocytes at the calcifying stage, BMPs stimulated alkaline phosphatase (ALPase) activity but TGF-beta inhibited it. In osteoblastic MC3T3-E1 cells, BMPs were found to be the most potent stimulator of ALPase activity thus far described: ALPase in the cells treated with approximately 100 ng/ml of BMPs reached 5- to 20-fold over the basal, whereas TGF-beta inhibited expression of ALPase activity in these cells. The stimulatory action of BMPs overrode the inhibition of ALPase activity by TGF-beta when the cells were incubated with TGF-beta and BMPs. BMPs also upregulated expression of the approximately 80 kD PTH receptor on the cells. These results suggest that BMPs have unique biologic activities in vitro that lead to growth and phenotypic expression of cells playing a critical role in endochondral bone formation.

We examined receptor occupation, calcium mobilization and amylase release for cholecystokinin octapeptide (CCK-8) within a 3-min incubation period at 37 degrees C using dispersed acini from rat pancreas. Analysis of competitive binding inhibition data obtained after a 3-min incubation revealed the presence of only a single class of CCK receptors, while two classes of CCK receptor, i.e., high-affinity and low-affinity CCK receptors, were detected when binding reached a steady-state after a 60-min incubation. The IC50 of CCK receptors calculated from the 3-min binding data was 19.0 +/- 0.5 nM (mean +/- S.D.), close to the Kd of the low-affinity CCK receptors determined by equilibrium binding studies. Exposure of fura-2-loaded acini to 10-1000 pM CCK-8 caused an immediate and dose-dependent increase in [Ca2+]i followed by a gradual decrease in [Ca2+]i. The CCK-stimulated amylase release after 3 min of incubation was biphasic; amylase release increased over the dose range of 3-300 pM CCK-8, peaked at 300 pM CCK-8 and decreased with supramaximal concentrations of CCK-8. Our data suggest that occupation of the low-affinity, but not the high-affinity, CCK receptors is more directly associated with calcium mobilization and subsequent stimulation of amylase release in rat pancreatic acini.

Purified native forms of human parathyroid hormone-related peptide (PTHrp) have recently been reported to display biological activities characteristic of transforming growth factor beta (TGF-beta). The TGF-beta-like property of PTHrp may reside within the amino N-terminal PTH-receptor binding region of the polypeptide, since a synthetic analog corresponding to amino acids 1-36 of human PTHrp is as active as purified native PTHrp in bioassays specific to TGF-beta. Complete lack of structural similarity between PTHrp and TGF-beta prompted us to address the question whether copresence of the TGF-beta-like and PTH-like biological activities in the N-terminal sequence of the PTHrp molecule is a general phenomenon observable with different N-terminal PTHrp peptides of varying amino acid chain length in a variety of target cells that respond in defined ways to TGF-beta in vitro. Two forms of synthetic N-terminal human PTHrp, PTHrp-(1-34) and [Tyr40]PTHrp-(1-40), which are fully active in conventional assays for PTH/PTHrp, were tested for effects in three in vitro bioassay systems for TGF-beta: 1) stimulation, and 2) inhibition, respectively, of epidermal growth factor-dependent soft-agar colony formation of either normal rat kidney-derived fibroblasts (NRK 49F) or human lung carcinoma cells (A549); and 3) biosynthesis of metabolically labeled fibronectin in both NRK 49F cells and clonal osteoblastic rat osteosarcoma cells (ROS 17/2.8). Human TGF-beta over the dose range of 2.5-80 pM significantly stimulated or inhibited soft-agar colony formation of either NRK 49F or A549 cells, respectively, and caused a severalfold increase in biosynthetically labeled [35S]fibronectin in NRK 49F and ROS 17/2.8 cells. In contrast, none of PTHrp-(1-34), [Tyr40]PTHrp-(1-40), and synthetic human PTH-(1-34), each tested at 0.1-10 nM, displayed detectable biological activity in any of the three assay systems. In addition, covalent cross-linking of intact NRK 49F and ROS 17/2.8 cells with either [125I]TGF-beta or 125I-[Tyr40] PTHrp-(1-40) revealed the presence of several distinct affinity-labeled receptor species for TGF-beta in both cell types and the 80K PTH/PTHrp receptors in ROS 17/2.8 cells. The affinity-labeled TGF-beta receptor species were insensitive to excess PTHrp and PTH peptides, and the 80K PTH/PTHrp receptors were insensitive to excess TGF-beta, indicating that PTHrp and TGF-beta do not cross-react with respect to receptor binding for interaction with these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

We examined the effects of epidermal growth factor (EGF) on the anchorage-dependent and -independent growth of four human squamous carcinoma cell lines that overexpress EGF receptors. While EGF inhibited anchorage-dependent growth, it stimulated anchorage-independent growth of all four cell lines tested. The results suggest that the proliferative responses to EGF are characterized by a preference for anchorage-independent, rather than -dependent growth, in cells overexpressing EGF receptors. Moreover, as EGF has been shown to stimulate the in vivo growth of squamous carcinoma cells overexpressing EGF receptors, it is also suggested that the in vitro EGF responsiveness of these cells in soft agar, but not in monolayer, better correlates with the in vivo EGF responsiveness.

Rapid measurement of serum intact parathyroid hormone concentration was achieved by modification of an immunoradiometric assay for the hormone. Incubation of serum samples for 15 min at 37 degrees C under shaking gave optimal results in terms of assay variance and reproducibility: intra-assay CVs were less than 10% over the hormone concentrations of 11-1,600 pg/ml; intra- and inter-assay CVs for two control sera at different hormone levels were less than 12%. The minimal detectable hormone concentration was found at 27.8 pg/ml. The serum hormone levels of 43 subjects (31 health subjects, 9 patients with primary hyperparathyroidism, and 3 patients with secondary hyperparathyroidism) determined by either rapid or regular assay well correlated with each other (r2 = 0.979, p less than 0.001). In two patients with parathyroid adenoma serum intact PTH levels fell rapidly to 12.1% of the preoperative values 20 min after ligation of the vascular pedicle to the hyperfunctioning glands. We conclude that the modified assay protocol allows rapid, accurate, and simple estimation of intact PTH concentrations, and can be used as an intraoperative measure to aid both diagnosis and surgical cure of hyperparathyroidism.

To evaluate the temporal features of physiological fluctuation in serum PTH concentration, we sampled peripheral blood at 4-min intervals for 24 h from five normal men (32.8 yr; range, 26-40 yr) and measured serum PTH levels using a two-site immunoradiometric assay with the exquisite sensitivity and specificity for human PTH-(1-84) (intact PTH). The resultant 24-h time series of serum intact PTH levels were assessed by contemporary techniques in chronophysiology for rhythmic and episodic peak detection. Cosinor analysis disclosed a significant circadian rhythm in serum intact PTH concentrations in all five men, with the mean circadian amplitude and acrophase of 7.2 +/- 4.4 ng/L and 2305 +/- 401 h, respectively (mean +/- SD; n = 5). No apparent fixed ultradian periodicity was found by autocorrelation and spectral analyses. Evaluation of episodic intact PTH pulsatility by Cluster analysis revealed 23.0 +/- 4.4 discrete PTH pulses/24 h (P less than 0.01 vs. signal-free noise), which occurred at an interpulse interval of 61.6 +/- 11.1 min. The average duration of a serum intact PTH peak was 42.8 +/- 7.3 min, and its mean incremental amplitude was 12.6 +/- 1.3 ng/L, which corresponded to a 31.8 +/- 5.2% increase above the preceding nadir. Discrete PTH peaks were separated by nonpulsatile valleys which lasted for 17.9 +/- 4.4 min. Cross-correlation between the time series of serum intact PTH and whole blood ionized calcium (Ca2+) was at its maximum (-0.5) at concurrent time points in three subjects, while significant positive correlation between serum intact PTH and simultaneous serum inorganic phosphorus concentrations was observed in four of five subjects. There was no apparent correlation between the levels of serum intact PTH and serum magnesium. Our data show that serum levels of intact PTH, the only biologically active form of PTH in the blood, is characterized by a significant circadian periodicity, spontaneous episodic pulsatility with distinct peak properties, and a significant temporal coupling with Ca2+ and inorganic phosphorus concentrations. We conclude that PTH secretion, as judged by the temporal pattern of serum intact PTH levels, is pulsatile in normal men.