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A tumor-derived factor believed to cause hypercalcemia by acting on the parathyroid hormone (PTH) receptor was recently purified, cloned, and found to have NH2-terminal sequence homology with PTH. The 1-34 region of this protein was synthesized, evaluated for its postreceptor effects on the ROS 17/2.8 cell line, and its properties were compared to 1-34 PTH. Both 1-34 human humoral hypercalcemia factor (HCF) and 1-34 PTH stimulated adenylate cyclase with an effective concentration (EC)50 of approximately 1 nM. The extent of stimulation by both peptides was equally enhanced by dexamethasone. They both had a pronounced inhibitory effect on growth in the presence of dexamethasone, with an EC50 of approximately 0.1 nM, reduced alkaline phosphatase (AP) activity by approximately 70% in the absence of dexamethasone and by approximately 80% in the presence of dexamethasone with an EC50 of 0.03 nM, and when present at a concentration of 10 nM, reduced AP mRNA levels (estimated by Northern analysis) by approximately 80% in the presence or absence of dexamethasone. Thus, in addition to similar dose-response curves for adenylate cyclase stimulation, both HCF and PTH produced identical postreceptor effects in ROS 17/2.8 cells. These effects of HCF are probably mediated by the interaction of the tumor-derived factor with the PTH receptor.

Reaction of Acetoacetamide (I) with α, β-unsaturated ketone (II) and aldehyde (XV) is described. Reaction of I with 3-buten-2-one (IIa) gives rise to 3-carbamoyl-2, 6-heptanedione (IIIa), 2-carbamoyl-5-hydroxy-5-methylcyclohexanone (IVa), 6-carbamoyl-3-methyl-2-cyclohexanone (Va), 3-acetyl-6-methyl-3, 4-dihydro-2 (1H)-pyridone (VIa), and 3, 9-dimethyl-2-azaspiro [5, 5] undeca-3, 8-diene-1, 7-dione (VII). Reaction conditions such as catalyst and reaction temperature influence the product formed. Similarly, reactions of I with 3-penten-2-one (IIb), 4-methyl-3-penten-2-one (IIc), 4-phenyl-3-buten-2-one (IId), and 2-benzylideneacetophenone (IIe) afford the corresponding cyclohexanone derivatives (Vb, c, d, e) besides the dihydropyridone derivatives (VIb, d, e). α, β-Unsaturated aldehyde such as crotonaldehyde (XVa) reacts with I to give 3-acetyl-6-hydroxy-4-methyl-2-piperidone (XVIa), 3-acetyl-4-methyl-3, 4-dihydro-2 (1H)-pyridone (XVIIa), and 3-acetyl-6-ethoxy-4-methyl-2-piperidone (XVIIa). Similar reaction of cinnamaldehyde (XVb) with I gives rise to the adduct (XVIb), dihydropyridone derivative (XVIIb), and piperidone derivative (XVIIIb).

Bone formation is precisely regulated by cell-cell communication in osteoblasts. We have previously demonstrated that genetic deletion of Col6a1 or Col12a1 impairs osteoblast connections and/or communication in mice, resulting in bone mass reduction and bone fragility. Mutations of the genes encoding collagen VI cause Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), which have overlapping phenotypes involving connective tissue and muscle. Recent studies have identified COL12A1 gene mutations in patients with UCMD- and BM-like disorders harboring no COL6 mutations, indicating the shared functions of these collagens in connective tissue homeostasis. The purpose of this investigation has been to test the hypothesis that collagens VI and XII have coordinate regulatory role(s) during bone formation. We analyzed the localization of collagens VI and XII relative to primary osteoblasts during osteogenesis. Immunofluorescence analysis demonstrated that collagens VI and XII colocalized in matrix bridges between adjacent cells during periods when osteoblasts were establishing cell-cell connections. Quantification of cells harboring collagen bridges demonstrated that matrix bridges were composed of collagens VI and XII but not collagen I. Interestingly, matrix bridge formation was impaired in osteoblasts deficient in either Col6a1 or Col12a1, suggesting that both collagens were indispensable for matrix bridge formation. These data demonstrate, for the first time, a functional relationship between collagens VI and XII during osteogenesis and indicate that a complex containing collagens VI and XII is essential for the formation of a communicating cellular network during bone formation.

Reaction of β-aminocrotonamide (I) with α, β-unsaturated ketones is described. Benzylideneacetophenone (IIa) reacts with β-aminocrotonamide (I) to give 2-methyl-4, 6-diphenylpyridine-3-carboxamide (IIIa), 3-acetimidoyl-4, 6-diphenyl-3, 4-dihydro-2 (1H)-pyridone (IV) and 6-carbamoyl-3, 5-diphenyl-2-cyclohexenonimine (V). Similar reaction of β-aminocrotonamide (I) with 3-buten-2-one (IIb) and 4-phenyl-3-buten-2-one (IIc) affords 2, 6-dimethylpyridine-3-carboxamide (IIIb) and 2, 6-dimethyl-4-phenylpyridine-3-carboxamide (IIIc), respectively.

The effect of transforming growth factor-beta (TGF beta) on bone in vivo was examined. Twelve daily injections of 1 microgram TGF beta directly onto the periostea of parietal bones of neonatal rats stimulated the formation of periosteal woven bone. The thickness of the treated parietal bones increased at least 2-fold in a dose-dependent manner. This TGF beta effect was localized at the sites of injection, and no change was observed in contralateral parietal bones and tibiae. The body weight in these growing rats was not affected by TGF beta 1. TGF beta 2 had effects similar to those of TGF beta 1 on the parietal bones in vivo. These results reveal for the first time that TGF beta stimulates bone formation in vivo and indicate its anabolic role in local bone metabolism.

Osteocalcin (OC) is one of the abundant non-collagenous bone matrix proteins produced exclusively by osteoblasts, and its serum level is used as an indicator of bone metabolism in patients. Transforming growth factor-beta (TGF beta) is abundant in bones and platelets, promotes wound healing in vivo, and is a potent stimulator of the production of extracellular matrix proteins in fibroblasts and osteoblasts. The effects of TGF beta on OC gene expression were examined in rat osteoblast-like cells, ROS17/2.8. TGF beta 1 decreased OC levels in the culture media 2- to 3-fold. TGF beta 1 also decreased the level of osteocalcin mRNA about 3-fold in a dose-dependent manner. TGF beta 2 and TGF beta 1,2 a heterodimeric form, showed similar effects on OC mRNA levels as TGF beta 1. The suppression of the OC message level was detectable at 24 h and lasted for up to 72 h. This effect on OC mRNA was blocked by cycloheximide. The stability of OC mRNA was not changed by TGF beta 1. On the other hand, the rate of OC gene transcription was reduced 4- to 5-fold, as estimated by in vitro nuclear transcription (run-on) assay. TGF beta 1 blocked the increase in the OC mRNA level induced by PTH or 1,25-dihydroxyvitamin D3. These results indicate that TGF beta inhibits osteocalcin gene expression at least in part through transcriptional control.

Reaction of β-aminocrotonamide (I) with α, β-unsaturated ketones is described. Benzylideneacetophenone (IIa) reacts with β-aminocrotonamide (I) to give 2-methyl-4, 6-diphenylpyridine-3-carboxamide (IIIa), 3-acetimidoyl-4, 6-diphenyl-3, 4-dihydro-2 (1H)-pyridone (IV) and 6-carbamoyl-3, 5-diphenyl-2-cyclohexenonimine (V). Similar reaction of β-aminocrotonamide (I) with 3-buten-2-one (IIb) and 4-phenyl-3-buten-2-one (IIc) affords 2, 6-dimethylpyridine-3-carboxamide (IIIb) and 2, 6-dimethyl-4-phenylpyridine-3-carboxamide (IIIc), respectively.

Controlled reduction of N-methyl-3-ethylpyridinium iodide afforded the corresponding 1,2-dihydropyridine which was stabilised by complexation to give 1,2- and 1,6-dihydro-3-ethyl-N-methylpyridinetr...

Osteocalcin (bone gamma-carboxyglutamic acid-containing protein) is exclusively produced by osteoblasts, which are the major target cells of parathyroid hormone (PTH) in bone. This study examined the effect of human (h) PTH(1-34) on osteocalcin gene expression in the rat osteoblast-like osteosarcoma cells ROS17/2.8. hPTH(1-34) increased in a dose-dependent manner the steady state levels of osteocalcin mRNA 2- to 3-fold with an ED50 of about 5 X 10(-10) M. This effect was detectable at 12 h, peaked at 24 h, and lasted at least up to 48 h. Forskolin, cyclic 8-bromo-AMP, isobutylmethylxanthine, cholera toxin, and (-)-isoproterenol similarly elevated osteocalcin mRNA. hPTH(1-34) did not alter the transcriptional rate of the osteocalcin gene, estimated by nuclear run-on assays, but increased the stability of osteocalcin mRNA. hPTH(1-34) also increased 2- to 3-fold the osteocalcin level in the culture media determined by radioimmunoassay. PTH, thus, promoted osteocalcin gene expression in these cells at least in part through mRNA stabilization via cyclic AMP mediation, a mechanism known only in few systems.

Hypercalcemia is frequently a serious complication of malignancy. Since in certain patients this condition resembles hyperparathyroidism, a PTH-like factor secreted by the tumor was implicated in this syndrome. Indeed, it was found that several tumors produce a peptide which has PTH-like activity: it stimulates renal and bone adenylate cyclase (Rodan et al., 1983; Stewart et al., 1983), produces bone resorptionin vitro and inhibits phosphate uptake in kidney cells (Strewler et al., 1987). Most recently several tumor-derived peptides were partially sequenced and were found to have sequence similarity to PTH (Moseley et al., 1987; Stewart et al., 1987; Strewart et al., 1987). A complementary DNA sequence cloned from a lung carcinoma cell line (BEN) confirmed the similarity of the deduced N-terminal amino acid sequence to that of PTH, and showed very little similarity to the rest of the molecule (Suva et al., 1987).

A synthetic route to the pyridocarbazole alkaloids, olivacine and guatambuine is described. The crucial step in the synthesis utilizes tricarbonylchromium(0) complexes of a suitable dihydropyridine...

Alkaline phosphatase [ALP; orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a ubiquitous enzyme of unknown function expressed at high levels in cells of mineralizing tissues. To study the structure, function, and expression of ALP, a full-length cDNA of rat ALP (2415 bases) was isolated from a ROS 17/2.8 osteosarcoma cell lambda gt10 cDNA library. The predicted amino acid sequence spans 524 residues and includes an N-terminal signal peptide of 17 amino acids, the phosphohydrolase active site, a rather hydrophilic backbone with five potential N-glycosylation sites, and a short hydrophobic C-terminal sequence. ALP negative CHO cells transfected with an expression vector containing the ALP coding sequences express ALP. The rat bone, liver, and kidney ALP shows remarkable 90% homology with the corresponding human enzyme, the most divergent region being the C-terminal hydrophobic domain through which the enzyme may be anchored to the plasma membrane. The rat ALP also shows 50% homology with the human placental and intestinal ALP and 25% homology with the Escherichia coli ALP. The amino acids involved in catalysis show nearly complete homology among all known ALP sequences, suggesting that these enzymes evolved from a common ancestral gene. The rat ALP cDNA pRAP 54, used as a hybridization probe in RNA blot analysis of several tissues that express ALP, revealed the presence of an ALP mRNA of approximately equal to 2500 bases. Furthermore, hybridization patterns derived from Southern blot analysis of rat chromosomal DNA offered molecular evidence that the ALP expressed in ROS 17/2.8 osteosarcoma and various rat tissues, excluding the intestine, is the product of the same single copy gene.

We established a clonal chondrocyte-like cell line (TC6, TC stands for large T immortalized chondrocyte-like cell line) derived from articular cartilage of transgenic mice harboring a temperature-sensitive simian virus 40 large T-antigen gene. TC6 cells exhibited spindle-like or polygonal morphology and grew well at 33 degrees C in alpha-minimal essential medium supplemented with 0.5% fetal bovine serum. After confluence, these cells formed nodules that were positive for staining with alcian blue. Northern blot analysis demonstrated that these cells expressed messenger RNAs (mRNA) of the genes encoding cartilage-specific proteins such as type II procollagen, link protein, and aggrecan. Furthermore, the expression of type II procollagen and link protein genes in TC6 cells was regulated by parathyroid hormone and basic fibroblast growth factor, suggesting the presence of the receptors for the hormone and cytokine. The expression of link protein mRNA in TC6 cells was regulated in a time-dependent manner and was enhanced in culture within a week and increased continuously up to 10-fold by the end of 4 weeks. Expression of mRNAs encoding type II procollagen and versican/PG-M also increased moderately during the culture period. TC6 cells expressed type I procollagen mRNA, however, its level declined along with time in culture in contrast to the enhancement of the genes encoding cartilage-specific molecules in these cells. Interestingly, alkaline phosphatase mRNA expression was barely detectable in the TC6 cells in their growing phase while it was enhanced dramatically more than 7-fold by day 14 in culture. These results indicate that the TC6 cells could serve as an excellent model for the studies on chondrocyte physiology.

Tibial shafts from 15-day-old chick embryos were cultured on chorioallantoic membranes of chick embryos. These bones were stimulated by either constant-direct or constant-alternating current via platinum wire electrodes. Around the anodes of 10 microA constant direct-current stimulation, the number of osteoclasts was significantly larger than that of the control, and numerous osteoblasts were observed. Around the cathodes of this current, periosteum was thickened and accompanied by proliferation of osteoblasts. Slight bone resorption in the immediate vicinity of the cathodes was also observed. The periosteum between the two electrodes was significantly thickened by this current. The comparative study between equivalent constant direct-current and alternating-current stimulation showed a dominance of direct current. This model could be useful in investigations on electrical stimulation of bone in vivo. It produces fewer artifacts and shows activation of osteoclasts in association with activation of osteoblasts in electrically induced reactions of bone tissue.

The aim of this study is to explore the expression of the BMP−2, −4 and −7 and their extracellular antagonists (noggin, chordin, follistatin and gremlin) in synovial membrane tissue during osteoarthritic processes of the knee joint in humans. The purpose of this investigation is to find the possible role of the BMPs antagonists in osteophytic outgrowth regulation. Since osteophytic growth is characteristic for the degenerative joint disease, we hypotheses that synovial membrane is the site of the molecular factors production which are proved to be stimulative for osteophytic growth. The study was performed on series of 14 synovial tissue samples collected during knee joint surgery of the individuals undergoing total joint replacement. As a control samples we used synovial tissue from the autopsy cases, without any degenerative or inflamatory changes in knee joint tissue. The tissue samples were embedded in parafin, cut on 5 micrometers thin slices and immunostained with specific antibodies against defined BMPs and BMPs antagonists. BMP−2 and−4 were not detected by immunohistochemistry in OA synovial tissue. This finding was additionally proved by postive (positively stained subependimal cells in mouse brain) and negative control (negatively stained synovial cells in immunoprotocol without primary antibodies). BMP−7 was strongly expressed in the lining layer cells (intracytoplasmatic brown staining). Noggin and chordin were expressed in both layers of the synovial epithelium and also in the stromal fibroblasts and extracellular matrix. Noggin expression was found in higher percentage of the cells when compared to chordin. Gremlin was expressed in the lining layer of the synovial epithelium and also inside the blood vessels. Imunostaining for gremlin was detected as a membranous staining. Distribution of the follistatin was not equally expressed in all the samples. But it was regularly positive in the wall of the blood vessels. The BMPs were differentially expressed in synovial tissue from normal and OA knee joint. In OA synovial membrane BMP−2 and −4 were negative in contrast to BMP−7 which is strongly expressed in the lining layer cells. BMP antagonists were also differentially expressed in OA synovial tissue: gremlin and noggin were predominantly expressed in the lining layer in contrast to follistatin and chordin which were predominantly expressed in perivascular synovial tissue fibroblasts.