Effects of interleukin-1 on syntheses of alkaline phosphatase, type X collagen, and 1,25-dihydroxyvitamin D3 receptor, and matrix calcification in rabbit chondrocyte cultures.

The effect of IL-1 on expression of the mineralization-related phenotype by chondrocytes was examined. In cultures of rabbit growth plate chondrocytes, IL-1 beta at 0.1 ng/ml caused 95% decreases in alkaline phosphatase activity, alkaline phosphatase mRNA levels, the incorporation of 45Ca into insoluble material, and the calcium content during the hypertrophic stage. These effects of IL-1 beta were dose-dependent and were observed in 24-48 h. Furthermore, IL-1 beta suppressed increase in cell size and the syntheses of 1,25-dihydroxyvitamin D3 receptor and type X collagen, other markers of hypertrophy, but had little effect on the synthesis of total protein including type II collagen. The inhibition of calcification was observed only when chondrocytes were exposed to IL-1 before the onset of calcification: IL-1 treatment from the mineralization stage had a marginal effect on 45Ca incorporation into insoluble material. These results suggest that IL-1 inhibits chondrocyte hypertrophy and the onset of calcification in ossifying cartilage.

Study of the optimum level of electrode placement for the evaluation of absolute lung resistivity with the Mk3.5 EIT system.

Inter-subject variability has caused the majority of previous electrical impedance tomography (EIT) techniques to focus on the derivation of relative or difference measures of in vivo tissue resistivity. Implicit in these techniques is the requirement for a reference or previously defined data set. This study assesses the accuracy and optimum electrode placement strategy for a recently developed method which estimates an absolute value of organ resistivity without recourse to a reference data set. Since this measurement of tissue resistivity is absolute, in Ohm metres, it should be possible to use EIT measurements for the objective diagnosis of lung diseases such as pulmonary oedema and emphysema. However, the stability and reproducibility of the method have not yet been investigated fully. To investigate these problems, this study used a Sheffield Mk3.5 system which was configured to operate with eight measurement electrodes. As a result of this study, the absolute resistivity measurement was found to be insensitive to the electrode level between 4 and 5 cm above the xiphoid process. The level of the electrode plane was varied between 2 cm and 7 cm above the xiphoid process. Absolute lung resistivity in 18 normal subjects (age 22.6 +/- 4.9, height 169.1 +/- 5.7 cm, weight 60.6 +/- 4.5 kg, body mass index 21.2 +/- 1.6: mean +/- standard deviation) was measured during both normal and deep breathing for 1 min. Three sets of measurements were made over a period of several days on each of nine of the normal male subjects. No significant differences in absolute lung resistivity were found, either during normal tidal breathing between the electrode levels of 4 and 5 cm (9.3 +/- 2.4 Omega m, 9.6 +/- 1.9 Omega m at 4 and 5 cm, respectively: mean +/- standard deviation) or during deep breathing between the electrode levels of 4 and 5 cm (10.9 +/- 2.9 Omega m and 11.1 +/- 2.3 Omega m, respectively: mean +/- standard deviation). However, the differences in absolute lung resistivity between normal and deep tidal breathing at the same electrode level are significant. No significant difference was found in the coefficient of variation between the electrode levels of 4 and 5 cm (9.5 +/- 3.6%, 8.5 +/- 3.2% at 4 and 5 cm, respectively: mean +/- standard deviation in individual subjects). Therefore, the electrode levels of 4 and 5 cm above the xiphoid process showed reasonable reliability in the measurement of absolute lung resistivity both among individuals and over time.

Purification and characterization of cytochrome P-450 induced by benz(a)anthracene in mouse skin microsomes.

Topical application of benz(a)anthracene to mouse skin elicited a 2-fold increase in cytochrome P-450 content, with accompanying increases in monooxygenase activities such as benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, and acetanilide 4-hydroxylation, in the microsomes. A major form of cytochrome P-450 was purified from skin microsomes of mice treated with polycyclic aromatic hydrocarbon. A specific content of 1.95 nmol/mg of protein, which corresponded to 48-fold purification from the microsomes, was observed. The purified protein produced a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a molecular weight of 55,000. Using Western blotting, the band immunochemically cross-reacted with antibody which had been raised against rat liver cytochrome P-450MC-1. The purified preparation efficiently catalyzed benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation when reconstituted with NADPH-cytochrome P-450 reductase. These activities were inhibited by 7,8-benzoflavone as well as anti-cytochrome P-450MC-1 antibody, but not by P-450PB-1 antibody. The results indicate that, in mouse skin microsomes, a cytochrome P-450 induced by benz(a)anthracene is enzymatically and immunochemically similar to rat liver cytochrome P-450MC-1. It is suggested that this enzyme plays an important role in the activation of carcinogenic polycyclic aromatic hydrocarbons.

Structure of the gene encoding human liver cholesterol 7 alpha-hydroxylase.

A human cholesterol 7 alpha-hydroxylase gene spanning 17 kb in length which includes the sequence 5' flanking region (6. kb) and that of the entire transcriptional region (10.5 kb) was obtained from two partially overlapping clones (i.e., HG 18 and HG 17) of a human genomic library. The exon-intron boundaries are completely identical with that of rat gene. Several transcriptional factor recognition sequences were observed in the 5' flanking region.

Structural and phylogenetic analyses of RGD-CAP/beta ig-h3, a fasciclin-like adhesion protein expressed in chick chondrocytes.

A cDNA for RGD-CAP/beta ig-h3 was cloned from a chick embryo chondrocyte cDNA library. The deduced amino acid sequence showed that the chick RGD-CAP/beta ig-h3 is 76-77% identical with human, mouse and pig forms of the protein, and 43% identical with human and mouse osteoblast specific factor 2 (OSF2). RGD-CAP/beta ig-h3 contained four internal repeat domains and two highly conserved sequences (H1 and H2) in each repeat. Chick RGD-CAP/beta ig-h3, as well as the mammalian RGD-CAP/beta ig-h3, contained an RGD sequence, which may serve as a recognition sequence for integrins, in the fourth repeat. Database searches revealed that the H1 and H2 sequences are conserved in some secreted or membrane proteins of several species including mammals, insects, sea urchins, plants, yeast and bacteria. Phylogenetic analysis showed that a portion of the common ancestor gene for RGD-CAP/beta ig-h3 and OSF2 was duplicated to form four repeat domains before the separation of the genes followed by the divergence of vertebrate species.

Membrane-bound transferrin-like protein (MTf): structure, evolution and selective expression during chondrogenic differentiation of mouse embryonic cells.

Mouse membrane-bound transferrin-like protein (MTf) cDNA was cloned to examine its expression during chondrogenic differentiation in the mouse embryonic cell line ATDC5, and to analyze the phylogenetic relationships among the MTfs of four animal species and 23 other transferrin members. Phylogenetic analysis indicated that the MTf gene diverged from the common ancestor gene earlier than the genes of the other transferrins such as serum transferrin, lactoferrin and ovotransferrin, and that the divergence occurred after the divergence of vertebrates and invertebrates. MTf, as well as the other transferrins, consists of two repeated domains. The similarity between the N-terminal and the C-terminal domains of MTf is much higher than that of the other transferrins, although the five amino acid residues required for iron binding were not conserved in the C-terminal domain of MTf in contrast to the conservation of these residues in both domains of the other transferrins. Among various adult mouse tissues, MTf mRNA was expressed at the highest level in cartilage and at a moderate level in the testis. MTf mRNA was expressed only at very low levels in the brain, spleen, thymus, muscle, lung, skin and intestine, and hardly detected in the heart, kidney, stomach and liver. In cultures of the mouse ATDC5 cell line, MTf is developmentally expressed in parallel with the expression of type II collagen and aggrecan, in the pattern commensurate with the onset of chondrogenesis to form cartilage nodules. The structural characteristics and the expression pattern suggest that during development and in adult tissues, MTf has some functions that are different from those of other transferrins.

RGD-CAP ((beta)ig-h3) enhances the spreading of chondrocytes and fibroblasts via integrin alpha(1)beta(1).

In previous studies, RGD-CAP (collagen-associated protein containing the RGD sequence) isolated from a collagen fiber-rich fraction of pig cartilage was found to be orthologous to human (beta)ig-h3, which is synthesized by lung adenocarcinoma cells in response to transforming growth factor-beta. In the present study, we examined the effect of recombinant chick RGD-CAP on the spreading of chondrocytes and fibroblasts using RGD-CAP-coated dishes. When rabbit articular chondrocytes, chick embryonic sternal chondrocytes, rabbit peritoneal fibroblasts or human MRC5 fibroblasts were seeded on plastic dishes coated with RGD-CAP, cell spreading was enhanced compared with that on control dishes (bovine serum albumin- or beta-galactosidase-coated dishes). The effect of RGD-CAP on the cell spreading required divalent cations (Mg(2+) or Mn(2+)), and was reduced by EDTA. Monoclonal antibodies (mAbs) to the human integrin alpha(1) or beta(1) subunit, but not to the alpha(2), alpha(3), alpha(5) or beta(2) subunits, suppressed the RGD-CAP-induced spreading of human MRC5 fibroblasts. In a parallel experiment, the mAb to the alpha(5) subunit, but not the mAb to the alpha(1) subunit, suppressed fibronectin-induced spreading of these cells. These findings suggest that RGD-CAP is a novel ligand for integrin alpha(1)beta(1) that dose not bind to the RGD motif. Accordingly, an RGD-CAP fragment, which carries a deletion in the C-terminal region containing the RGD motif, was still capable of stimulating cell spreading.

Role of chondroitin sulfate-hyaluronan interactions in the viscoelastic properties of extracellular matrices and fluids.

The purpose of this study was to investigate the role of chondroitin sulfate-hyaluronan interactions in the viscoelastic properties of tissues and fluids, using capillary and cone-on-plate viscometers. Chondroitin sulfate markedly increased the viscosity of hyaluronan solutions at a wide range of hyaluronan mass (50-1900 kDa) under physiological conditions of pH, temperature, ionic strength and glycosaminoglycan concentration (0.5-40 mg/ml), although the viscosity of the chondroitin sulfate solutions themselves was very low. In the assay using a cone-on-plate viscometer, chondroitin sulfate increased the viscosity of hyaluronan solutions at various shear rates. At low shear rates, the viscosity of a chondroitin sulfate (5 mg/ml)-hyaluronan (0.5 mg/ml) mixture was about 40% of that of an aggrecan (5 mg/ml)-hyaluronan (0.5 mg/ml) mixture, and at 2.8-fold higher concentrations, chondroitin sulfate elicited the same effect on the viscosity of hyaluronan solutions (5 mg/ml) as an aggrecan monomer. In the presence of oscillatory motion, the addition of aggrecan increased the elasticity (storage) modulus G' and the viscosity (loss) modulus G" of hyaluronan solutions and markedly decreased the loss tangent G"/G' at frequencies corresponding to normal joint movements. In contrast, chondroitin sulfate had only a marginal effect on the loss tangent G"/G', although it increased G' and G". These findings demonstrated that chondroitin sulfate, as well as aggrecan, increases the viscosity of hyaluronan solutions, although chondroitin sulfate has less effect on the elasticity of hyaluronan solutions than that of aggrecan, and suggest that chondroitin sulfate may play an important physiological role in determining the viscoelastic properties of extracellular matrices and fluids.

Characterization of a cartilage-derived 66-kDa protein (RGD-CAP/beta ig-h3) that binds to collagen.

A 66-kDa collagen fiber-associated protein (RGD-CAP) was isolated from a fiber-rich fraction of pig cartilage by ultrafiltration and collagen-affinity chromatography. Amino acid sequencing and cDNA cloning indicated that the RGD-CAP is identical or closely related to beta ig-h3 protein which is induced in human adenocarcinoma cells by transforming growth factor-beta (TGF-beta) (Skonier, J., Neubauer, M., Madisen, L., Bennett, K., Plowman, G.D., and Purchio, A.F. (1992) DNA Cell. Biol. 11, 511-522). The RGD-CAP, as well as beta ig-h3, has the RGD sequence in the C-terminal region. The native RGD-CAP bound to type I, II, and IV collagens even in the presence of 1 M NaCl. A recombinant preparation of RGD-CAP expressed in Escherichia coli cells also bound to collagen but not to gelatin. The RGD-CAP mRNA was expressed in chondrocytes throughout all stages, although the expression level was highest during the prehypertrophic stage. In addition, TGF-beta increased the RGD-CAP mRNA level in chondrocyte cultures. Since RGD-CAP transcripts were found in most tissues, this novel collagen-binding protein may play an important role in cell-collagen interactions in various tissues including developing cartilage.

Clock gene expression in the submandibular glands.

Clock genes, which mediate molecular circadian rhythms, are expressed in a circadian fashion in the suprachiasmatic nucleus and in various peripheral tissues. To establish a molecular basis for circadian regulation in the salivary glands, we examined expression profiles of clock-related genes and salivary gland-characteristic genes. Clock-related genes-including Per1, Per2, Cry1, Bmal1, Dec1, Dec2, Dbp, and Reverbalpha-showed robust circadian expression rhythms in the submandibular glands in 12:12-hour light-dark conditions. In addition, a robust circadian rhythm was observed in amylase 1 mRNA levels, whereas the expression of other salivary-gland-characteristic genes examined was not rhythmic. The Clock mutation resulted in increased or decreased mRNA levels of Per2, Bmal1, Dec1, Dec2, and Dbp, and in Cry1-/- background, Cry2 disruption also increased or decreased mRNA levels of these clock-related genes and the amylase 1 gene. These findings indicate that the Clock- and Cry-dependent molecular clock system is active in the salivary glands.

Detection of emboli in vessels using electrical impedance measurements--phantom and electrodes.

A phantom was constructed to simulate the electrical properties of the neck. A range of possible electrode configurations was then examined in order to improve the sensitivity of the impedance measurement method for the in vivo detection of air emboli. The neck phantom consisted of simulated skin, fat and muscle layers made of agar and a conductive rubber tube mimicking the common carotid artery. The ring-shaped electrodes with a guard electrode showed the highest sensitivity to emboli at short distances.

Induction of basic helix-loop-helix protein DEC1 (BHLHB2)/Stra13/Sharp2 in response to the cyclic adenosine monophosphate pathway.

DEC1 (BHLHB2)/Stra13/Sharp2, a basic helix-loop-helix (bHLH) transcription factor has been suggested to be involved in the control of proliferation and/or differentiation of several cells including nerve cells, fibroblasts and chondrocytes. In the present study, we examined the effect of parathyroid hormone (PTH), dibutyryl cAMP (Bt2cAMP) and forskolin on the expression of DEC1 in various cells. In rabbit chondrocyte cultures, PTH or Bt2cAMP increased the DEC1 mRNA level within 1 h. Thereafter, the DEC1 mRNA level rapidly decreased to the basal level at 3 h, and increased at 6-24 h. In cultures of a mouse embryo prechondrogenic cell line ATDC5, PTH or forskolin, an activator of adenylate cyclase, also increased the DEC1 mRNA level within 1 h. Furthermore, in all evaluated cell lines of human fibroblasts, canine epithelial cells, human carcinoma, human glioblastoma and human melanoma, Bt2cAMP increased the DEC1 mRNA level within 1-3 h. Studies with actinomycin D and cycloheximide indicated that the enhancement of DEC1 mRNA by cAMP was not due to mRNA stabilization and did not require new protein synthesis. These findings suggest that DEC1 is a novel direct target for cAMP in wide types of cells, and that the bHLH protein is involved in the control of gene expression in cAMP-activated cells.

Retinol-binding protein is produced by rabbit chondrocytes and responds to parathyroid hormone (PTH)/PTH-related peptide-cyclic adenosine monophosphate pathway.

PTH and dibutyryl cAMP [(Bu)2cAMP] induced the expression of a 19-kDa protein in the conditioned media of rabbit growth plate chondrocyte cultures. The 19-kDa protein was identified as plasma retinol-binding protein (RBP) by aminoterminal sequence analysis and immunoblot analysis with an anti-RBP monoclonal antibody. Northern blot analysis showed that PTH, PTH-related peptide (PTHrP), and (Bu)2cAMP increased the RBP messenger RNA (mRNA) level in chondrocyte cultures. Further, both PTH and (Bu)2cAMP markedly induced the expression of RBP mRNA by about 10-fold at 3 h and by about 40-fold at 24 h, indicating a pretranslational regulation. The level of the mRNA expression induced by PTH, PTHrP, and (Bu)2cAMP was as high as that by retinoic acid (RA), known as a potent inducer of RBP in hepatoma cells. RBP mRNA was also detected in cartilage tissues at higher levels than in the other tissues examined except liver. Both RBP and PTH/PTHrP inhibited the dedifferentiative activity of RA on growth plate chondrocytes when added to the culture medium. These results demonstrate that chondrocytes synthesize and secrete RBP in vivo and in vitro and suggest that PTH/PTHrP modulates the effect of RA by means of RBP production in chondrocytes.

Regulation of messenger ribonucleic acid expression of 1 alpha,25-dihydroxyvitamin D3-24-hydroxylase in rat osteoblasts.

We have reported that PTH inhibits 25-hydroxyvitamin D3-24-hydroxylase messenger RNA (mRNA) expression induced by 1 alpha,25-dihydroxyvitamin D3 [1 alpha, 25-(OH)2D3] in rat kidney but not intestine. In the present study, we examined whether the suppression of 24-hydroxylase mRNA expression by PTH occurs commonly in tissues and cells which have PTH receptors. Administration of 1 alpha, 25-(OH)2D3 into rats fed a synthetic vitamin D-repleted diet containing adequate calcium greatly increased serum levels of calcium and 1 alpha, 25-(OH)2D3. Also, there was a 4-fold increase in bone 24-hydroxylase activity in response to 1 alpha, 25-(OH)2D3 administration. In rats fed a low calcium diet, renal 24-hydroxylase activity was suppressed probably due to secondary hyperparathyroidism. In contrast, the low calcium feeding did not suppress bone 24-hydroxylase activity. The expression of 24-hydroxylase mRNA in rat osteoblastic C-26 and C-11 cells was similar and attained maximal levels 24 h after cells were incubated with 10(-8) M 1 alpha, 25-(OH)2D3. Induction of 24-hydroxylase mRNA expression by 1 alpha, 25-(OH)2D3 was much greater and earlier in immature C-26 cells than mature C-11 cells. Simultaneous addition of PTH, prostaglandin E2, or cAMP together with 1 alpha, 25-(OH)2D3 did not down-regulate mRNA expression of 24-hydroxylase induced by the vitamin in both C-26 and C-11 cells. Of the three osteoblastic cells (C-26, C-20, and C-11) examined, C-26 cells showed the least mRNA expression of vitamin D receptors, in spite of the highest expression of 24-hydroxylase mRNA. These results suggest that unlike in the kidney, bone 24-hydroxylase mRNA expression is not down-regulated by PTH despite of the presence of PTH receptors. They also suggest that the degree of the induction of 24-hydroxylase mRNA by 1 alpha, 25-(OH)2D3 is not explained simply by the vitamin D receptors content.

Effects of cyclic adenosine 3',5'-monophosphate on chondrocyte terminal differentiation and cartilage-matrix calcification.

We examined the effects of cyclic AMP on terminal differentiation and calcification in rabbit growth plate chondrocyte cultures. Dibutyryl cAMP (dbcAMP), as well as 8-bromo-cAMP abolished the increases in chondrocyte size, alkaline phosphatase activity, type X collagen synthesis, 1 alpha, 25-dihydroxyvitamin D3 receptor synthesis, the incorporation of 45Ca into insoluble material, and the calcium content. All of these occurred in parallel untreated cultures during the hypertrophic (terminal) stage. The inhibition of alkaline phosphatase by dbcAMP was detectable after 24 h, and this effect was reversible. dbcAMP and 8-bromo-cyclic AMP inhibited alkaline phosphatase induction and calcification at low concentrations (3-5 microM), whereas 10-30-fold higher concentrations were required to stimulate proteoglycan synthesis. These findings suggest that cAMP plays a crucial role in suppressing terminal differentiation of chondrocyte and cartilage-matrix calcification.

Effects of parathyroid hormone (PTH) and PTH-related peptide on expressions of matrix metalloproteinase-2, -3, and -9 in growth plate chondrocyte cultures.

The roles of PTH and PTH-related peptide (PTH-rp) in the expression of matrix metalloproteinases (MMPs) during endochondral bone formation were investigated, using various cartilages obtained from young rabbits and rabbit chondrocyte cultures. Immunohistochemical, immunoblotting, zymographical, and/or Northern blot analyses showed that MMP-2 and -9 levels were much higher in the growth plate than in permanent cartilage in vivo. In growth plate chondrocyte cultures, PTH, PTH-rp, and (Bu)2cAMP increased the amount of MMP-2 present in the culture medium, as revealed by zymograms and immunoblots, whereas the other tested growth factors or cytokines, including bone morphogenetic protein-2 and interleukin-1, did not increase the MMP-2 level. PTH also increased the MMP-2 messenger RNA level within 24 h. In addition, PTH increased MMP-3 and -9 levels in the growth plate chondrocyte cultures. However, in articular chondrocyte cultures, PTH had little effect on the levels of MMP-2, -3, and -9. In contrast to PTH, interleukin-1 induced MMP-3 and -9, but not MMP-2, in growth plate and articular chondrocytes. These findings suggest that in ossifying cartilage, PTH/PTH-rp plays a pivotal role in the induction of various MMPs, including MMP-2 (which is considered to be a constitutive enzyme), and that PTH/PTH-rp is involved in the control of cartilage-matrix degradation during endochondral bone formation.

Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase.

A complete cDNA clone encoding human cholesterol 7 alpha-hydroxylase has been isolated using a rat P-450ch7 alpha cDNA insert [(1989) FEBS Lett. 257, 97-100] as a probe and totally sequenced. The cDNA contained 1512-base pair open reading frame encoding 504 amino acid residues (Mr 57,630), 39-base pair 5'-untranslated region 1322-base pair 3'-ultranslated region including 20 nucleotides of poly A tail in the total length of 2873 base pairs. The deduced amino acid sequence showed 82% similarity to rat P-450ch7 alpha. Unique amino acid residues were observed in putative binding domains for heme and steroid which are highly conserved in most steroidogenic P-450s.

Cloning and expression of cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase.

A cDNA encoding 25-hydroxyvitamin D3 24-hydroxylase (P450cc24) was isolated from a rat kidney cDNA library using specific antibodies to the enzyme. The isolated cDNA was 3.2 kbp long and contained a 1542-bp open reading frame encoding 514 amino acids. The deduced amino acid sequence contained a presequence typical of mitochondrial enzymes in the N-terminal region. The amino acid sequence shows less than 30% similarity to those of any other cytochrome P450s so far reported and, therefore, P450cc24 constitutes a novel family of P450. COS-7 cells transfected with the cDNA produced a protein that was reactive with the antibodies and catalyzed NADPH-dependent 24-hydroxylation of 25-hydroxyvitamin D3 in the presence of adrenodoxin and NADPH-adrenodoxin reductase. Using the cDNA as a probe we demonstrated that the increase of 24-hydroxylation activity caused by administration of vitamin D3 into rats was accompanied by an increase of the mRNA.

Molecular cloning of cDNA for vitamin D3 25-hydroxylase from rat liver mitochondria.

A cDNA clone encoding mitochondrial vitamin D3 25-hydroxylase was isolated from a rat liver cDNA library by the use of specific antibodies to the enzyme. The isolated cDNA clone was 1.9 kbp long and contained a 1599 bp open reading frame encoding 533 amino acid residues. The deduced primary structure contained a presequence typical for mitochondrial enzymes in the N-terminal region. The N-terminal sequence of the mature enzyme was determined to be Ala-Ile-Pro-Ala-Ala, which agrees perfectly with a portion of the deduced sequence, establishing the cleavage point of the precursor.

Molecular cloning and sequence analysis of cDNA encoding delta 4-3-ketosteroid 5 beta-reductase of rat liver.

A cDNA clone encoding delta 4-3-ketosteroid 5 beta-reductase was isolated from rat liver cDNA libraries using antibodies specific for the enzyme and oligonucleotides as probes. The cDNA contained 981-base pair open reading frame encoding 327 amino acid residues (Mr 37,376) and an unusually long 3'-untranslated region rich in AT sequence in the total length of 3189 base pairs. The predicted amino acid sequence contains the sequences similar to the putative NADPH- and steroid-binding regions.